Quality Assurance and

Calibration Methods
Chapter 5

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 1
Chapter Outline
• Section 5-1 Basics of Quality Assurance
• Section 5-2 Method Validation
• Section 5-3 Standard Addition
• Section 5-4 Internal Standards

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 2
Case Study: Misconduct at the Gothan Crime Lab?

How to determine if data was falsified?

• Senior technician is accused of
falsifying methamphetamine assays
• Accuser is a visiting scientist and
may/may not have ulterior motives
• How do you determine (1) if mistakes
are made and (2) if mistakes are
intentional?
• Conclusion could force re-examination of 3 000+ drug cases
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 3
Section 5-1
Basics of Quality Assurance

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 4
Quality Assurance
Quality assurance is what we do to get the right answer.

• Answer should have sufficient Data quality standards:

accuracy and precision to
• Get the right data.
support subsequent decisions
• Get the data right.
• No point in obtaining more
accurate/precise answer than • Keep the data right.
necessary (Nancy W. Wentworth, U.S. EPA)

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 5
Table 5-1 Quality assurance process
Question Actions
Use Objectives
Why do you want the data and results, and • Write use objectives
how will you use the results?

Specifications
How good do the numbers have to be? • Write specifications
• Pick methods to meet specifications
• Consider sampling, precision, accuracy, selectivity, sensitivity, detection
limit, robustness, and rate of false results
• Employ blanks, fortification, calibration checks, quality control samples,
and control charts to monitor performance
• Write and follow standard operating procedures

Assessment
Were the specifications achieved? • Compare data and results with specifications
Is the method fit for purpose? • Document procedures and keep records suitable to meet use objectives
• Verify that use objectives were met

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 6
Use Objectives
The goal of quality assurance is making sure that results meet the
customer’s needs. Write clear, concise use objectives and keep in mind:

Raw data: measurements

Treated data: concentrations derived from raw data by use of
calibration methods
Results: quantities reported after statistical analysis of treated data

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 7
Specifications
Specifications: state how good the numbers need to be and what
precautions are required in the analytical procedure
• How shall samples be taken?
• How many are needed?
• Are special precautions needed to avoid degradation?
• What are practical restraints (cost, time, material)?
• What level of accuracy will satisfy the use objectives?
• What rate of false negatives or positives is acceptable?

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 8
 Box 5-1 Medical Implication of False Positive Results
False positive: concentration exceeds the legal limit
when, in fact, the concentration is below the limit
False negative: concentration is below the legal limit
when it is actually above the limit

Even well-executed procedures

produce some false conclusions due
to random error.

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 9
When Choosing a Method Consider
Selectivity: ability to distinguish analyte from other species in sample (avoid
interference)
Sensitivity: ability to respond reliably and measurably to change in analyte
concentration (slope of the calibration curve)
Blanks: sample not intended to contain analyte
• Account for interference by other species in the sample
• Account for trace amount of analyte found in reagents
• Frequent measures of blanks detect whether analyte from previous
sample is carried into subsequent analysis by vessels or instruments
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 10
Types of Blanks
Method blank
• All components except analyte
• Taken through all steps of the analytical procedure
• Subtract the response of the method blank from the response of sample
before calculating the quantity of analyte
Reagent blank
• Similar to a method blank, but it has not been subjected to all sample
preparation procedures
Field blank
• Indicates if analyte is inadvertently picked up by exposure to field conditions
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 11
Spike Recovery
Sometimes response to analyte can be decreased or increased by
something else in the sample

Matrix: everything in the unknown, other than the analyte

Spike (or fