Analyst® 1.6.

2 Software

Advanced User Guide

RUO-IDV-05-0271-A
Release Date: April 2013
This document is provided to customers who have purchased AB SCIEX equipment to use in the
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Foreword. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Access System Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Contact Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Chapter 1 General Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Analyst Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
API Instrument Project Folders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Program Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Projects and Subprojects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Subprojects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Project Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Software Security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Workspaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Chapter 2 Tune and Calibrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Automatic Tuning and Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Back up Instrument Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Restore Instrument Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Compound Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Flow Injection Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
T-Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Chapter 3 Batches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Batch Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Batch Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Build a Batch as a Text File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Import a Batch as a Text File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Set Quantitation Details in the Batch Editor (Optional) . . . . . . . . . . . . . . . . . .21
Chapter 4 Device Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Devices in Acquisition Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Add or Remove an LC Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Set the LC Pump Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
Set the Autosampler Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
Set the Integrated Syringe Pump Properties . . . . . . . . . . . . . . . . . . . . . . . . . .25
Set the Column Oven Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
Set the Switching Valve Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
Set the Diode Array Detector Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Set the Analog-to-Digital Converter Properties . . . . . . . . . . . . . . . . . . . . . . . .26
Dynamic Fill Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Experiments and Periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Information Dependent Acquisition Methods . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Solvent Compressibility Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

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Syringe Size Versus Flow Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

Chapter 5 Qualitative Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Chromatograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32
Background Subtraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32
Perform a Background Subtraction from a Spectrum . . . . . . . . . . . . . . . . . . .32
Unlock the Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Background Subtract to File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Perform a Background Subtraction to File . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Baseline Subtract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
Calculators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
Elemental Composition Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
Hypermass Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
Elemental Targeting Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
Mass Property Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
Isotopic Distribution Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
Centroided Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
Calculate the Centroid of a Peak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
Total Ion Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
Extracted Ion Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
Base Peak Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
Extracted Wavelength Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
Diode Array Detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
Total Wavelength Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
Overlay Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
Cycle Between Overlaid Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
Sum Overlays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
Graphs Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
Add Captions to a Graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
Add Text to a Graph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
Compound Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
Contour Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
View a Contour Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Select an Area in a Contour Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Set the Intensity and Absorbance in a Contour Plot . . . . . . . . . . . . . . . . . . . .42
Change Colors in a Contour Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Dynamic Background Subtraction Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Fragment Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Connect the Fragment Interpretation Tool to a Spectrum . . . . . . . . . . . . . . . . . .43
Match Fragments with Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
Select a Bond in a Molecular Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
View Isotopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
Display a Formula Difference in a Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . .44
Display a Formula Difference in the Fragment List . . . . . . . . . . . . . . . . . . . . .45
Display a Formula Difference in a Molecular Structure . . . . . . . . . . . . . . . . . .45
IDA Explorer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
Library Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Switch Between Existing Library Databases . . . . . . . . . . . . . . . . . . . . . . . . . .47

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Connect to a Local Library Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48

Connect to a Server Library Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
View All Library Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
Add a Record to the Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
Search Library Records with Constraints . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
Library Search Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Search for a Similar Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
View a Compound from the Search Results . . . . . . . . . . . . . . . . . . . . . . . . . .53
Processed Data Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
Save a Processed Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
Open a Processed Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
Qualitative Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
Signal-to-Noise Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54
Smoothing Algorithms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54
Smooth Data using the Smooth Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . .55
Smooth Data using Gaussian Smoothing . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
Spectral Arithmetic Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
Toolbar Icons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
Chapter 6 Quantitative Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Calibration Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
About Calibration Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
Select the Best Regression Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
Integration Algorithms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60
Analyst Classic and IntelliQuan Integration Algorithms . . . . . . . . . . . . . . . . . .61
Quantitation Method-Creation Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62
Wizards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62
Find Peaks Using an Automatic Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
Quantitation Method Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
The Semi-Automatic Method Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
Find Peaks Using a Semi-Automatic Method . . . . . . . . . . . . . . . . . . . . . . . . .64
Metric Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
Generate a Metric Temporary Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65
Generate a Metric Plot and Save the Plot Criteria . . . . . . . . . . . . . . . . . . . . . .65
Save Default Plot Criteria for Future Results Tables . . . . . . . . . . . . . . . . . . . .67
Noise and Area Threshold Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
Recalculate the Noise and Area Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . .68
Peak Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
Peak Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
Peak Review Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69
Detect Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69
Find the Potential Peak Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70
Confirm the Peak Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70
Find the Peak Top . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
Find the Peak End . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
Separate Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
Queries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75
Queries on Sample Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
Default Queries and Table-Specific Queries . . . . . . . . . . . . . . . . . . . . . . . . . .76
How Accuracy Variations Affect the Results . . . . . . . . . . . . . . . . . . . . . . . . . .76

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Regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
Linear Regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
Linear Through Zero Regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
Mean Response Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
Quadratic Calibration Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
Report Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79
Customize Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81
Preview, Print, and Export Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81
Results Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82
Define the Layout of Results Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82
Sort Data in Results Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
Sort a Results Table and Save the Sort Criteria . . . . . . . . . . . . . . . . . . . . . . .83
Save Default Sort Criteria for Future Results Tables . . . . . . . . . . . . . . . . . . .84
Sort a Results Table using Preset Sort Criteria . . . . . . . . . . . . . . . . . . . . . . . .85
About using Queries with Results Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
Compare Results Between Batches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86
How Concentration Levels Affect Results . . . . . . . . . . . . . . . . . . . . . . . . . . . .86
Results Table Layouts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86
Summary Layout View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87
Analyte Layout View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87
Analyte Group Layout View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .88
Results Table Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .88
Results Table Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94
Weighting Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94
Appendix A PPG Exact Mass Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97

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Foreword

This Advanced User Guide provides information about the Analyst® software features.

Access System Documentation

The Help, guides, and tutorials for the mass spectrometer and the software are installed
automatically.
• Click Start > All Programs > AB SCIEX > Analyst. A complete list of the available
documentation can be found in the Help.
• Press F1 to open the Help.

Contact Technical Support

The manufacturer and its representatives maintain a staff of fully-trained service and technical
specialists located throughout the world. They can answer questions about the system or any
technical issues that might arise. For more information, visit the Web site at www.absciex.com or
contact Technical Support using [email protected].

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Foreword

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General Information
1
The software is divided into discrete functional areas called modes. Modes allow the user to
perform activities related to a main task. Modes are accessed through the Navigation bar or the
Mode list in the toolbar. Users can switch from one mode to another without any loss of work. For
more information, refer to the Analyst® software Show Me tutorial.
Figure 1-1 Analyst Software Window

Item Description
1 Mode list
2 Navigation bar

Analyst Service
The Analyst Service is the communication path between the mass spectrometer and attached
devices. The Analyst Service is started each time the Analyst software is started. In general the
Analyst Service starts automatically when the user logs on to Windows. If the service is not
running when the Analyst software is started, then the Analyst Service will start automatically.

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General Information

API Instrument Project Folders

The API Instrument project folder is important for the instrument to function properly. The API
Instrument project contains the information required to tune and calibrate the system. The API
Instrument project folder also contains data files for a manual tune that was performed using the
Start button instead of the Acquire button. These data files are saved automatically in the API
Instrument project folder in the Tuning Cache folder and named with the date and time they were
created. Empty the Tuning Cache folder regularly.
The following are some of the folders found in the API Instrument project.
• Bundler: Contains a program that takes all aspects of a data file (.wiff file) and
automatically combines them when the sample is completed.
• Configuration: Contains all the hardware profiles (.hwpf files).
• Example Scripts: Contains some of the scripts that are used with the software.
• Instrument Data: Contains a file called InstrumentData.ins. The file stores all the
critical calibration information and more.
• Method Tables: Contains all instrument parameters that define the enhanced scan
functions. Do not change the files in this folder. Changing the contents of this folder
will affect the performance of the enhanced scan modes.
• Parameter Settings: Contains all the instrument parameters and linkages.
Instrument parameters are saved as ParamSettingsdef.psf files.
• Preferences: Contains the Tunedata.tun file. All settings (parameter, tuning,
instrument, processing, appearances, and queue) are saved as Tunedata.tun in this
folder.
• Processing Scripts: Contains the scripts for data processing in Explore mode.
Scripts are found in the Script menu.
• Queue Data: Contains information from the queue.
• Tuning Cache: Contains all the data created in Manual Tuning by clicking Start
instead of Acquire. Files are saved with a generic time and date stamp for their
names. The Tuning Cache folder holds a limited number of files and will overwrite
files as needed. Save the files with a new name and move the files immediately if
they need to be saved.

Program Files
The following folders are found within the Program Files\Analyst folder.
• bin: Contains the software program files. Contents of this folder should not be
changed as this will affect the software functionality.
• Firmware: Contains the instrument system controller software (scu21.exe) and the
instrument firmware files. Use these files to download new firmware to the
instrument when required. For more information, refer to the software installation
guide included with the software.
• Help: Contains the help files, guides, tutorials, release notes and software
installation guide.

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 General Information

• Scripts: Contains all the scripts that can be installed. Research-grade scripts are
available to extend the functionality of the software. Some scripts are installed
automatically and some scripts can be installed individually. For more information,
refer to the Scripts User Guide.
• Simulation: Contains the instrument data files required to run the software in
simulation mode.

Projects and Subprojects

Decide where to store the files related to the experiment before starting the experiment. Use
projects and subprojects for each experiment to manage the data better and compare the results.

Subprojects
A subproject contains a subset of the folders in the project. All subprojects must contain the same
folders. Subprojects are useful for organizing data.
For example, if samples of various compounds from different laboratories are run using the same
acquisition method, then create subprojects to store the results for each laboratory, but leave the
acquisition method folder in the project. The acquisition method is then available for use in the
subproject or laboratory. Alternatively, if samples are being analyzed over a period of several
weeks, then the results from each day can be stored in a separate subproject. Refer to
Figure 1-2 on page 12.

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General Information

Figure 1-2 Example of a Project and Subproject Folder Structure

Project Organization
A project is a folder structure for organizing and storing sample information, data, quantitation
information, and so forth. Within each project there are folders that can contain different types of
files. For example, the Data folder contains acquisition data files. Table 1-1 on page 12 describes
the contents of the different folders.
The software can access a project only if it is stored in a root folder. Users cannot create projects
in a folder that has not been defined as a root folder.
The preset root folder is Analyst Data on the drive where the software is installed. To store
projects in other locations, create new root folders. For more information about root folders, refer
to the Help.
Table 1-1 Project Folders
Folder Contents
\Acquisition Methods Contains all acquisition methods used. Acquisition methods have the
.dam extension.
\Acquisition Scripts Contains all the acquisition batch scripts available.

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 General Information

Table 1-1 Project Folders (Continued)

Folder Contents
\Batch Contains all the acquisition batch files used. Acquisition batches have
the .dab extension. It also contains a subfolder, Templates, that contains
acquisition batch templates. Batch templates have the .dat extension.
\Data Contains the acquisition data files, which have the .wiff extension.
\Log Contains results of quantitation and compound optimization.
\Processing Methods Contains all qualitative data processing methods used.
\Processing Scripts Contains all data processing scripts available. Processing scripts stored
in the API Instrument project are shown in the Scripts menu.
\Project Information Contains all project information and settings for the project. This folder
cannot be stored in a subproject.
\Quantitation Methods Contains all quantitation methods used, which have a .qmf extension.
\Results Contains all quantitation Results Table files, which have the .rdb
extension.
\Templates Contains report templates, which have the .rpt extension.

Software Security
The software has a number of functions for configuring and managing security. The Analyst®
software administrator can:
• Choose a security mode to best suit the needs of the operating environment.
• Add and delete users and roles.
• Set access rights to users and roles as required.
• Control access to remote instrument stations.
• Control access to project files.
For more information about Analyst software security, refer to the Laboratory Director’s Guide.

Workspaces
A workspace is a particular arrangement of windows and panes, including any associated file or
files. For example, while working on a particular data set, users can open and size various
windows to help with the analysis. This arrangement, or workspace, can be saved so that the
next time users look at the data, the window arrangement is identical.
In Quantitate and Explore modes, users can have multiple workspaces per session. This means
that different workspaces can be designed to suit different tasks within these modes, and then
save them for future use. When in one of these two modes, a particular workspace can be
opened without exiting that mode.

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Tune and Calibrate
2
Tuning the instrument is the process of optimizing the resolution and instrument parameters to
attain the best sensitivity and performance of the mass spectrometer. Optimizing the resolution
means adjusting the peak width and peak shape. Users can tune and calibrate the instrument
either automatically or manually.

Caution: Potential Calibration Error. If the temperature changes by more than 2°C, then
the resolution and mass calibration might be affected.

Tip! Clean the Q0 region regularly to minimize the impact of charging (a significant
loss of sensitivity of the ions of interest over a short period of time) on the quadrupoles.
Refer to the Qualified Maintenance Person Guide.

Automatic tuning: The software performs resolution optimization and mass calibration, using
the Instrument Optimization wizard. For linear ion trap (LIT) instruments, MS3 optimizations are
also performed.
Manual tuning: Users can perform many of the instrument resolution optimizations and
calibrations manually.

Automatic Tuning and Calibration

Instrument Optimization is automatic instrument tuning software that tunes both quadrupole and
LIT modes and performs mass calibration. For quadrupole mode, it adjusts the resolution offsets.
For LIT mode, it optimizes AF3 and EXB. For MS3, it adjusts the excitation and isolation
coefficients. Select one of the following instrument performance options:
• Verify instrument performance: Tests the instrument performance but leaves the
instrument settings unchanged. A report is generated at the end of the test. Use this
option weekly to check how well the instrument is performing.
• Adjust mass calibration only: Automatically checks and adjusts the mass
calibration. If the mass calibration has changed, then the software corrects it. Use
this option weekly for LIT instruments or monthly to check and adjust the mass
calibration if required.
• Adjust instrument settings: Checks and adjusts the instrument settings and mass
calibration. The instrument settings are updated from the current settings to optimal
settings. Use this option if instrument performance is poor or if the peak shape is
bad. Only experienced users should adjust the instrument settings.

Note: Old LIT methods must be updated with the new settings. Toggle the
LIT speed in the advanced MS tab and then save the method.

• Reset selected scan modes to default values and adjust instrument settings:
Resets the instrument values to the factory preset values. Select this option if a
major component of the instrument is replaced or after the first installation. Only
FSEs should use this feature.

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Tune and Calibrate

Back up Instrument Parameters

Back up the current instrument parameters in case they must be restored later. The preset
location for the instrument parameters is C:\Analyst Data\Projects\API Instrument\Instrument
Optimization\Instrument Settings Backups\User Created Backups.
1. In the Navigation Bar, double-click Instrument Optimization.
2. Click File > Backup Instrument Settings.
3. Type a file name.
4. Click Save.

Restore Instrument Parameters

1. In the Navigation Bar, double-click Instrument Optimization.
2. Click File > Restore Instrument Settings Files.
3. Navigate to the instrument settings.
4. Click Open.

Compound Optimization
The Compound Optimization software wizard automatically optimizes an analyte. Samples can
be introduced using infusion or FIA (flow injection analysis.) When the SelexION™ device is
installed on the mass spectrometer, samples can also be introduced using the T-infusion
option.The software first checks for the presence of the compounds. The voltages of the various
ion path parameters are gradually increased or decreased to determine the maximum signal
intensity (Q1 scan) for each ion. A text file is generated and then displayed during the
optimization process. This file records the various experiments performed and the optimal values
for each ion optic parameter. A file folder containing all the experiments performed is also
generated and can be found by opening the data file folder in Explore mode. For each
experiment performed, an acquisition method is also generated and saved in the acquisition
method folder.

Flow Injection Analysis

Flow Injection Analysis (FIA) is the injection of a small quantity of a sample by an autosampler
into the LC stream. During the FIA optimization process, multiple sample injections are
performed for various source- or compound-dependent, or both, parameter types that are
changed between injections. FIA optimizes for declustering potential, collision energy, and
collision cell exit potential by performing looped experiments in succession, that is, one
compound-dependent parameter followed by then next compound-dependent parameter. It
optimizes for source-dependant parameters by making an injection for each parameter.
Use FIA optimization to optimize both compound- and source-dependent parameters using LC at
higher flow rates.

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 Tune and Calibrate

Infusion
Infusion is the continuous flow of the sample at low flow rates into the ion source using a syringe
pump. During the infusion optimization process, the software can select precursor and product
ions and optimize for declustering potential, collision energy, and collision cell exit potential for
both. The voltages of these ion path parameters are gradually increased or decreased to
determine the maximum signal intensity for the precursor and product ions.
Use infusion optimization to optimize compound-dependent parameters only at much lower flow
rates than those used during LC/MS analysis.

T-Infusion
T-infusion (or split infusion) is the continuous flow of the sample at low rates into the ion source
through a three-way grounding union on the ion source. The three-way grounding union is
connected to a syringe pump with a red PEEK tubing and an LC pump.
Use T-infusion to automatically optimize DMS parameters (SV, COV, and DMO) and compound
parameters.

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Batches
3
A batch is a collection of information about the samples to be analyzed. Samples are usually
grouped into sets to make it easier to submit them. Grouping the samples into a set also reduces
the amount of data that must be typed manually. A set can consist of a single sample or multiple
samples. All of the sets in a batch use the same hardware profile, however, samples in a set can
have different acquisition methods. A batch can be submitted only from an acquisition station.
Batches link together:
• Sample information, such as name, ID, and comment.
• Autosampler location (rack information).
• Acquisition methods.
• Processing method or script (optional).
• Quantitation information (optional).
• Custom sample data (optional).
• Set information.

Batch Editor
Use the Batch Editor to create or modify batches and to create batch templates. To run samples,
each using different acquisition methods, select multiple acquisition methods in the same set.
An acquisition method can also be used as a template. In this case, the same method is used for
each sample, but the user can select different masses or mass ranges for each sample. The
Batch Editor can also be used to import sample lists created in external programs, such as
Microsoft Excel.
The user can modify every detail of the batch before submitting it for processing. When a batch is
submitted for analysis, the user can submit the entire batch, specific sets within the batch, or
specific samples within a set.
For example, to analyze ten samples, five using one acquisition method and five using a different
acquisition method, create a batch of two sets, one for each method used.
Table 3-1 Batch Editor Tabs
Tab Description
Sample Used to create the sample list and to select sample details such as the sample
name and the acquisition method to be used to acquire the sample.
Locations Used to select the positions of samples in the autosampler. Sample locations can
be specified numerically in the Sample tab, however, the Locations tab provides
a graphical interface for selecting sample locations.
Quantitation Used to select the sample types and concentrations for quantitation batches.
Because quantitation information can be specified post-acquisition in the
quantitation Results Table, users do not have to use the Quantitation tab in the
Batch Editor. Instead, the Quantitation Wizard can be used.

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Batches

Table 3-1 Batch Editor Tabs (Continued)

Tab Description
Submit Used to verify sample information and to submit samples to the acquisition
queue. The Queue Manager shows queue, batch, and sample status and allows
users to manage samples in the queue.

Batch Files
To make data entry easier, batch file information from other applications can be imported. The
Batch Editor can import text and other files that are formatted correctly. For examples of correctly
formatted files, refer to the Batch folder in the Example project.
The information in a batch file can also be exported for use with other applications, such as
Microsoft Excel, Microsoft Access, and certain LIMS (Laboratory Information Management
System) software.

Build a Batch as a Text File

Users can export a batch only if it contains at least one set with at least one sample. If the text file
is saved, it can be used again later as a template.
1. Make sure that the active hardware profile includes all the devices needed to acquire
the samples.
2. In the Navigation Bar, under Acquire, double-click Build Acquisition Batch.
3. Create a single-set, single-sample batch.
4. Click File > Export.
5. Name the file.
6. Click Save.
7. Open the text file in a spreadsheet program.
8. Type, or copy and paste, the details for the samples: one sample per row, with the
details under the appropriate headings.

Note: Do not delete any of the columns. The columns in the spreadsheet
must match the columns in the Batch Editor.

9. Save the modified text file as a .txt or .csv file.

10. Close the spreadsheet program.
The text file can now be imported into the Batch Editor.

Import a Batch as a Text File

Before batch information is imported from a text file, make sure the data in the file is organized
and formatted correctly. In particular, the column headings in the spreadsheet must match the
Batch Editor column headings.
1. In the Navigation Bar, under Acquire, double-click Build Acquisition Batch.

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 Batches

2. In the Sample tab, right-click and then click Import From > File.
3. Click the text file containing the batch information.

Note: If the saved text file is not visible, then in the Files of type list, select
Microsoft Text Driver (*.txt; *.csv). Files with the extension .txt appear in the
field.

4. Click Open.
If an autosampler is being used, then the Select Autosampler dialog opens.
5. In the autosampler list, select the autosampler.
6. Click OK.
The sample table fills with the details from the text file.

Set Quantitation Details in the Batch Editor (Optional)

If users do not want select quantitation details post-acquisition, then Quantitation methods can
be included with a batch and used to define the quantitation details prior to submitting a batch.
Internal Standard and Standard columns are shown in the Quantitation tab according to the
quantitation method selected in the Sample tab.
1. With a batch file open in the Batch Editor window, click the Quantitation tab.
2. Select the set.
3. In the cell, select a Quant Type for all the samples.
4. If applicable, type the peak concentration in the Analyte column.
5. If applicable, type the Internal Standard.
6. Repeat the preceding steps for each set in the batch.
7. Save the file.

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Batches

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Device Methods
4
If creating an acquisition method file from an existing file, the user can use some or all of the
device methods in the acquisition method. Use the Acquisition Method Editor to customize the
acquisition method by adding or removing device methods. If the required device icon is not in
the Acquisition Method Browser pane, then users can add the device only if it is included in the
active hardware profile.
It is recommended that only users who are proficient in method development create or modify
acquisition and quantitation methods. Refer to the About People and Roles section in the
Laboratory Director’s Guide for more information about roles and security.

Devices in Acquisition Methods

Create an acquisition method for a device by selecting the operating parameters for that device.
Acquisition methods can be created for any of the following devices if they are configured in the
active hardware profile:
• Pumps.
• Autosamplers.
• Syringe pumps.
• Column ovens.
• Switching valves.
• Diode array detector.
• Analog-to-digital converters.
• Integrated systems.
For information about setting properties for devices, refer to the Peripheral Devices Setup Guide.

Note: The available parameters for the LC devices vary depending on the
manufacturer.

Add or Remove an LC Device

1. With a method file open in the Acquisition Method Editor, in the Acquisition method
pane, right-click Acquisition Method and then click Add/Remove Device Method.

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Device Methods

Figure 4-1 Add/Remove device methods Dialog

2. Select or clear the check boxes beside the device method to add or remove the
device method.
3. Click OK.

Set the LC Pump Properties

1. With an acquisition method file open in the Acquisition Method Editor, in the
Acquisition method pane, click the Pump icon.
The tab for the properties of the selected pump opens in the Acquisition Method
Editor pane.
2. Edit the fields as required.
3. Save the file.

Set the Autosampler Properties

1. Make sure that on the Acquisition Properties tab, the Synchronization Mode field
is set to LC Sync. The device and the instrument will start simultaneously.
2. With a method file open in the Acquisition Method Editor, in the Acquisition method
pane, click the Autosampler icon.
The Autosampler Properties tab opens in the Acquisition Method Editor pane.
3. Edit the fields as required.
4. Save the file.

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 Device Methods

Set the Integrated Syringe Pump Properties

This procedure is for systems with built-in syringe pumps.
1. With an acquisition method file open in the Acquisition Method Editor, in the
Acquisition Method Browser pane, click the Syringe Pump icon.
The Syringe Pump Method Properties tab appears in the Acquisition Method Editor
pane.
2. Edit the fields as required.
3. Save the file.

Set the Column Oven Properties

1. With a method file open in the Acquisition Method Editor, in the Acquisition method
pane, click the Column Oven icon.
The Column Oven properties tab opens in the Acquisition Method Editor pane.
2. Edit the fields as required.
3. Save the file.

Set the Switching Valve Properties

The switching valve can be used as a diverter or injection valve. Select the Manual Sync with
Valve synchronization mode if using the valve as an injector. Select any other mode if using the
valve as a diverter.
1. With a method file open in the Acquisition Method Editor, in the Acquisition method
pane, click the Valve icon.
The Valve Properties tab opens in the Acquisition Method Editor pane.
2. Change the position names from their preset names, if required.
The switching valve is sometimes used to switch the flow of solvent to waste, or to a
different column. The preset position names are A and B.
• In the Change Position Names list, select a position.
• In the Change Position Names list, rename the preset position names A and
B to Inject and Divert or to Column and Waste, depending on how the valve is
plumbed.
3. In the Total Time (min) column, click a cell, and then type the total time the valve will
remain in this position.
4. In the Position column, click a cell and then, in the Position list, select the valve
position.
5. Repeat the steps 3 and 4 for each switch of the valve required during acquisition.
6. Save the file.

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Device Methods

Set the Diode Array Detector Parameters

1. With a method file open in the Acquisition Method Editor, in the Acquisition method
pane, click the Diode Array Detector (DAD) icon.
The DAD Method Editor tab opens in the Acquisition Method Editor pane.
2. Set the desired acquisition parameters and save the file.

Set the Analog-to-Digital Converter Properties

1. With a method file open in the Acquisition Method Editor, in the Acquisition method
pane, click the Analog to Digital Converter (ADC) icon.
The Analog/Digital Convertor Properties tab opens in the Acquisition Method Editor
pane.
2. In the Sample section, in the Rate (pts/sec) field, type the rate.

Note: The interval and rate are proportional to each other. When the rate
is changed, the software automatically calculates the interval again.

3. Do the following to set the channel details:

• In the Channels field, click the channel name, and then select the check box
beside the name to include it in the method.
• In the Interpreted Value @ Full Scale field, type the appropriate value.
• In the Interpreted Unit field, type the appropriate unit.
The number of available channels is specified when setting up the ADC in the
hardware profile.
4. Save the file.

Dynamic Fill Time

Dynamic Fill Time (DFT) is a feature specifically designed to optimize the data obtained in every
spectrum for the linear ion trap scan functions. DFT will automatically adjust the fill time used to
fill the ion trap based on the ion flux coming from the source. For more intense ions, the fill time
will be automatically reduced to ensure the trap is not overfilled with ions.
For less intense ions, the fill time will be automatically increased, ensuring that good ion statistics
are obtained in the spectrum. DFT is applicable for the following scan types:
• Enhanced MS (EMS)
• Enhanced Resolution (ER)
• Enhanced Product Ion Scan (EPI)
• MS/MS/MS (MS3)
Adjust the DFT settings by selecting Tools > Settings > Method Options in the software.

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 Device Methods

Experiments and Periods

The mass spectrometer acquisition method consists of experiments and periods. In the
Acquisition Method Browser pane, create a sequence of acquisition periods and experiments for
the instrument. Users can also open a method previously created in the Tune Method Editor.

Experiments
An experiment includes the instrument settings and the scan type during an MS scan. A set of
MS scans performed for a specific amount of time is called a period. An acquisition method in
which the MS parameters and actions are the same through the entire duration is called a single-
period, single-experiment method.
In looped experiments, MS settings are changed on a scan-by-scan basis. For example, if the
sample contains two compounds, A and B, users may want to loop an MS/MS experiment of
compound A with an MS/MS experiment of compound B to obtain information about both
compounds in the same run. The mass spectrometer method will alternate between the two scan
types. Other examples of looped experiments include alternating between positive and negative
modes in a run and Information Dependent Acquisition (IDA) methods.

Periods
A period can contain one or more looped experiments. In a multi-period acquisition method,
experiments are performed for a specified amount of time and then switch to another set of
experiments. Periods are useful when the elution time of the compounds in an LC run is known.
The instrument can perform different experiments according to when the compounds elute to
obtain as much information as possible in the same run.
Figure 4-2 shows a three-period method. The first period has a duration of 3.015 minutes; the
second period is 4.986 minutes; and the third period is 7.000 minutes.
Figure 4-2 Example of a Multi-Period Experiment

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Device Methods

Information Dependent Acquisition Methods

Information Dependent Acquisition (IDA) is an acquisition method that analyzes data during
acquisition. IDA is used to change the experimental conditions depending on the analysis results.
These real-time changes are controlled by criteria set in the acquisition method, including:
• Ion intensity and charge state.
• Inclusion and exclusion lists.
• Isotope pattern.
Optimizing data acquisition settings while the data is being acquired allows users to conserve
both the sample and working time on an instrument.
Create an IDA method with up to two survey scans and eight dependent scans in a single
experiment. A survey scan is used in IDA to trigger additional experiments. The following scan
types can be used as a survey scan:
• Enhanced Multiply-Charged (EMC).
• Enhanced Product Ion (EPI) (second survey scan).
• Enhanced MS (EMS).
• MRM.
• Neutral Loss (NL).
• Precursor Ion (Prec).
• Q3 MS.
The following scan types can be used as dependent scans:
• EPI.
• MS/MS.
In an IDA experiment, the instrument actions are varied from scan type to scan type based on the
data acquired in a previous scan. The software analyzes data as it is being acquired and then
determines the masses on which to perform dependent scans. Uses can set the criteria that will
activate an IDA experiment and the method parameters to be used.
IDA experiments modify experiments and improve results based on the following user-defined
criteria:
• Ion intensity and charge state.
• Inclusion and exclusion lists.
• Isotope pattern.
• Dynamic exclusion.
• Rate of change in ion intensity (refer to Contour Plots on page 40).

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Solvent Compressibility Values

Table 4-1 Solvent Compressibility Values
Solvent Compressibility (10–6/bar)
Acetone 126
Acetonitrile 115
Benzene 95
Carbon Tetrachloride 110
Chloroform 100
Cyclohexane 118
Ethanol 114
Ethyl acetate 104
Heptane 120
Hexane 150
Isobutanol 100
Isopropanol 100
Methanol 120
1-Propanol 100
Toluene 87
Water 46

Syringe Size Versus Flow Rate

The flow rate of a syringe pump depends on the syringe installed in the pump. The following
tables show the relationship between flow rate and syringe size.
Table 4-2 Syringe Size and Flow Rate at L/hour
Syringe size L/hour
(µL) Minimum Maximum
0.5 .002 23.8
1.0 .003 47.8
2.0 .006 95.2
5.0 .015 238.0
10.0 .029 474.0
25.0 .073 1193.0

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Table 4-3 Syringe Size and Flow Rate at µL/minute

Syringe size µL/minute
(µL) Minimum Maximum
50 .002 39.7
100 .005 79.7
250 .012 197.8
500 .024 397.0
1000 .048 795.0
1.0 .049 805.0

Table 4-4 Syringe Size and Flow Rate at mL/hour

Syringe size mL/hour
(mL) Minimum Maximum
2.0 .011 186.8
2.5 .010 168.2
3.0 .011 181.4
5.0 .019 317.0
10.0 .028 461.0
20.0 .050 821.0
30.0 .074 1208.0

Table 4-5 Syringe Size and Flow Rate at mL/minute

Syringe size mL/minute
(mL) Minimum Maximum
50.0 .002 28.40
100.0 .003 47.60
140.0 .004 55.10

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5
Users can view the information contained in a data file in table or graph form. Graphical data is
presented either as a chromatogram or as a spectrum. Data from either of these displays can be
viewed as a table of data points and various sorting operations can be performed on the data.
The software stores data in files with a .wiff extension. Wiff files can contain data for more than
one sample. In addition to .wiff files, the software can open .txt files; .txt files contain data for only
one sample. When a data file is opened in the software, different panes appear depending on the
type of experiment that was performed.
If the MCA check box is selected in the Tune Method Editor, the data file will open to the MS
(mass spectrum). If the MCA check box is not selected, then the data file opens with the Total Ion
Chromatogram (TIC). Users can select a range and then double-click in the TIC pane at a
particular time to show the MS for this range.

Chromatograms
A chromatogram displays the variation of some quantity with respect to time in a repetitive
experiment; for example, when the instrument is programmed to repeat a given set of mass
spectral scans several times. Chromatographic data is contiguous, even if the intensity of the
data is zero. Chromatograms are not generated directly by the instrument, but are generated
from mass spectra.
In the chromatogram display, the intensity, in counts per second (cps), is shown on the y-axis
versus time on the x-axis. Peaks are automatically labeled.
In the case of LC/MS, the chromatogram is often displayed as a function of time, the time at
which a particular scan was obtained, which can be derived from the scan number.
When data is viewed as a spectrum, mass-specific information about a compound is obtained. A
chromatogram provides a general view of the data, usually time dependent when using an LC
column, but it does provide information about the components of a peak. A spectrum, however,
looks at a particular peak and provides the molecular weight of the corresponding compound,
which can be used to find more specific information. For example, while a chromatogram may
show only one peak, that peak can represent more than one compound; that is, different masses.
A spectrum shows all of the masses that make up a peak, including the intensity of each mass.
Chromatographic data can change in both time and intensity if there is a change in the
chromatographic conditions in a given sample. Spectral intensities may change, but the masses
are fixed because the mass of a compound does not change.
There are two ways to generate spectral data:
• If only one scan is acquired, then the data is shown as a spectrum.
• From a chromatogram.
A typical spectrum is shown with the molecular weight, labeled with the m/z (mass-to-charge
ratio), on the x-axis. The intensity is shown on the y-axis.
A chromatogram is a graphical display of the data obtained from the analysis of a sample. It plots
the signal intensity along an axis that shows either time or scan number.

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The software plots intensity, in counts per second (cps), on the y-axis against time on the x-axis.
Peaks above a set threshold are labeled automatically. In the case of LC/MS, the chromatogram
often is shown as a function of time.

Spectra
A spectrum is the data that is obtained directly from the instrument and normally represents the
number of ions detected with particular mass-to-charge (m/z) values. It is displayed as a graph
with the m/z values on the x-axis and intensity (cps) represented on the y-axis.
In the case of MS/MS data, the intensity is associated with two masses, the precursor ion mass
(Q1) and the product ion mass or masses (Q3).

Background Subtraction
Background subtraction reduces the amount of noise in a spectrum by subtracting either one or
two ranges that contain noise from a range that contains a peak. Users can move the ranges
independently or lock them and move them as a single entity within the graph to optimize peak
isolation, or to isolate another peak. Locked Background Subtract is the preset setting.

Perform a Background Subtraction from a Spectrum

1. Open a data file.
2. Select a background range.
Figure 5-1 XIC

3. Hold down the Shift key and then select another background range.
4. To set the subtract range, click Explore > Background Subtract > Set Subtract
Range.

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5. Select the peak of interest.

6. Click Explore > Background Subtract > Perform Background Subtract.
The background is subtracted from the peak and a new spectrum is generated.
7. To isolate another peak, drag the locked ranges in the chromatogram and repeat the
background subtract.

Tip! To clear the background subtract region, click Explore >

Background Subtract > Clear Subtract Range.

8. To save the background subtracted spectrum as a processed data file, click File >
Save.

Unlock the Ranges

The selected subtraction range is set to locked.
• Click Explore > Background Subtract > Subtract Range Locked.
The ranges are unlocked and each one can be moved independently.

Background Subtract to File

Use Background Subtract to File to save a new file that has a defined noise region subtracted
from each scan. When a range is selected in the TIC, all the scans from that selection are
internally averaged, and the resulting spectrum is subtracted from all the scans. Use the
Background Subtract to File dialog to select where the file is to be saved. In some cases, the
resulting file might look close to the original scan.

Caution: Potential wrong result. Be careful when applying background subtraction as

this operation will modify the data.

Perform a Background Subtraction to File

1. Open a data file.
2. Select the background range within the TIC and set it as background.
3. Click Explore > Background Subtract to File.

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Figure 5-2 Background Subtract to File Dialog

4. In the Output Project and Filename section, type the project and file names for the
resulting file.
5. Click Start Processing.
The progress bar displays the progress of the subtraction process. If the Open the
new file immediately in Analyst check box is selected, then when the subtraction
is complete, the file opens.
6. If the check box is cleared, when the subtraction is complete, click Finish.

Baseline Subtract
Baseline subtract removes a constant or slowly varying offset from a set of data. This is useful in
locating small peaks that are obscured by noise. The software uses the following algorithm in
performing a baseline subtraction.
• Every data point in the data set is considered as the center of a window (in mass or
time) with a user-definable width measured in amu or minutes.
• The minimum values on either side of the current data point (minima) within the
window are located.
• A straight line is fitted between the two minima and the height (intensity) of the
current data point above the line is calculated. The end points of the data are
regarded as minima.
• The data point is replaced with the new calculated value.

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Calculators
Users can perform calculations on the basis of collected data. Although the calculator is a
separate window, it is connected to the active graph within the software.
The following calculators are available.
• Elemental Composition Calculator
• Hypermass Calculator
• Elemental Targeting Calculator
• Mass Property Calculator
• Isotopic Distribution Calculator
Users can cut and paste from one text box to another between the different windows in the
calculators. Data from any of the calculators can be printed by clicking the Print icon in the top left
corner of the window. For more information about using calculators, refer to the Help.
Data from the Elemental Composition, Mass Property, and Isotopic Distribution calculators can
be exported to a separate file. Use the Elemental Targeting calculator to modify the data within
the active graph. Data from the HyperMass and Isotopic Distribution calculators can be overlaid
on the active spectrum.

Tip! Set the precision of calculator data in the Calculators tab of the Appearance
Options dialog. To open the dialog, click Tools > Settings > Appearance Options.

Elemental Composition Calculator

The Elemental Composition calculator determines potential molecular or amino acid
compositions based on a target mass-to-charge ratio. Type this ratio manually or select it from an
active spectrum. This calculator creates a table with the possible element or amino acid
combinations making up the mass of interest and the characteristics of each.
Type or select values for such parameters as tolerance, electron state, and number of charges.
Users can also type a list of possible elements and put a limit on the number of each.

Hypermass Calculator
The Hypermass calculator determines the distribution of a multiply charged envelope based on
an uncharged mass. Users can select the uncharged mass, including the adduct and its polarity.
The calculator displays a graphical representation of the Hypermass series, which can be
overlaid onto the active spectrum. A list of the Hypermass data is also available.

Elemental Targeting Calculator

The Elemental Targeting calculator reduces the data spectrum based on a specific pattern,
primarily one corresponding to isotopic distributions. It can also search an MS data spectrum for
a specific pattern of peaks, which can be entered either as a formula or as an isotopic
distribution.
If the calculator finds a match, it creates a reduced plot containing only data pertaining to the
specified pattern. For a spectrum, the calculator removes all unmatched data. For a

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chromatogram, the calculator calculates the elemental target for each of the underlying spectra
and regenerates each point in the chromatogram on the basis of these new spectra.

Mass Property Calculator

The Mass Property calculator determines various properties such as exact mass, the average
mass, the mass accuracy, and the mass defect of a mass of interest. The results generated by
this calculator depend upon the number of input fields completed.

Isotopic Distribution Calculator

The Isotopic Distribution calculator determines the isotopic distribution based on an entered
formula. This allows users to distinguish between compounds with the same mass based on
relative intensities of isotopes.
The calculated isotopic distribution can be displayed in graphical or text format on the Isotopic
Distribution pane, overlaid on the active spectrum, or exported to a separate file.

Centroided Peaks
Calculating the centroid of a peak converts peak distribution values into a single value of m/z and
intensity that represents the peak. Centroided data collected in profile mode simplifies the data
and reduces the file size. Centroided data provides more accurate peak assignment and reduces
the amount of data, but it also removes the information about the peak shape.
The centroid algorithm converts peaks to single values by using an intensity weighted average to
calculate the center of gravity of the peak. The output of the algorithm is a list of peaks with
parameters, as shown in Table 5-1 on page 36.
Table 5-1 Peak Parameters
Parameter Definition
Centroid Value The value of the centroided data in units of mass or time.
Intensity The intensity of each peak in cps.
Width The width of the centroided peak in amu.

Data is automatically calculated as a centroid when added to a library or when a search is

conducted.

Calculate the Centroid of a Peak

1. Select a pane containing a spectrum.
Calculating the centroid of the peak changes the appearance of the existing graph.
To compare the result with the original data, make a copy of the graph before
calculating the centroid.
2. Click Explore > Centroid.

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Figure 5-3 Analyte Centroid Location

Centroid Location

A1=A2

A1 A2

Time

Data Analysis
Users can open files containing existing data or data that is currently being acquired. All
experiment-related data can also be viewed in tabular form. The table pane consists of two tabs,
the Data List tab and the Peak List tab.The Data List tab contains experiment-related information,
such as acquisition time and scan intensity. The Peak List tab displays peak-related information
such as peak height, peak area, and baseline type.

Total Ion Chromatogram

A Total Ion Chromatogram (TIC) is created by summing the intensity contributions of all ions from
a series of mass scans. Users can use the TIC to view an entire data set in a single pane. It
consists of the summed intensities of all ions in a scan plotted against time in a chromatographic
pane. If the data contains results from multiple experiments, individual TICs (Total Ion
Chromatograms) for each experiment and another TIC that represents the sum of all
experiments can be created. The preset TIC that represents the sum of all of the experiments is
shown with a splitter tool below the center of the x-axis.

Extracted Ion Chromatogram

An Extracted Ion Chromatogram (XIC) is an extracted ion chromatogram created by taking
intensity values at a single, discrete mass value, or a mass range from a series of mass spectral
scans. It shows the behavior of a given mass, or mass range, as a function of time. The intensity
of the ion, or the summed intensities of all ions in a given range, is plotted in a chromatographic
pane.

Base Peak Chromatogram

A Base Peak Chromatogram (BPC) displays the intensity of the most intense ion in every scan
as a function of scan number or retention time. It is useful in instances where the TIC is so
dominated by noise that there is a large offset and chromatographic peaks are hard to
distinguish. It is also helps to distinguish between co-eluting components. BPCs only be
generated from single period, single experiment data.

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The graph uses two colors, alternating each time the mass of the base peak changes. The color
changes are maintained when the data is manipulated by scrolling or zooming. For information
about selecting the colors used in the graph, refer to the Help.

Extracted Wavelength Chromatogram

An Extracted Wavelength Chromatogram (XWC) is a wavelength chromatogram created by
taking intensity values at a single wavelength, or by the sum of the absorbance for a range of
several wavelengths

Diode Array Detector

Users can view the Diode Array Detector (DAD) spectrum for a single point in time, or for a range
of time as a Total Wavelength Chromatogram.

Total Wavelength Chromatogram

A Total Wavelength Chromatogram (TWC) is a less commonly used chromatogram. It displays
the total absorbance (mAU) as a function of time. The TWC provides a way of viewing an entire
data set in a single pane. It consists of the summed absorbances of all ions in a scan plotted
against time in a chromatographic pane. If the data contains results from multiple experiments,
individual TWCs for each experiment and another TWC that represents the sum of all
experiments can be created.

Overlay Graphs
Two or more sets of data can be visually compared by overlaying graphs created by similar
methods. Each individual spectrum is distinguished by the color of its trace. For full scan data,
this allows users to visualize the differences between several sample spectra.
If one or more panes are chosen, then each XIC will open in a separate pane.

Tip! To overlay fewer than four graphs in the same pane, press Ctrl + right-click in
a pane and then click Appearance Options. In the Appearance Options dialog,
Multiple Graph Options tab, select Yes for the Overlay Multiple Panes fields for
Spectrum and Chromatogram.

1. Select the first pane to be overlaid.

2. Click Explore > Overlay.
3. Click in the second pane.
The graphs are overlaid showing the two traces in different colors.

Tip! To view a color-coded list of the overlaid graphs, right-click the title
bar of the pane.

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Cycle Between Overlaid Graphs

1. Select a pane that contains overlaid graphs.
2. Click Explore > Cycle Overlays.
The display changes so that the next graph in sequence is shown in the foreground.

Sum Overlays
If two or more graphs are overlaid, users can sum the graphs to get a new trace. Each point on
the new trace is the sum of the points from the graphs. Summing several overlays of similar data
type can make subsequent processing operations easier and faster. For example, users can
overlay several XICs, sum them, and then smooth the summed overlay to remove noise.
Summing overlays is similar to generating a TIC with the benefit of being able to choose which
graphs to overlay. For example, if ten experiments are being viewed, the TIC will add all ten
experiments together. If overlays are summed, then users have the option of adding only nine of
the ten overlaid graphs. This procedure can be used if the data collected in the one experiment is
just noise.
1. Overlay the graphs that are to be summed.
2. Click Explore > Sum Overlays.
The overlaid graphs are added together.

Graphs Labels
Graphs can be customized using the preset style for labels on graphs and chromatograms.
Users can select the fonts to use for peak and axis labels, and the colors to use for the traces.
Users can also add axis labels and the type of label and precision for the peaks.

Add Captions to a Graph

Use captions to label peaks of interest or significant points on the graph. When a caption is
placed beside a peak, the caption stays with the peak when the graph is zoomed in or out.
Captions also stay with the original sample when users navigate between samples in a data file.
A caption contains one line of text, with a maximum of 128 characters.
1. In the spectrum, right-click, and then click Add Caption.
The Add Caption dialog opens.
2. In the Caption box, type the text.
3. To change the size and style of the caption, click Font.
4. To place the caption, click OK.

Tip! If the position of the caption is not satisfactory, then drag the caption
to a different position. The caption stays in the same place relative to the x-
and y-axes when the graph is zoomed in or out. To edit or delete the
caption, right-click the caption and then click the appropriate command.

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Add Text to a Graph

Use text to add multiple lines of information to a graph. Unlike captions, which are associated
with a specific peak and move with it as the graph is zoomed, text labels remain in their original
location as the graph is zoomed. They do not stay with the original sample when users navigate
between samples in a data file.
1. In the graph, right-click and then click Add User Text.
The Add User Text dialog opens.
2. In the User Text field, type the text.
3. To center the text, select the Center Text check box.
4. To change the size and style of the caption, click Font.
5. To insert the text, click OK.

Tip! If the position of the text is not satisfactory, then drag the text to a
different position. To edit or delete the text, right-click the text and then
choose the appropriate command.

Compound Database
The compound database stores information about compounds, including optimization
specifications. Use the compound database when there is a large numbers of samples and a
large number of compounds need to be optimized quickly. The Compound Database window
stores optimized conditions for compounds that can be retrieved to run samples.

Contour Plots
A Contour Plot is a color-coded plot of a complete data set that uses color to represent a third
dimension in the plot. In a Contour Plot of a TIC, the x-axis represents retention time or scan
number, the y-axis represents mass, and the color represents the intensity of the data at that
point. In a Contour Plot of a TWC for DAD data, the x-axis represents retention time or scan
number, the y-axis represents wavelength, and the color represents absorbance. The Contour
Plot is a post-acquisition tool that does not function in a real-time scan acquisition.

Note: The Contour Plot does not support MI or MRM scans, but it does support DAD
scans.

Color is the third axis in Contour Plot, and it represents either intensity or absorbance. Users can
change the high and low intensity or absorbance values in Contour Plot using the control
triangles on the color bar above the Contour Plot. The percentage parameters at the top of the
Contour Plot pane indicate the values held by the low and high sliders. The actual values are
based on a percentage of the maximum intensity or absorbance within the selected area. The
value is shown in the top right corner of the Contour Plot pane.
The controls shown in Figure 5-4 on page 41 change the colors in a Contour Plot.

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Figure 5-4 Buttons Controlling Contour Plot Colors

Users can define the colors on a Contour Plot graph to provide better contrast and display data
specifications according to their needs. For example, setting the intensity/wavelength and
changing the color of the values for Below Low Data and Above High Data can eliminate
background noise in a Contour Plot.
The Below Low Data and Above High Data buttons shrink and expand on the color bar if the
slider controls are moved. When the contour plot colors are changed, the new colors become the
preset colors for all subsequent graphs.
Table 5-2 Right-Click Menu for Contour Plot Panes
Menu Function
Show DAD Spectrum Opens a new pane with the DAD spectrum.
Extract Wavelengths Extracts up to three wavelength ranges from a DAD spectrum to
(Use Range) display the XWC.
Extract Wavelengths Extracts wavelength ranges using the maximum wavelengths.
(Use Maximum)
Zoom to selection Zooms in on the selected area.
Add User Text Adds a text box at the position of the cursor.
Undo Zoom Returns the graph to the original scale
Delete Pane Deletes the selected pane
Show Cross-Hair Shows the Cross-Hair (nm/min)

View a Contour Plot

A Contour Plot can be viewed only after acquisition. Users can view a Contour Plot from TIC,
XIC, TWC, or XWC graphs. TICs and XICs are available for all .wiff data files. TWCs and XWCs
are available only for data acquired by a DAD.
1. In Explore mode, open a data file as a TIC, XIC, TWC, or XWC graph.
2. Highlight the range to be viewed in the Contour Plot. If a selection is not made, the
entire range is viewed.
3. Click Explore > Show > Show Contour Plot.
A Contour Plot of the selected area opens in a separate pane.

Select an Area in a Contour Plot

To zoom in on a particular selection, or view the corresponding mass spectrum for that selection,
do one of the following:

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• To select a standard area within a box, drag the pointer to create a box around
an area in the Contour Plot.
• To make a vertical selection, press Ctrl and drag the pointer vertically.
• To make a horizontal selection, press the space bar and drag the pointer
horizontally.

Set the Intensity and Absorbance in a Contour Plot

• Do one of the following:
• To set the low intensity/absorbance value in a Contour Plot, from the color bar
above the Contour Plot, drag the left triangular slider to the required position.
Contour Plot automatically adjusts the color of values below the setting to
indicate they are outside the range.
• To set the high intensity/absorbance value in a Contour Plot, from the color bar
above the Contour Plot, drag the right triangular slider to the required position.
The Contour Plot automatically adjusts the color of values above the setting to
indicate they are outside the range.

Change Colors in a Contour Plot

Tip! By using the Define Custom Colors palette, users can create customized colors
for use in a Contour Plot.

1. In the Contour Plot pane, click one of the color buttons.

The Color dialog opens.
2. Click a color.
3. Click OK.
The graph changes to reflect the color change.

Dynamic Background Subtraction Algorithm

Dynamic Background Subtraction™ algorithm improves detection of precursor ions in an IDA
(Information Dependant Acquisition) experiment. When the algorithm is activated, IDA uses a
spectrum that has been background subtracted to select the ion of interest for MS/MS analysis,
as opposed to selecting the precursor from the survey spectrum directly. Because this process
takes place during LC analysis, the algorithm enables detection of species as their signal
increases in intensity, therefore focusing on detection and analysis of the precursor ions on the
rising portion of the LC peak, up to the top of the LC peaks (maximum intensity).

Fragment Interpretation
The Fragment Interpretation tool helps the user interpret MS/MS data. Given the chemical
structure of a molecule, this tool can generate a list of theoretical fragment masses from single

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non-cyclic bond cleavage of that molecular structure. The tool can then match the theoretical list
with peaks in the current mass spectrum.
The Fragment Interpretation Tool generates a list of theoretical fragment masses from single,
non-cyclic bond cleavage of a molecular structure. The molecular structure can be created in a
third-party drawing program and then saved as a .mol file. Fragment Interpretation displays the
theoretical fragments in the fragment list and compares the fragment masses to peaks in the
mass spectrum. Peaks above the threshold intensity and within the user-defined mass tolerance
(maximum 2 amu) of fragment masses are considered matched and are shown in bold text in the
fragment list.

Note: The Fragment Interpretation tool cannot be used with the following scan types:

• Precursor Ion
• Neutral Loss
• Q1 Multiple Ion
• Q3 Multiple Ion
• Multiple Reaction Monitoring (MRM)

Connect the Fragment Interpretation Tool to a

Spectrum
When a single, non-cyclic bond in the molecular structure is selected, the Fragment
Interpretation tool highlights the two fragments created when the bond is cleaved and matching
peaks in the connected spectrum are displayed.
If multiple spectrum panes are being viewed, then the Fragment Interpretation tool connects to
the active spectrum. If the data file contains more than one sample, then the Fragment
Interpretation tool connects to the active spectrum.
If a spectrum is open when the Fragment Interpretation tool is opened, then the active panel links
to the open spectrum automatically.
1. Click Explore > Show > Show Fragment Interpretation Tool.
2. From the lower right corner of the Fragment Interpretation pane, click the connect
button.
The pointer changes to the connecting tool.
3. Click the spectrum graph that is to be connected to the Fragment Interpretation tool.
The connected graph indicator in the lower left corner contains the name of the
graph connected to the Fragment Interpretation pane. The connection is broken
when either the graph or Fragment Interpretation is closed. If the connected .wiff file
has more than one sample, the Fragment Interpretation pane updates automatically
as users scroll through the samples.

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Match Fragments with Peaks

1. Click Explore > Show > Show Fragment Interpretation Tool.
2. With a .mol file in the Fragment Interpretation pane, select a cell in the Fragment List
that is shown in bold.
In the spectrum, the software highlights the matching spectral peak in the color
selected under the Options tab. In the molecular structure, the bond is highlighted.
3. If a row that has more than one matching fragment is clicked, the spectral peak that
is closest to its monoisotopic mass is highlighted in the mass spectrum in the color
specified in the Options tab.

Select a Bond in a Molecular Structure

1. Click Explore > Show > Show Fragment Interpretation Tool.
2. With a .mol file opened in the Fragment Interpretation pane, click a single, non-cyclic
bond in the molecular structure.
The two resulting fragments are shown as highlights in the fragment list. The masses
of the two fragments are shown on either side of the bond.
If a spectrum is connected, then the Fragment Interpretation tool displays any
matching peaks in the graph. If a fragment in the list is selected and the fragment is
matched to a peak, then the Fragment Interpretation window zooms in on that peak.

View Isotopes
The Fragment Interpretation tool can display the theoretical isotopic distribution for a peak
matching a fragment in the fragment list.
1. Click Explore > Show > Show Fragment Interpretation Tool.
2. In the Fragment Interpretation pane, click the Options tab.
3. Select the Show Isotopes check box.
4. Click Apply.
5. In the fragment list, select a fragment that matches a peak.
The isotopic distribution for matched peaks is shown in the spectrum.

Display a Formula Difference in a Spectrum

The formula and monoisotopic mass difference between two related hypothetical fragments can
be displayed. The formula difference is shown when two peaks are selected. The formula and
monoisotopic mass difference is shown when two fragments are selected, or two single, non-
cyclic bonds.
1. Click a fragment peak.
2. Press the Shift key and then click another fragment peak.
If the formula difference is equal to a fragment from the fragment list, the fragment
highlights in the list. Otherwise, the formula difference between the matching
fragments of the peaks is shown in a message box.

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Display a Formula Difference in the Fragment List

1. Click the row number for one fragment.
2. Press the Ctrl key and then click another fragment.
The formula and monoisotopic mass difference is shown in a message box if the
fragments are related.

Display a Formula Difference in a Molecular Structure

1. Click a single, non-cyclic bond. The preset fragment (of the two highlighted
fragments) is selected. To select the other fragment of the cleaved bond, CTRL+click
the bond.
2. Select a second non-cyclic bond. To select the preset fragment, press the Shift key
and then click the bond. To select the other fragment of the cleaved bond, press
Ctrl+Shift+click the bond.
Fragment Interpretation calculates the formula and monoisotopic mass difference
between the fragment selected in step 1 and the fragment selected in step 2, if the
fragments are related. The formula and monoisotopic mass difference is shown in a
message box.

IDA Explorer
The Information Dependent Acquisition (IDA) Explorer is used to display data acquired through
an IDA method.
The IDA Explorer can be turned off and on in the IDA Explorer tab in the Appearance Options
dialog. Columns present in the List View can be defined in this tab as well.
The left side of the viewer shown in Figure 5-5 on page 46 displays the masses on which a
product ion scan was performed. In this area, users can examine the mass, intensity, time, and
collision energy of ions on which product ion scans were performed in either a list view or a tree
view. In list view, the list can be sorted by double-clicking on any column header. Use the
Appearance Options dialog to customize the columns in the list view.
On the right side, the viewer is split into four panes. The top left pane displays the survey TIC
data. The bottom left pane shows the XIC of the mass. The top and bottom right panes show the
survey and product scans, respectively.
The IDA viewer lists all the masses on which Enhanced Product Ion scans or Enhanced
Resolution scan types were performed. In the IDA viewer, users can do the following:
• Click a mass in the list or tree view to display plots relevant to that mass.
• View the survey spectrum from which the mass was identified and the product
spectrum of that mass.
• Display the TIC of the survey scan and the XIC for each mass.

Note: Brackets around a mass indicate that the mass is merged. A merged mass is
contiguous across a number of cycles. When a merged mass is displayed, it indicates
an averaged spectrum, containing the average of all contiguous spectra.

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Figure 5-5 IDA Viewer

Library Databases
The Library Search feature compares unknown spectra to known MS spectra contained in a
library database and generates a list of possible matches. Use Library Search to create and
manage a mass spectra database that can be used to search for and match unknown spectra
against the mass spectra stored in the database.
With Library Search users can:
• Compare library contents against an unknown spectrum.
• Add records to the library.
• Edit existing records.
Library data can be stored in the following locations:
• MS Access on a local database.
• MS SQL Server.
Before using the Library Search feature, determine where the library database is stored and
connect the computer to that location. Library databases can be stored locally and over a
network.
Use an alias to connect to a database. In this case, the alias specifies a connection to a specific
database and may include the user name and password required to access the database. For
example, a user may have a small library database of identified compounds on a computer and
the company may have a central database that is used occasionally by the users. Creating

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aliases for each database allows the user to switch between them quickly. For information about
creating aliases and connecting to databases, refer to the Help.

Switch Between Existing Library Databases

Users can connect to any databases that have aliases that are already set up.
1. Click Tools > Settings > Optimization Options.
The Optimization Options dialog opens.
2. Click the Library Manager tab.
Figure 5-6 Library Manager Tab

3. In the Available Libraries section, click the alias of the database to connect to and
then click Connect.
4. To allow other users to access the database, select the Available to all users of
this machine check box.
5. Click OK.

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Connect to a Local Library Database

1. Click Tools > Settings > Optimization Options.
The Optimization Options dialog opens.
2. Click the Library Manager tab.
3. In the Available Libraries section, click New.
Figure 5-7 Add Library Dialog

4. Type a name for the library.

5. In the Database Information section, select MS Access (local).
6. Type the database location.
7. In the Security Information section, if required, type a user name and password to
access the database.
8. Click Save.

Connect to a Server Library Database

1. Click Tools > Settings > Optimization Options.
The Optimization Options dialog opens.
2. Click the Library Manager tab.
3. In the Available Libraries section, click New.

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4. Type a name for the library.

5. In the Database Information section, select MS SQL Server (server).
Figure 5-8 Add Library Dialog

6. Type the name of the database server.

7. Type the name of the database.
8. Do one of the following:
• If a specific user name and password are required to access this database,
then type the user name and password.
• If Windows security is used, then in the Security Information section, select
the Use Windows integrated security option.
9. Click Save.

View All Library Records

• Click Explore > Library Search > List.
The Librarian dialog opens with all records in the database.

Add a Record to the Library

1. Right-click an active spectrum, and then click Add a Record.

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The spectrum is automatically calculated as a centroid. The Add a Record dialog

opens with data from the spectrum.
2. In the Mass Spectral Information tab, type a name in the Compound Name field.
The compound name is mandatory and must uniquely identify the compound within
the library.
3. Edit any of the other fields. Many of the fields are filled in automatically from the data
associated with the spectrum.
4. Click the General Information tab.
5. Edit the fields as required.
6. Click OK.

Search Library Records with Constraints

1. Click Explore > Library Search > List With Constraints.
Figure 5-9 List Constraints Dialog

2. In the Field Name list, select a field on which to base a constraint.

3. In the Relation list, select the relation (operator) that applies to the field name.
4. In the Value field, type the value of the field name based on the relation.
5. To add the selected constraint to the Conditions list, click Add.
6. Continue to add constraints to the conditions list as required.
7. Coupling distinct constraints within the Conditions list creates more specific
conditions that enhance the search. To group constraints, select the constraints and
then click Group. To separate grouped constraints, click the group, and then click
Ungroup.

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8. To change the relationship between constraints, click the relationship, and then click
And or Or.
9. To exclude compounds containing a certain number of atoms of specific elements,
select or type the elements in the Elements Included table, and then type a
minimum and maximum number of atoms of the element.

Note: Element symbols are case-sensitive. For example, Hydrogen is H,

not h and Sodium is Na, not NA or na.

10. To exclude compounds containing certain elements, select or type the elements in
the Excluded table.
11. To search for compounds fitting the criteria, click List.
Records that match all the constraints are shown in the Records table. Listing
constraints are saved.

Library Search Tips

Table 5-3 Library Search Tips
To do this... ... do this
Group conditions. Select the conditions to group and then click Group. This
function behaves like parentheses in formulas.
Search without using Right-click an active spectrum, and then click Search Library.
constraints. The Search Results dialog opens.

Search for a Similar Spectrum

The user can search the library for a spectrum and its related compound information that
matches, or is similar to, an active spectrum. Searches can be performed with or without
constraints. When the user searches with constraints, only those records that match all the
criteria are listed. The results are shown in a ranked list. The first item on the list is the best fit to
the active spectrum. Entries lower in the list do not match as well.
The more constraints that are selected, the more precise the list becomes and fewer, more
relevant matches are listed. After a set of constraints is defined, they will apply to all subsequent
searches, unless they are edited. When a user searches without constraints, there is a much
larger list of suggested spectra because the library makes fewer specific matches to the spectral
data.
Only peaks above the threshold are used in the search. When selecting search constraints, The
user can also add or subtract peaks from the active spectrum.
For example, if the user thinks a peak is actually a background or noise spike, the peak should
not be used for the search because it could produce inaccurate results.
1. Right-click on an active spectrum and then click Search With Constraints.
The software calculates the centroid of the spectrum automatically.

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Figure 5-10 Search Constraints Dialog

2. In the Maximum Number of Match field, type the maximum number of compounds
to be returned by the search.
3. In the Preselect Constraints section, select the check boxes for the constraints to
apply.
4. For each constraint selected, in the Preset Tolerance section, type the tolerance.
5. If required, select a method of sorting records from the Result Sorted by list.
6. If required, type text in the Comment Contains field.
7. If required, type text in the Keyword Contains field.
8. To apply peak constraints by adding and removing peaks, click Peak Constraints.
The Peaks Included table opens.
9. To add peaks to the list to search against, click Add and then type the m/z and the
corresponding intensity in the empty cell.
10. To remove peaks so that they will not be included in the search, select the peaks and
then click Remove.
11. Click Search to save the constraints and begin the search.

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View a Compound from the Search Results

If several spectra match the unknown spectrum, the user may want to view other spectra and
compare them to the unknown.
1. In the Search Results dialog, in the list of compounds, select the row number of the
compound.
2. Click the spectrum pane of one of the known compounds.
The spectrum of the selected compound opens.

Processed Data Files

The user can save processed data, such as specific layouts and captions, that can be opened in
Explore mode only. These files also contain history information and are similar to data files
except that they will contain only the data from the active pane in Explore. These files have the
.pdt extension and are stored in the Data folder in the current project.

Save a Processed Data File

1. Select the pane.
2. Click File > Save Processed Data File.
3. In the File name field, type a name.
4. Click Save.

Open a Processed Data File

1. In Explore mode, click File > Open Processed Data File.
2. Select a file.
3. Click Open.

Qualitative Data
The user can view the information contained in a data file in table or graph form. Graphical data is
shown as a chromatogram or as a spectrum. Data in a table is shown as data points. The user
can perform various sorting operations on the data.
When the user opens a data file, different panes appear depending on the type of experiment
performed.
If the MCA check box is selected in the Tune Method Editor, the data file opens with the mass
spectrum. If the MCA check box is not selected, the data file opens the TIC.
A wiff file can contain data for more than one sample. In addition to .wiff files, the software can
open .txt files. A .txt file contains data for only one sample.

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Signal-to-Noise Ratio
The signal-to-noise ratio is the peak height divided by the noise.
To calculate the noise, the software uses the standard deviation (using a mean of zero) of all data
points in the chromatogram from the Background Start to Background End time (both shown in
the advanced parameters for the Quantitation Method Editor and Peak Review window.) These
times are set when a new background range is defined.
If the user builds a method without defining a new background range, which is possible if the
preset integration is accepted with no changes, then the value for both the Background Start and
the Background End is shown as N/A. As a result, the signal-to-noise ratio is not calculated and
the corresponding field in the Results Table is shown as N/A.

Smoothing Algorithms
The user can select either the smoothing algorithm or the Gaussian smoothing algorithm as the
smoothing method. The smoothing operation involves replacing each data point with the average
of the data point before and after it. The smoothed data set replaces the old set.
Data can be smoothed more than once, but the software can undo only the last smooth.
Smoothing is not available for multiple ion or MRM spectra.

Smooth Algorithm
When smoothing data, the user sets the point weighting values for three data points: the current
point, the preceding point, and the following data point. The smooth algorithm multiplies the data
points by the assigned weighting values, sums these values, and then divides the total by the
sum of the point weight values. It is a gentler smooth than the Gaussian algorithm and it takes a
long time to smooth very noisy data.

Gaussian Smoothing Algorithm

Gaussian smoothing involves replacing each data point with the weighted average of a number
of data points on either side of it. The weighting for each new data point is calculated on the basis
of a Gaussian curve. It is a coarser smooth than the smooth algorithm, but it is good for
smoothing very noisy data.
Set two values when using the Gaussian smoothing method:
Gaussian filter width (% of minimal distance between points): This value shows the width
used to calculate the weighting of neighboring points. The width is described in terms of
percentages of the distance between two points in the scan, where the preset width of 100%
gives a distribution that is as wide as the distance between data points.
Limit of Gaussian filter (number of minimal distance between points): This value
corresponds to the limits of the Gaussian curve, shown in multiples of the distance between
points. For example, the preset value of 10 creates a Gaussian curve that truncates after ten data
point widths on either side of the center.

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Smooth Data using the Smooth Algorithm

Tip! To undo smoothing, click Edit > Undo. The software supports one level of undo.

1. Select a pane containing a chromatogram or spectrum.

2. Click Explore > Smooth.
Figure 5-11 Smoothing Options Dialog

3. Type the weighting factor in the following fields.

• Previous Point Weight field
• Current Point Weight field
• Next Point Weight field
4. Click OK.
The data set is smoothed, replacing the current data set in the pane.

Smooth Data using Gaussian Smoothing

Tip! To undo smoothing, click Edit > Undo. The software supports one level of undo.

1. Select a pane containing a chromatogram or spectrum.

2. Click Explore > Gaussian Smooth.

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Figure 5-12 Gaussian Smooth Options Dialog

3. In the Gaussian filter width field, type the width used to find the weighting of
neighboring points as a percentage of the distance between the two points.
4. In the Limit of gaussian filter field, type the limit of the Gaussian curve, given in
multiples of the distance between points.
5. Click OK.
The data set is smoothed, replacing the current data set in the pane.

Spectral Arithmetic Wizard

The Spectral Arithmetic Wizard performs arithmetic operations using two complete
chromatograms to create a new chromatogram, which is saved as a new data file. For more
information, refer to the Help.
• Subtraction: Use the subtraction option to subtract a control or background from a
sample.
• Addition: Use the addition option to add separate chromatograms together to create
a single, more complete profile for a compound. For example, create several
chromatograms using different collision energies and then add the chromatograms
together to create a single profile.
• Average: Use the average option to statistically improve the profile for a compound
and to generate more reliable spectra.

Toolbar Icons
Table 5-4 Toolbar Icons
Icon Name Function
Perform Background Performs a background subtract after the
Subtract background ranges have been selected.

Subtract Range Locked Locks the selected background ranges. If the

background ranges are unlocked, then users can
move each range independently.

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Table 5-4 Toolbar Icons (Continued)

Icon Name Function
Background Subtract to File Saves a new file that has a defined noise region
subtracted from each scan.

Spectral Arithmetic Wizard Uses the Spectral Arithmetic Wizard to perform

arithmetic operations using two complete
chromatograms to create a new chromatogram,
which users can save as a new data file.
Centroid Calculates the centroid of the data.

Home Graph Returns the graph to the original scale.

Overlay Overlays graphs.

Cycle Overlays Cycles between overlaid graphs.

Sum Overlays Adds the graphs together.

Show Fragment Opens Fragment Interpretation tool, which calculates

Interpretation Tool the single, non-cyclic bond cleavage fragments from
a .mol file.
Smooth Smooths data using the smooth algorithm.

Gaussian smooth Smooths data using Gaussian smoothing.

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6
Calibration Options
The calibration options define the parameters for a calibration curve, which are used to
determine the calculated concentration of the samples. The curve is a plot of the concentration of
the standard against the area or height of the standard if no internal standard is used. If an
internal standard is used, the curve is a plot of the concentration ratio against the area or height
ratio. This curve is used, along with the area (or height) for the unknowns, to interpolate the
calculated concentration.
Choose the best regression type or fit to fit the curve to the points and the best weighting factor
for the project.

About Calibration Curves

The calibration curve is used to determine the calculated concentration of samples, including QC
samples. It is a curve that results from plotting the concentration of the standard versus its area
or height, or ratios, if an internal standard is used. The area or height of a sample is then applied
to this curve to determine the sample concentration, as displayed in the Results Table. The
regression equation generated by this calibration curve is used to calculate the concentration of
the unknown samples.
The software places the known concentrations (or ratios) on the x-axis and the calculated area or
height (or ratios) on the y-axis. It then plots the points for all the standards in the batch. The
system produces a best-fit curve to those points through regression and weighting type that is
selected. This curve is used, along with the area (or height), for the unknowns to interpolate the
concentration.

Select the Best Regression Type

After selecting a regression type (fit), the user cannot see the calibration curve from the wizard.
Instead, use the preset values, experience, or corporate policy to choose a regression type.
After changing the fit, check the Accuracy column in the Results Table for changes. The better
the fit is, the better the accuracy of the quantitative analysis will be.
The calibration curve plots the standards concentration against its peak area or height (or ratios,
if an internal standard is used). When the points for the standards are plotted, determine the best
fit for the curve to these points and indicate the choice in the Specify Calibration dialog of the
wizard. The preset fit is linear, which assumes that all the standards will fall on a straight line.
Select from the types of fit in Table 6-1 on page 59.
Table 6-1 Types of Fit
Fit Description
Linear Linear regression assumes that the standard points fall on a straight
line.

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Table 6-1 Types of Fit (Continued)

Fit Description
Linear Through Zero Linear Through Zero regression assumes that the standard points fall
on a straight line and that the points line up with the zero point on the
x- and y- axes. Use this setting to force the line to go through the zero
point.
Quadratic If the standard points do not fall on a straight line, use quadratic
regression to produce a quadratic fit to the data points.
Mean Response Factor If the standard points fall on a straight line, then to average the points,
use mean response factor regression to produce an average of the
slope for every point on the curve.
Power If there is some linear and some curvature in the line of points, use
power regression instead of linear or quadratic regression to produce
a line somewhere between these fits.

Choosing the Best Weighting Factor

The calibration curve plots the standards concentration against its peak area or height. When the
points for the standards are plotted, determine the best weighting factor for these points and
indicate it in the Specify Calibration dialog. The preset fit is None, which assumes that all points
along the curve have the same importance. Select from the types of weighting in Table 6-2 on
page 60.
Table 6-2 Types of Weighting
Weighting Description
1/x To place some additional emphasis on lower-value points, use a weighting
factor of 1/x.
1/x2 Use a weighting of 1/x2 to place much higher emphasis on lower-value points.
1/y Use a weighting factor of 1/y when calibrating by the area (y-axis) rather than by
the concentration (x-axis), and some emphasis needs to be placed on lower-
value points. A weighting of 1/y is a variant of 1/x where y and x should be
proportional to each other.
1/y2 Use a weighting factor of 1/y2 when calibrating by the area (y-axis) rather than
by the Concentration (x-axis), and a much higher emphasis needs to be placed
on lower-value points. A weighting of 1/y squared is a variant of
1/x squared where y and x should be proportional to each other.
In x Use the logarithm of x to place more emphasis on higher-value points.
In y Use the logarithm of y to place more weight on higher-value points. Use when
calibrating by the area (y-axis) rather than by the concentration
(x-axis).

Integration Algorithms
The Analyst® software has two integration algorithms: the original Analyst Classic integration
algorithm and the IntelliQuan integration algorithms. The IntelliQuan algorithm provides more
consistent peak-finding and integrated functionality, with fewer parameters that require
adjustment.

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Analyst Classic and IntelliQuan Integration Algorithms

The IntelliQuan algorithm uses one of two peak-finding parameters: Automatic IQA II, which is a
parameterless setting, or Specify Parameters MQ III. After integrating peaks using the IntelliQuan
algorithm, choose which peak-finding parameter best fits the data set. This is done in the
displayed peak integration parameters in the Peak Review pane or window.
Table 6-3 on page 61 shows the parameters available with the Analyst Classic algorithm.
Table 6-3 Analyst Classic Algorithm
Parameter Definition
Default Bunching Factor The number of points to be averaged together and
considered as a single point for the purpose of peak-finding.
Default Number of Smooths The number of times to smooth the chromatogram.
Default Void Volume Retention Any peaks that appear before this time are ignored.
Time
Default Concentration Units The concentration units used to describe the sample
concentration, for example, pg/µL.
Default Calculated Concentration The concentration units used to describe the calculated
Units sample concentration, for example, pg/µL.
Default RT Window The time window centered at the expected retention time for
peak-finding. For example, a 30 second retention time
window gives an additional 15 seconds before and after the
expected retention time.

Table 6-4 on page 61 shows the parameters available with the MQ III algorithm, but not the IQA II
algorithm.
Table 6-4 MQ III Algorithm
Parameter Definition
Default Noise Percentage The threshold used in peak-finding. Only peaks higher than
this specified percentage will be detected.
Default Baseline Subtraction A time window around each data point that is used to
Window determine the height of the baseline correction to be applied
to that point. This time window helps remove excessive
noise from the chromatogram. The baseline is defined as
the line connecting the point of minimum intensity on the left
side of a given data point to the point of minimum intensity
on the right side, within the specified window.
Default Peak-Splitting Factor Controls whether a given peak cluster consists of multiple
adjacent peaks or one (possibly noisy) peak. If the intensity
dip is less than the value specified, then a single peak is
reported; otherwise the point with minimum intensity in the
dip splits the cluster into two separate peaks. Setting a large
factor will prevent clusters from being split into more than
one peak.
Default Void Volume Retention Any peaks that appear before this time are ignored.
Time

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Table 6-4 MQ III Algorithm (Continued)

Parameter Definition
Report Largest Peak Selecting this parameter returns the largest peak in the
retention time window. If this parameter is not selected, the
closest peak to the expected retention time is found. The
expected retention time is automatically calculated in the
Quantitation Wizard.

Table 6-5 on page 62 shows the parameters available for use with both IntelliQuan algorithms.
Table 6-5 IntelliQuan Algorithm
Parameter Definition
Default Minimum Peak Height The minimum height of a peak required for peak integration.
Default Minimum Peak Width The minimum width of a peak required for peak integration.
Default RT Window Specifies the time window centered at the expected
retention time for peak-finding. For example, a 30 second
retention time window gives an additional 15 seconds before
and after the expected retention time.
Default Smoothing Width The number of points used in data smoothing.
Default Concentration Units The concentration units used to describe the sample
concentration, for example, pg/µL.
Default Calculated Concentration The concentration units used to describe the calculated
Units sample concentration, for example, pg/µL.

Quantitation Method-Creation Tools

The software offers four quantitation method-creation tools, each of which creates a fully
functional method. The best choice of tool depends on the tasks to accomplish.
It is recommended that only users who are proficient in method development create or modify
acquisition and quantitation methods. Refer to the About People and Roles section in the
Laboratory Director’s Guide for more information about roles and security.

Wizards
There are two available method-creation wizards: the Standard Quantitation wizard and the
Automatic Quantitation wizard. Both allow the user to select the batch or batches to be
quantified, create or select a quantitation method, and then integrate the sample data.
The difference between the two is the type of method created. The standard wizard creates a
standard method, while the automatic wizard creates an automatic method. Peaks are not
verified as part of the method creation (the peaks can still be reviewed after integration has taken
place).
There is only one common occasion for which peaks do not need to be verified: when
quantitation is done simply to integrate, and not to find concentrations. This may need to be
done, for example, for a batch that contains different compounds in every sample, or when the
mass is not the same from sample to sample. If this is the case, use the automatic wizard.
Otherwise, to perform quantitation, use the standard wizard.

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Use the Standard Quantitation wizard after acquiring the sample to do the following:
• Choose a representative sample.
• Select analyte and internal standard peaks.
• Adjust peak-finding and integration parameters.
• Review peaks during method creation.
• Select calibration.
Use the Automatic Quantitation wizard to select a batch, create a method (without peak
confirmation), and then integrate the sample data. This wizard is quicker than the Standard
wizard and does not require that the masses scanned be the same for all samples. It does not,
however, allow selecting an internal standard—all ions are treated as analytes.
Use the Automatic Quantitation wizard after acquiring the sample in the following scenarios:
• Want to select calibration.
• Do not want to adjust peak-finding and integration parameters.
• Do not want to select analyte peak names.
• Do not want any internal standards.
• Do not want to review peaks during method creation, or have different compounds in
every sample.
When peaks are only being integrated, they do not need to be verified as no concentration
calculation is required. In this case, use the Automatic quantitation method creation, which allows
reviewing the peaks after integration has taken place.

Find Peaks Using an Automatic Method

The software uses the standard peak-detection process, with the following exceptions:
• It uses the bunching factor and the number of smooths (from the wizard) as is.
• It calculates the expected retention time and noise and area thresholds separately
for every peak.

Quantitation Method Editor

Use this option after acquiring the sample to do the following:
• Adjust peak-finding and integration parameters.
• Select analyte and internal standard peaks.
• Select calibration.
Use the Quantitation Method Editor to do three additional tasks:
• Sum ions for integration.
• Use an internal standard from a different period or experiment (if the internal
standard was acquired in a different period or experiment than the analyte).
• Edit an existing method.

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The Semi-Automatic Method Editor

The Semi-Automatic Quantitation Method Editor is part of the Batch Editor. Use the Semi-
Automatic Quantitation Method Editor to select quantitation information, such as sample type and
sample concentration, prior to data acquisition. This preparation makes performing subsequent
quantitative analysis easier. Alternatively, a full method can be selected in the Batch Editor, which
is then automatically applied at the end of the batch run to generate the quantitation Results
Table.
Use this option in the following scenarios:
• Have not yet acquired any samples.
• Want to select names and masses for analyte and internal standard peaks.
• Want to select concentrations and sample types on the Quantitation tab in the Batch
Editor, but do not have any other quantitation method.
• Want to edit the quantitation method, if necessary, at a later time.

Find Peaks Using a Semi-Automatic Method

The software uses the standard peak-detection process, with the following exceptions:
• It uses the bunching factor (from the Quantitation Method Options dialog) and the
number of smooths (from the Create Semi-Automatic Quantitation Method dialog) as
is.
• It uses the most concentrated standard as the representative sample. To establish a
retention time, it uses the largest peak within that chromatogram.
• To set noise and area thresholds, it uses the resulting baseline noise. (This process
is identical to how the preset values are set for peaks in normal methods.) These
integration parameters are applied to all other samples.
• If the batch being examined does not contain quantitation information (sample type
and concentrations), the retention time and thresholds are calculated separately for
every peak (as for fully automatic methods).

Metric Plots
A metric plot graphically shows the data in a Results Table column plotted against the x-axis or
the y-axis, or the data in two columns plotted against each other. This section describes how to
generate and work with metric plots.
A few predefined metric plots are also included:
• Int_Std_Response (to locate problem sample)
• Analyte_Area versus Height (to verify chromatography behavior)
• PK profile (conc. versus time point, to run after Sample query)
Use metric plots to plot a given column, such as Analyte Peak Area, Accuracy, or Calculated
Concentration, from the Results Table. Two Results Table fields can also be plotted against each
other. Then points that are shown outside the normal range can be investigated. Metric plots are
often used with queries. For more information about queries, refer to the Help.
Generate metric plots in the following ways:

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• Use the Plot button to plot a column or columns of the current Results Table, but not
save the plotting criteria.
• Create a table-specific plot to save the plot criteria with the current table.
• Create a global plot to save the plotting criteria for use with future Results Tables.
QC, unknown, blanks, double blanks, or solvents cannot be seen on the calibration curve, but
metric plots can be generated of them.
Figure 6-1 Example of a Metric Plot

Item Description
1 Double blanks

Generate a Metric Temporary Plot

1. With a Results Table open, do one of the following:
• To plot the data on the y-axis with the x-axis as an index, click the heading of
the column for the data to be plotted.
• To plot the data from the first selected column on the x-axis and the second
selected column on the y-axis, select two columns by pressing the Ctrl key
and clicking the column headings.
2. Above the Results Table, click the Metric Plot by Selection icon.
The metric plot opens.
3. Right-click in the plot pane and then click Data Legend to view an explanation of the
colors used by the plot.
4. Right-click in the plot pane and then click Point Legend to view an explanation of
the symbols used by the plot.

Generate a Metric Plot and Save the Plot Criteria

1. Open an appropriate Results Table.

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2. Right-click in the Results Table and then click Metric Plot > New.
Figure 6-2 Metric Plot Dialog

3. In the Name field, type the name for the new plot criteria.
4. In the X-Axis section, in the Group list, to plot a field in the y-axis using the x-axis as
an index, select Index and leave the Column list blank.
5. If required, to plot two columns against each other, in the Y-axis section, in the
Group list, select Internal Standard, and then, in the Column list, select IS Peak
Area.
6. If required, in the Regression list, select the type of regression to be used, and then
select the appropriate regression settings.
7. To generate the plot and save the plot criteria, click Save/Execute.
The metric plot opens. For more information, refer to Figure 6-1 on page 65.
8. Right-click in the plot pane and then click Data Legend to view an explanation of the
colors used by the plot.
9. Right-click in the plot pane and then click Point Legend to view an explanation of
the symbols used by the plot.
This set of criteria is now available for future plots of this Results Table. Right-click in
the Results Table to access the criteria. The plotting criteria can also be edited.
10. To view the problem sample, try plotting the concentration of the unknown against
time or plotting the area of the internal standard against the index.

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Save Default Plot Criteria for Future Results Tables

1. Right-click in the Results Table and then click Table Settings > Export To New
Table Settings.
This will export the table settings from the .rdb so that it can be reused in other
quantitation runs within the project.
2. To export table settings to another project, click Tools > Project > Copy Data.
Figure 6-3 Copy Data Dialog

Noise and Area Threshold Parameters

To identify peaks, the software requires a set of noise and area threshold parameters. The
software sets these parameters initially, but they can be changed later. It sets the parameters as
follows:
1. The software calculates the largest intensity difference between any two sequential
data points. This number represents the difference between two intensities, not the
actual intensity itself.
2. For each sequential pair with an intensity difference of less than 5% of the value
calculated in Step 1, it calculates the standard deviation (using a mean of zero) of
the intensity differences. (The software does not use those pairs of points with an
intensity difference larger than 5% of the maximum.)
It sets the noise and area thresholds as follows:
3. The noise threshold is equal to the standard deviation calculated in step 4.
4. The area threshold is equal to five times the noise threshold.

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Note: The minimum value for both the noise and area thresholds is 0.000001. If the
calculations produce a value that is lower than this minimum, then the software resets
the value of that threshold at 0.000001.

Recalculate the Noise and Area Threshold

If a new background area is defined, the software recalculates the noise and area thresholds as
follows. For each sequential pair of data points, the software calculates the standard deviation,
using a mean of zero, of the intensity difference. The Analyst software uses all points within the
selected range because it is explicitly being told that the selected area is background noise.
It sets the noise and area thresholds as follows:
1. The noise threshold is equal to the standard deviation calculated for the selected
range.
2. The area threshold is equal to five times the noise threshold.

Note: The minimum value for both the noise and area thresholds is 0.000001. If the
calculations produce a value that is lower than this minimum, then the software resets
the value of that threshold at 0.000001.

Peak Integration
The following are integration types by which the baseline was found and integrated when the
peak was found.
• Manual: The peak was manually integrated by the user.
• Automatic: The peak was automatically integrated as follows:
• Baseline-to-baseline: The peak area is defined by vertical droplines at the
beginning and end of the peak which extend to the baseline. This integration
type is possible only for peaks that do not have another peak immediately
preceding or following.
• Valley: Same as baseline-to-baseline, except that it applies only to peaks that
do have another peak immediately preceding or following.
• Exponential Skim: The peak area is the main or parent peak in an
exponential skim.
• Exponential Child: The peak area is the child peak resulting in an
exponential skim.

Peak Review
During peak review users can survey the peaks that the software selected and then redefine the
peak or the start and end points where necessary.
In general, the software is adept at accurately identifying analyte and internal standard peaks.
For a variety of reasons, including sample acquisition and quantitation method definition,
sometimes the software misses the correct peak, chooses the wrong one, or is unable to locate a

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peak at all. Other times, although the software may correctly identify the peak, users may not
agree with the start or end points selected.

Peak Review Tips

Table 6-6 Peak Review Tips
To do this... ... do this
Peak Integration: To review To review all peaks, make sure that all samples are listed in the
peaks Results Table.
The Peak Review window contains the peaks listed in the
Results table. If some samples are hidden in the table (for
example, if a query is applied), then they are also hidden in peak
review.
Peak Integration: To move to Right-click anywhere in the Peak Review pane and then click
the first peak in the batch Show First Page. To move to the last peak in the batch, right-
click anywhere in the Peak Review pane and then click Show
Last Page.

Detect Peaks
The software detects peaks in four stages.
1. First, it finds the potential peak start by examining the distance between each
bunched point and the preceding one. When the distance exceeds the current noise
threshold, a potential peak start has been found.
2. Then it confirms the peak start by making sure that enough points exist in a row to
exceed the area threshold.
3. Next, it finds the peak top by searching for a point that is lower than the previous
point.
4. Finally, it finds the end of the peak by identifying the place where the distance
between one bunched point and the next falls below the noise threshold. If
necessary, it then separates peaks.

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Find the Potential Peak Start

To find the potential start of a peak, the software measures the intensity difference between
sequential pairs of bunched points, starting at the first point. When it finds a difference that
exceeds the current noise threshold, the software declares the first point a potential peak start.
Figure 6-4 Find the Potential Peak Start

Item Description
1 Exceeds noise threshold
2 Potential peak start
3 Does not exceed noise threshold

Confirm the Peak Start

To make sure that it has found a real peak, the software moves along the curve, adding the
intensity difference between each bunched data point from the intensity at the potential peak start
to a total sum. This process stops when the intensity difference between successive points is less
than the noise threshold. This sum is an approximation of the area of the leading edge of the
peak. If this sum exceeds the area threshold, then the software confirms the peak start.
Next, the software determines the actual start of the peak by moving backward from the potential
peak start until it finds the lowest point in the peak. It moves back through five bunches of raw
data. This point is the actual peak start.

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Figure 6-5 Confirm the Peak Start

Item Description
1 Sum of area slices greater than threshold

Figure 6-6 Confirm the Actual Peak Start

Item Description
1 Look through data points in this region
2 Minimum data point
3 Potential peak start

Find the Peak Top

To find the peak top, the software first looks for a point that is lower than the preceding point.
Then, to confirm that it has found the top correctly, it sums the intensity differences between the
potential top and subsequent bunched points until it reaches the end of the peak. If the total
distance between points exceeds two-thirds of the area threshold, then the peak top is

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confirmed. That is, the software makes sure it has a peak first, and then works backward to find
the top of it.
If, however, the software finds a higher bunched point before the area test has been passed, then
it identifies a new top and restarts the area test.

Note: The actual retention time for a peak is not simply the point identified as
described previously. Instead, it is determined from a quadratic fit based on the three
highest data points.

Figure 6-7 Find the Peak Top

Item Description
1 Sum of area slices is greater than two thirds of the area threshold

Figure 6-8 Identify a New Peak Top

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Item Description
1 The shoulder has a maximum, but the cumulative crest area is not greater than two
thirds of the area threshold

Find the Peak End

The software declares a peak end point when one of the following occurs:
• The difference between two consecutive points fails the noise threshold test.
• The software detects the start of a new peak.
In either case, the lowest bunched point from the last five bunches is considered to be the actual
end point of the peak.
In general, the software finds several peaks for each chromatogram. The peak it selects is the
one whose retention time is the closest to the expected retention time, specified in the method. If
no peak has a retention time within the specifications, the software marks the peak as not found.
Figure 6-9 Find Peaks

Item Description
1 The shoulder has no separate maximum point

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Figure 6-10 Find the Peak End: Case 1

2
3

Item Description
1 Exceeds noise threshold
2 Peak end
3 Does not exceed noise threshold

Figure 6-11 Find the Peak End: Case 2

Item Description
1 Peak end

Separate Peaks
If a new peak begins before the current peak hits the baseline, the software decides, based on
the following criteria, whether to resolve the baseline by using exponential skims. The skim
passes under one or more peaks that follow the precursor. These peaks are called product
peaks.

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When the software performs an exponential skim, it subtracts the area below the skim from the
product peaks and gives it to the precursor peak. It then subtracts the small area above the skim
from the precursor peak and adds it to the first product peak.
The software uses the following criteria to determine whether it will use exponential skimming:
• Exponential Peak Ratio.
• Exponential Adjusted Ratio.
• Exponential Valley Ratio.
Figure 6-12 Separate Peaks: An Exponential Skim

4 4

Item Description
1 This area is subtracted from the precursor peak and added to the first product peak
2 Exponential skim
3 Cluster baseline
4 These areas are subtracted from the product peaks and added to the precursor peaks

Queries
A query is a method of selecting only those records that meet certain criteria. Users can use
queries to view particular parts of the data in the Results Table that interests them, based on
textual or mathematical selections.
When a query is used, the table displays only the rows of data that meet the selected criteria. All
columns are displayed. Selections can be further refined by running a second query on the rows
displayed by the first query.
Use pre-defined choices and typed entries to create a query that can be executed, saved, or
modified. Each line of the query works like a Boolean search that runs against Results Table
columns to determine which records to display. Each line of the query selects only the records for
display that meet its criteria. A preset or table-specific query can be defined.
It is recommended that the user validate any queries that are used to analyze data in a Results
Table.

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Queries on Sample Type

For a query designed to select only the standard sample type, the Results Table displays only
those rows of data that contain Standard in the Sample Type column.

Default Queries and Table-Specific Queries

A default query is generally used to identify samples that do not meet certain criteria. A table-
specific query is generally used to identify records that meet certain criteria.
The default query is generally used to find problems with quality control or standards. If the
concentration and maximum variation of the QCs and standards are selected in the Quantitation
Method Wizard, then the Results Table displays only those samples that lie outside this range. If
the Results Table displays nothing, then the samples are all right.
Table-specific queries are run against a displayed Results Table to select records that meet
certain criteria. Design these queries through the menu that is available by right-clicking in the
table. Save and export a query to make it available for future Results Tables.

Table-Specific or Global Settings

When working with table setting options, procedures can be table-specific or global.
• Table-specific settings: When table settings are modified on a table itself, the
changes to the settings are available only to that table; however, they can be
exported as global settings.
• Global settings: Modifying global settings involves making changes to a group of
settings that can be applied to future Results Tables. To customize a Results Table
that is being created, choose a group of settings on the Create Quantitation Set:
Select Settings & Query page. If a group of settings is not selected, the software
automatically uses the preset settings.

How Accuracy Variations Affect the Results

For preset queries, accuracy is expressed as a percent and implemented as plus or minus that
number. For example, if 10 is typed for Maximum Variation for standards in the Create Default
Query dialog, then all records containing standards whose accuracy falls outside 90% and 110%
are displayed in the Results Table. If 5 is typed, only standards whose accuracy is less than 95%
and greater than 105% would appear in the Results Table. For more information, refer to Results
Tables on page 82.

Regression
This section gives the equations for each of the regression types. In the following equations, x, y,
and w are as described in Weighting Factors on page 94.
In the following equations, x represents the analyte concentration for standard sample types, and
y represents the corresponding peak area or height. The exact variables used for the regression
depend on whether or not an internal standard is being used and whether the peak area or the
peak height is used as shown in the following table.

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Table 6-7 Regression Formulae

Internal Standard Used? Area Used? x y
Yes Yes Ca/Cis/DF Aa/Ais
Yes No Ca/Cis/DF Ha/His
No Yes Ca/DF Aa
No No Ca/DF Ha

Where:
• Ca = actual analyte concentration
• Cis = internal standard concentration
• DF = dilution factor
• Aa = analyte peak area
• Ais = internal standard peak area
• Ha = analyte peak height
• His = internal standard peak height

Linear Regression
Linear regression assumes that the points of the standard fall on a straight line.
The linear calibration equation is calculated as:
y=mx+b
The slope and intercept are calculated as:
m =  wwxy – wxwy   D x
b =  wx 2 wy – wxwxy   D x
The correlation co-efficient is calculated as:
r =  wwxy – wxwy    D x D y 
where:
D x = wwx 2 –  wx  2
D y = wwy 2 –  wy  2

Linear Through Zero Regression

Linear through zero regression assumes that the points of the standard fall on a straight line and
that the points do line up with the zero point on the x and y axes. Use this setting to force the line
to go through the zero point.
The linear through zero calibration equation is calculated as:
y=mx
The slope is calculated as:

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m = wxy  wx 2
The correlation co-efficient is calculated as:
r = wxy   wx 2 wy 2 
If the standard points fall on a straight line, then to average the points use mean response factor
regression to produce an average of the slope for every point on the curve.

Mean Response Factor

The mean response factor calibration is calculated as:
y=mx
This is the same equation as for the linear though zero case. However the slope is calculated
differently as:
m = w  y  x   w
The standard deviation of the response factor as:
 =  nD   n – 1   w
Where:
D = w wy 2  x 2 –  wy  x 2

Note: Points whose x value is zero are excluded from the sums.

If there is some linear and some curvature in the line of points, then use power regression
instead of linear or quadratic regression to produce a line somewhere between these fits.

Power
The power function calibration equation is calculated as follows:
y = ax n
The equations for the linear calibration are used as described previously to calculate the slope
(m) and intercept (b), except that x in those equations is replaced by ln x and y replaced by ln y.
When this is done a, p and the correlation co-efficient (r) are calculated as:
a = exp ( b )
p=m
r=r
If the points of the standard do not fall on a straight line, use quadratic regression to produce a
quadratic fit to the data points. This setting is frequently used.

Quadratic Calibration Equation

The quadratic calibration equation is calculated as:
y = a2x2 + a1x + a0
The polynomial co-efficients are calculated as:

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a2 =  b 2  b 0 – b 5  b 3   b 1  b 0 – b 4  b 3
a1 = b5  b3 – a2b4  b3
a 0 =  wy – a 1 wx – a 2 wx 2  w
The correlation co-efficient as:
r =  1 – w w  y – a 2 x 2 – a 1 x – a 0 2  Dy
Where:
D y = wwy 2 –  wy 2
b 0 = wx  w – wx 2  wx
b 1 = wx 2  w – wx 3  wx
b 2 = wy  w – wxy  wx
b 3 = wx 2  wx – wx 3  wx 2
b 4 = wx 3  wx – wx 4  wx 2
b 5 = wxy  wx – wx 2 y  wx 2

Report Templates
Add the following information to report headers and footers.

Tip! Make a backup of the existing report templates before editing them.

Table 6-8 Basic Design Elements

Element Definition
Printing Date The date the document was printed.
Printing Time The time the document was printed.
Operator The operator who printed the document.
Workstation The workstation from which the document was printed from.
Page n of N Indicates the page number of total number of pages.
Custom Field Create custom text here.
Analyst Version Indicates the version of the Analyst® software.
User Type Indicates the user type (Security).
Electronic Signature Indicates whether the electronic signature feature (security) is
enabled or disabled.

Table 6-9 Acquisition Elements

Element Definition
Acquisition File The name of the data file with the sample acquisition information.
Acquisition Date The date the sample acquisition was run.

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Table 6-9 Acquisition Elements (Continued)

Element Definition
Acquisition Time The time of the sample acquisition was run.
Operator The name of the operator who ran the sample batch.
Batch Name The name of the batch.
Sample Number The number related to the sample.
Sample Name The name of the sample.
Sample Comment Shows any comments about the sample entered through the
Acquisition Method Editor.
Sample ID The identification number of the sample.
Scan Mode Indicates the method in which the system calculates the mass
points for a scan for a full mass range scan.
Scan Type and Polarity Indicates the acquisition scan type (Q1, Q3, MRM, Product ion,
Precursor ion, neutral loss/gain) and acquisition method polarity
(positive or negative).
Scan Mass(es) Ions or ion fragments to be scanned.
Dwell Time The time the system takes to scan a particular mass.
Pause Time Indicates the pause between the scanning of mass ranges or
between experiments.
Ion Energy Ion energy comes from the acquisition method and is related to the
IonSpray™ ion source voltage or the collision energy.
Collision Energy Collision energy comes from the acquisition method and is related
to the IonSpray ion source voltage.
Period and Experiment A period contains a collection of experiments; an experiment
contains a number of properties, such as Scan Type, Scan Mode,
Resolution, Ion Source Parameters, and a collection of mass
ranges or masses.
State Table Parameters The instrument parameters used in the experiment.
Pump The name of the pump used for the experiment.
Autosampler The name of the autosampler used for the experiment.
Custom Annotation The custom text added via the Batch Editor.
Collected By The name of the person who collected the data.

Table 6-10 Quantitation Elements

Element Definition
Results Table Name The name of the Results Table.
Results Table Path The location of the Results Table.
Method Name The name of the quantitation method.
Method Path The location of the method file.
Project Name The name of the project.
Results Table Name The name of the Results Table.

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Customize Reports
The Report Template Editor provides a way to customize reports by setting up headers, footers,
and page layouts. Use report templates with both printed output and data exported to another
application.
Printed output includes several types of elements.
• Window: Windows in the Analyst software appear in the working area of the
software window, below the toolbar and to the right of the Navigation bar. Printing a
window will print everything that is shown in that space.
• Pane: Panes are parts of a window arranged in such a way that they do not overlap
and are always fully visible. For example, the Method Editor window contains two
panes: the Browser pane and the Method Editor pane. Information from each pane in
the window can be printed.
• Report: Reports are structured sets of information created in the software. Some
reports can be directly printed, such as calibration reports; other information must be
exported, such as batches and quantitation Results Tables.
• Workspace: A workspace is a particular arrangement of windows and panes along
with an associated file or files. Printing a workspace involves printing each open
window and pane in the current mode.

Preview, Print, and Export Reports

Acquisition methods, batches, quantitation Results Tables, and graph Results Tables can be
exported as reports. Other forms of information, such as calculator data, can be exported but
cannot be customized with a report template.
Most areas seen on the screen can be printed. Graphs can be previewed, scaled, or copied
using the Print Preview feature.
An exported report is saved in a file format that is appropriate for programs such as Notepad,
Microsoft Word or Excel, or certain LIMS (Laboratory Information Management System)
software.
Export reports in the following formats:
• .csv
• .doc
• .pdf
• .txt
The formats available depend on the information being exported. For example, a graph can be
exported as a .pdf; a table of data can be exported as a .txt file.
To include additional information in the header and footer of the report, print the report using an
appropriate template.
Table 6-11 Preview, Print, and Export Reports
To do this... ...do this
Preview a graph 1. Click File > Print Preview > Pane.
2. Edit the Print Preview dialog.
3. Click Print.

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Table 6-11 Preview, Print, and Export Reports (Continued)

To do this... ...do this
Print a report without a Click File > Print, and then click the report.
template
Print a report with a template 1. Click File > Print & Report Setup.
2. In the Report Template section, select the template.
3. Click OK.
Export a report 1. Click File > Export.
2. In the File field, type the name of the file.
3. In the Save as type list, select the appropriate file type.
4. If exporting a report in Quantitate mode, select either All
Columns or Visible Columns from the Export section and
then click Save.

Results Tables
Results Tables summarize the calculated concentration of analyte in each unknown sample
based on the calibration curve. They also include the calibration curves and statistics for the
results.
Export the data from a Results Table to a .txt file for use in other applications, such as Microsoft
Excel. All possible data in the table or just the data in the visible columns can be exported.
The data in a Results Table can be sorted in three different ways:
• Quickly sort the table on one to three columns, using one of the Sort buttons. This
sort criteria cannot be saved.
• Create a table-specific sort to save the sort criteria with the current table. Table-
specific sorts enable sorting the current table on one to three columns and saving
the criterion for use with that table.
• Use a previously created preset sort. Create and save a sort and later apply it to a
Results Table.

Tip! To save a sort or any other table setting, right-click in the table and
then click Table Settings > Export To New Table Settings. The sort and
other parameters can be used in the current project. To use the table
settings in a different project, copy it to another project. Click Tools >
Project > Copy Data. A new project must be created and selected to be
available for use.

Define the Layout of Results Tables

Four predefined views of the Results Table are available.
• Right-click in the Results Table and then click one of the following:
• To view the Full layout, click Full.
All the analytes are displayed.
• To view the Summary layout, click Summary and then click a field name.

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• To view the Analyte layout, click Analyte and then click a single analyte if
more than one analyte exists.
• To view the Analyte Group layout, click Analyte Group and then click an
analyte group.

Tip! A new analyte group must be created first. To do this, right-click in

the Results Table and then click Analyte Group > New.

The table opens with the selected layout.

Tip! To go back to the full view, right-click and click Full.

Sort Data in Results Tables

1. Select up to three columns in the Results Table in the order they need to be sorted.
2. Do one of the following:
• To sort in ascending order, click A-Z.
• To sort in descending order, click Z-A.

Sort a Results Table and Save the Sort Criteria

1. Right-click in the Results Table and then click Sort > New.
Figure 6-13 Sort Dialog

2. In the Name field, type the name for the new sort.

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3. For each sorting rule to be set, in the Sort By section, do the following:
a. In the Group list, select the type of column.
b. In the Column list, select the column.
c. Select the direction of the sort: Ascending or Descending.
4. Do one of the following:
• To perform the sort, save the sort criteria, and close the Sort dialog, click
Save/Execute.
• To perform the sort and close the Sort dialog without saving the sort criteria,
click Execute.

Save Default Sort Criteria for Future Results Tables

1. Click Tools > Settings > New Quantitation Results Table Settings.
Figure 6-14 Table Settings Dialog

2. Expand the Table Settings folder and then double-click the Default folder.
3. From the expanded Default folder, select the Sorts folder.
4. Click New.

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Figure 6-15 Sort Dialog

5. In the Name field, type a name.

6. For each sorting rule to be set, in the Sort By section, do the following:
a. In the Group list, select the type of column.
b. In the Column list, select the column.
c. Select the direction of the sort: Ascending or Descending.
7. To save the criteria and close the Sort dialog, click OK.
8. Click Done.

Sort a Results Table using Preset Sort Criteria

• Right-click in the Results Table, click Sort and then select the name of the sort.

About using Queries with Results Tables

A query is a request for records in a Results Table that the certain conditions set using textual or
mathematical selection criteria. Apply a query either during the process of generating a Results
Table or after one has been generated. These two types of queries are called default and table-
specific queries. For more information, refer to Default Queries and Table-Specific Queries on
page 76.
It is recommended that the user validate any queries that are used to analyze data in a Results
Table.

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Compare Results Between Batches

When more than one Results Table is displayed, obtain statistical information about the
standards and QCs for additional batches in the Statistics window. Normally results are
compared between batches to look for trends in the standards or QCs or to verify that the method
is valid.
For two or more open Results Tables, compare results in the Statistics window. Both sets of
statistics appear in the Statistics window.

Note: The number of analytes and the number of analyte names must be the same for
the data to be combined in the Statistics window.

How Concentration Levels Affect Results

The concentration is defined for all QCs and standards. If there is a change in the accuracy of the
concentration level by more than the amount defined in the Max. Variation field in the Create
Default Query dialog, then this information is shown in the Results Table.

Results Table Layouts

The software has four predefined views of the Results Table.
• Full Layout View
• Summary Layout View
• Analyte Layout View
• Analyte Group Layout View
Each analyte from a multiple analytes sample can be seen in the Analyte Layout view. The preset
view is Full layout.

Full Layout View

The preset Full Layout view shows the data for all analytes in the quantitation batch. The
columns that are displayed depend on the columns selected in the Results Table Columns
dialog, and the settings selected on the second page of the Quantitation Method Wizard.

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Figure 6-16 Sample Full Layout View

Summary Layout View

The Summary Layout view contains the locked columns and the chosen field for each analyte in
the remaining columns. For example, if Analyte Peak Area is selected from the menu for two
analytes, then the Sample Name and Analyte Peak Area columns for those analyte names are
seen. The Summary Layout view also includes the Formula and Custom columns, if these exist.
Figure 6-17 Sample Summary Layout View

Analyte Layout View

The Analyte Layout view contains the data for a particular analyte; all other analytes are hidden.
For example, if analyte A is selected, all the data for analyte A is seen. The columns that are
displayed depend on the columns selected in the Results Table Columns dialog, and the settings
selected on the second page of the Quantitation Method Wizard.
An Analyte Layout view, with Peak 1 selected, might look like Figure 6-18 on page 88. If this
layout is compared with the Full Layout view, notice that every other row has been excluded.

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Figure 6-18 Sample Analyte Layout View

Analyte Group Layout View

The Analyte Group Layout view contains the data for the analytes that belong to a particular
group. Columns that are selected as shown in the Results Table Columns dialog appear in the
Results Table as shown in Figure 6-19 on page 88. Display the Analyte Peak Name column in
the Results Table to show the names of the analytes that belong in the group.
Figure 6-19 Sample Analyte Group Layout View

Results Table Fields

Add columns to the standard Results Table to display DAD (diode array detector) data for the
Analyte, Internal Standard, and Record fields.

Formula Fields
The Formula fields display the result of a spreadsheet-style formula defined by users. The
Formula field located at the top of the Results Table is shown only if at least one Formula column
is in the Results Table. The Formula field becomes active when Formula column cells are
selected. The Delete Formula Column button below the Formula field also becomes available
when the Formula column is selected.
It is recommended that the user validate the results if a formula column is used.

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Custom Fields
Custom fields contain information defined during the acquisition process. When acquiring
samples, users can create custom columns and define the type of data that goes in them. Once
the custom column is part of the Results Table, it can be treated like any other column (for
example, move it, hide it, base a formula on it).

Internal Standards Column Fields

The Internal Standard Results fields display information about the internal standard after
analysis. Table 6-12 on page 89 shows the available fields.
Table 6-12 Internal Standards Column Fields
Field Definition
IS Peak Name The name of the internal standard peak.
IS Units The units in which the internal standard is given.
IS Peak Area The area of the internal standard peak.
IS Peak Height The height of the internal standard peak.
IS Concentration The known concentration of the internal standard, this
applies to standard and quality control sample types.
Zeroes are displayed for solvent, blank, and double blank
sample types; N/A is displayed for unknowns.
IS Retention Time The chromatographic retention time as determined by the
software.
IS Expected Retention Time The retention time of the representative sample. Taken
from the quantitation method.
IS Retention Time Window The retention time window as specified in the quantitation
method.
IS Centroid Location The intensity-weighted average retention time for the
analyte. The peak areas up to and after this time are
identified.
IS Start Scan The cycle number associated with the period/experiment
combination where the peak begins.
IS Start Time The time associated with the period/experiment
combination where the peak begins.
IS Stop Scan The cycle number associated with the period/experiment
combination where the peak ends.
IS Stop Time The time associated with the period/experiment
combination where the peak ends.
IS Integration Type The method by which the baseline was found and
integrated when the peak was found. The types are:
manual, automatic (Baseline-to-Baseline, Valley,
Exponential Skim, and Exponential Child).
IS Signal to Noise The signal-to-noise ratio of the peak.
IS Peak Width The ratio of the peak height to its width.
IS UV Range The UV range of the internal standard.

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Table 6-12 Internal Standards Column Fields (Continued)

Field Definition
IS UV Channel The UV channel of the internal standard.
IS Peak Width at 50 Percent (min.) A read-only column displaying the peak width at 50% of
peak height.

IS Baseline Slope (%/min.) A read-only column displaying the slope of the baseline.
IS Peak Asymmetry A read-only column displaying the peak asymmetry which
is calculated by the following formula:
[(Peak End Time) – (Retention Time)] / [(Retention Time) –
(Peak Start Time)]
Values near 1.0 indicate symmetric peaks, values greater
than 1.0 indicate tailing peaks, and values less than 1.0
indicate fronting peaks.
IS Processing Alg A read-only column displaying the processing algorithm
used.
IS Integration Quality The Integration Quality™ Index indicates how well the
peak is integrated. Values closer to 1 indicate well-
integrated peaks and values closer to 0 indicate poorly
integrated peaks.

Record Fields
The Record fields display additional information about each sample record (information that is
applicable only to the analyte, not the internal standard). Table 6-13 on page 90 shows the
available fields.
Table 6-13 Record Fields
Field Definition
Use Record Indicates whether this record should be included for calibration.
Applies to standards and QCs. If the check box is cleared, then the
unused standards and QCs are struck out in the Statistics Table.
Record Modified Indicates whether the quantitation method used for the record was
modified in any way from the original.
Calculated Concentration The calculated concentration of the analyte as calculated using the
calibration curve.
Relative Retention Time The ratio of the retention times of the internal standard and the
analyte.
Accuracy The calculated concentration divided by the known concentration
(as a percentage).
Response Factor The peak area or height (depending on the regression option)
divided by the analyte concentration.

Sample Column Fields

The Analyte Results fields display information about each analyte and internal standard (if one
was used) after analysis. Table 6-14 on page 91 shows the available fields.

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Table 6-14 Results Tables: Sample Column Fields

Field Definition
Analyte Peak Name The name of the analyte.
Analyte Units The units in which the analyte concentrations are given.
Analyte Peak Area The area of the analyte.
Analyte Peak Height The height of the analyte peak.
Analyte Concentration The actual, known concentration of the analyte, this applies to
standard and quality control sample types. Zeroes are
displayed for solvent, blank, and double blank sample types;
N/A is displayed for unknowns.
Analyte Retention Time The chromatographic retention time as determined by the
software.
Analyte Expected Retention The retention time of the representative sample as taken from
Time the quantitation method.
Analyte Retention Time Window The retention time window as specified in the quantitation
method.
Analyte Centroid Location The intensity-weighted average retention time for the analyte.
The peak areas up to and after this time are identified.
Analyte Start Scan The cycle number associated with the period/experiment
combination where the peak begins.
Analyte Start Time The time associated with the period/experiment combination
where the peak begins.
Analyte Stop Scan The cycle number associated with the period/experiment
combination where the peak ends.
Analyte Stop Time The time associated with the period/experiment combination
where the peak ends.
Analyte Integration Type The method by which the baseline was found and integrated
when the peak was found. The types are: manual, automatic
(Baseline-to-Baseline, Valley, Exponential Skim, and
Exponential Child).
Analyte Signal to Noise The signal-to-noise ratio of the peak compared to the
baseline.
Analyte Peak Width The ratio of the peak height to its width.
Analyte UV Range The UV range of the analyte.
Analyte UV Channel The UV channel of the analyte.
Analyte Peak Width at 50 A read-only column displaying the peak width at 50% of peak
Percent (min.) height.
Analyte Baseline Slope (%/ A read-only column displaying the slope of the baseline.
min.)

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Table 6-14 Results Tables: Sample Column Fields (Continued)

Field Definition
Analyte Peak Asymmetry A read-only column displaying the peak asymmetry which is
calculated by the following formula:
[(Peak End Time) – (Retention Time)] / [(Retention Time) –
(Peak Start Time)]
Values near 1.0 indicate symmetric peaks, values greater
than 1.0 indicate tailing peaks, and values less than 1.0
indicate fronting peaks.
Analyte Processing Alg A read-only column displaying the processing algorithm used
Analyte Integration Quality Integration Quality™ Index indicates how well the peak is
integrated. Values closer to 1 indicate well-integrated peaks
and values closer to 0 indicate poorly integrated peaks. This
facilitates peak review as users can see the peaks with low
Analyte Integration Quality values for manual review. In
addition, users can query the data for the Analyte Integration
Quality values that are less than a value they consider
acceptable in order to display and manually review a subset
of the data.

Sample Column Fields

The Sample Column fields display information about the sample that is common to all analytes.
Blank and double blank can be defined differently from lab to lab. Table 6-15 on page 92 shows
the available fields:
Table 6-15 Sample Column Fields
Field Definition
Sample Name The name that the user assigned to the sample when it was
acquired.
Sample ID A user-defined identifier associated with the sample.
Sample Type All analytes within a sample must have the same sample type.
One of the following sample types is displayed:
Unknown: Contains analytes whose concentrations are to be
determined.
Standard: A sample with known analyte concentration; used
for calibration purposes.
Quality Control: A sample with known analyte concentration;
used to check the accuracy of the standard curve.
Solvent: Confirms that the instrument is clean. Solvents are
not run through the sample preparation process.
Blank: A zero concentration sample that is not used in
regression.
Double Blank: A sample prepared without an internal standard
or sample analyte; confirms that the extraction process added
nothing.

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Table 6-15 Sample Column Fields (Continued)

Field Definition
Sample Comment A comment describing the sample.
Set Number A number that identifies a subset of an entire batch.
Acquisition Method The name of the method used to acquire the samples.
Acquisition Date The date and time when the acquisition was run.
Rack Type An identifier associated with the kind of autosampler rack used
(if any).
Rack Number The rack position in which the sample was placed when it was
acquired. (In single-rack autosamplers, this is always 1.)
Vial Position The position in the autosampler plate where the vial was
located.
Plate Type An identifier for the kind of plate used (multiplate racks only.)
Plate Number The plate position on the rack (multiplate racks only.)
File Name The name of the raw data file. This name is not unique because
the data from many samples may be contained in a single data
file.
Dilution Factor The amount by which the sample was diluted; used to
determine the calculated concentration.
Sample Annotation Additional comments describing the sample.
Weight-to-Volume Ratio Gives the weight to volume ratio for the sample.

Table 6-16 DAD Fields

Field Definition
Analyte Peak Area for DAD The area of the analyte peak (mAU/min).
Analyte Peak Height for DAD The height of the analyte peak (mAu).
Analyte Wavelength Ranges The range of wavelengths (nm).
IS Peak Area for DAD The area of the internal standard peak (mAU/min).
IS Wavelength Ranges The range of wavelengths (nm).
IS Peak Height for DAD The height of the internal standard peak (mAU)
Calculated Concentration for DAD.

Table 6-17 on page 93 shows the fields that can be added to the Results Table for data acquired
by an ADC (analog-to-digital converter).
Table 6-17 ADC Fields
Field Definition
Analyte Channel The ADC channel from which the analyte was acquired.
Analyte Wavelength Ranges The range of wavelengths (nm).
IS Channel The ADC channel from which the internal standard was
acquired.

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Table 6-17 ADC Fields (Continued)

Field Definition
IS Wavelength Ranges The range of wavelengths (nm).

Results Table Tips

Table 6-18 Results Table Tips
To do this... ... do this
Table-specific queries: To Right-click anywhere in the Results Table and then click Query >
view the entire table again Show All. The query can be applied again or edited.
To examine calibration Right-click anywhere in the curve, click Active Plot, and select
curves the curve to be plotted on top.
Sample statistic review: To Select the Display the Data Set(s) check box, and then, in the
review an individual peak Data Point column, double-click the data point that represents
the peak. The software displays the Peak Review window with
the user-selected peak.
Results Tables: To return the Right-click on the Results Table and click Sort > Sort By Index.
Results Table to its original
order

Weighting Factors
The following table shows how the weighting factor (w in the equations below) is calculated for
each of the seven weighting types. Refer to Regression on page 76.
Table 6-19 Weighting Factors
Weighting Type Weight (w)
None Always 1.0
1/x If |x| < 10–5 then w = 105, otherwise w = 1 / |x|. To place some additional
emphasis on lower-value points, use a weighting factor of 1/x.
1 / x2 If |x| < 10–5 then w = 1010, otherwise w = 1 / x2. Use a weighting of 1/x
squared to provide a much higher emphasis on lower-value points.
1/y If |y| < 10–8 then w = 108, otherwise w = 1 / |y|. Use a weighting factor of
1/y when calibrating by the area (y-axis) rather than by the concentration
(x-axis), and some emphasis needs to be placed on lower-value points. A
weighting of 1/y is a variant of 1/x where y and x should be proportional to
each other.
1 / y2 If |y| < 10–8 then w = 1016, otherwise w = 1 / y2. Use a weighting factor of
1/y squared when calibrating by the area (y-axis) rather than by the
concentration (x-axis), and extra emphasis needs to be placed on lower-
value points. A weighting of 1/y2 is a variant of 1/x2 where y and x should
be proportional to each other.

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Table 6-19 Weighting Factors (Continued)

Weighting Type Weight (w)
ln x If x < 0, then an error is generated, otherwise if x < 10–5 then w = ln 105,
otherwise w = |ln x|. Use the logarithm of x to place more weight on
higher value points.
ln y If y < 0, then an error is generated, otherwise if y < 10–8 then w = ln 108,
otherwise w = |ln y|. Use the logarithm of y to place more weight on
higher value points. Use when calibrating by the area (y-axis) rather than
by the concentration (x-axis).

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PPG Exact Mass Table
A
Table A-1 on page 97 lists the exact monoisotopic masses and charged species (positive and
negative) observed with the PPG (polypropylene glycol) calibration solutions. The masses and
ions were calculated using the formula M = H[OC3H6]nOH, while the positive ion MS/MS
fragments used the formula, [OC3H6]n(H+). In all calculations, H = 1.007825, O = 15.99491, C =
12.00000, and N = 14.00307.

Note: When performing calibration with the PPG solutions, make sure that the correct
isotope peak is used.

Table A-1 PPG Exact Masses

n Exact mass (M + NH4)+ MS/MS (M + 2NH4)2+ (M + COOH)–
(M) fragments
1 76.052 94.087 59.0 56.061 121.050
2 134.094 152.129 117.1 85.082 179.092
3 192.136 210.171 175.1 114.102 237.134
4 250.178 268.212 233.2 143.123 295.176
5 308.220 326.254 291.2 172.144 353.218
6 366.262 384.296 349.2 201.165 411.259
7 424.304 442.338 407.3 230.186 469.301
8 482.346 500.380 465.3 259.207 527.343
9 540.388 558.422 523.4 288.228 585.385
10 598.430 616.464 581.4 317.249 643.427
11 656.471 674.506 639.4 346.270 701.469
12 714.513 732.548 697.5 375.291 759.511
13 772.555 790.590 755.5 404.312 817.552
14 830.597 848.631 813.6 433.333 875.594
15 888.639 906.673 871.6 462.354 933.636
16 946.681 964.715 929.7 491.373 991.678
17 1004.723 1022.757 987.7 520.396 1049.720
18 1062.765 1080.799 1045.7 549.417 1107.762
19 1120.807 1138.841 1103.8 578.438 1165.804
20 1178.849 1196.883 1161.8 607.459 1223.845
21 1236.890 1254.925 1219.9 636.480 1281.887
22 1294.932 1312.967 1277.9 665.501 1339.929
23 1352.974 1371.009 1335.9 694.521 1397.971
24 1411.016 1429.050 1394.0 723.542 1456.013
25 1469.058 1487.092 1452.0 752.563 1514.055

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Table A-1 PPG Exact Masses (Continued)

n Exact mass (M + NH4)+ MS/MS (M + 2NH4)2+ (M + COOH)–
(M) fragments
26 1527.100 1545.134 1510.1 781.584 1572.097
27 1585.142 1603.176 1568.1 810.605 1630.138
28 1643.184 1661.218 1626.2 839.626 1688.180
29 1701.226 1719.260 1684.2 868.647 1746.222
30 1759.268 1777.302 1742.2 897.668 1804.264
31 1817.309 1835.344 1800.3 926.689 1862.306
32 1875.351 1893.386 1858.3 955.710 1920.348
33 1933.393 1951.428 1916.4 984.731 1978.390
34 1991.435 2009.469 1974.4 1013.752 2036.431
35 2049.477 2067.511 2032.5 1042.773 2094.473
36 2107.519 2125.553 2090.5 1071.794 2152.515
37 2165.561 2183.595 2148.5 1100.815 2210.557
38 2223.603 2241.637 2206.6 1129.836 2268.599
39 2281.645 2299.679 2264.6 1158.857 2326.641
40 2339.687 2357.721 2322.7 1187.878 2384.683
41 2397.728 2415.783 2380.7 1216.899 2442.724
42 2455.770 2473.805 2438.7 1245.920 2500.766
43 2513.812 2531.847 2496.8 1274.940 2558.808
44 2571.854 2589.888 2554.8 1303.961 2616.850
45 2629.896 2647.930 2612.9 1332.982 2674.892
46 2687.938 2705.972 2670.9 1362.003 2732.934
47 2745.980 2764.014 2729.0 1391.024 2790.976
48 2804.022 2822.056 2787.0 1420.045 2849.017
49 2862.064 2880.098 2845.0 1449.066 2907.059
50 2920.106 2938.140 2903.1 1478.087 2965.101
51 2978.147 2996.182 2961.1 1507.108 3023.143
52 3036.189 3054.224 3019.2 1536.129 3081.185
53 3094.231 3112.266 3077.2 1565.150 3139.227
54 3152.273 3170.307 3135.2 1594.171 3197.269
55 3210.315 3228.349 3193.3 1623.192 3255.311
56 3268.357 3286.391 3251.3 1652.213 3313.352
57 3326.399 3344.433 3309.4 1681.234 3371.394
58 3384.441 3402.475 3367.4 1710.255 3429.436
59 3442.483 3460.517 3425.5 1739.276 3487.478
60 3500.525 3518.559 3483.5 1768.297 3545.5202

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Table A-1 PPG Exact Masses (Continued)

n Exact mass (M + NH4)+ MS/MS (M + 2NH4)2+ (M + COOH)–
(M) fragments
61 3558.566 3576.601 3541.5 1797.318 3603.562
62 3616.608 3634.643 3599.6 1826.339 3661.604
63 3674.650 3692.685 3657.6 1855.359 3719.645
64 3732.692 3750.726 3715.7 1884.380 3777.687
65 3790.734 3808.768 3773.7 1913.401 3835.729
66 3848.776 3866.810 3831.7 1942.422 3893.771
67 3906.818 3924.852 3889.8 1971.443 3951.813
68 3964.860 3982.894 3947.8 2000464 4009.855
69 4022.902 4040.936 4005.9 2029.485 4067.897
70 4080.944 4098.978 4063.9 2058.506 4125.938
71 4138.985 4157.020 4122.0 2087.527 4183.980
72 4197.027 4215.062 4180.0 2116.548 4242.022
73 4255.069 4273.104 4238.0 2145.569 4300.064
74 4313.111 4331.145 4296.1 2174.590 4358.106
75 4371.153 4389.187 4354.1 2203.611 4416.148
76 4429.195 4447.229 4412.2 2232.632 4474.190
77 4487.237 4505.271 4470.2 2261.653 4532.231
78 4545.279 4563.313 4528.3 2290.674 4590.273
79 4603.321 4621.355 4586.3 2319.695 4648.315
80 4661.363 4679.397 4644.3 2348.716 4706.357
81 4719.404 4737.439 4702.4 2377.737 4764.399
82 4777.446 4795.481 4760.4 2406.758 4822.441

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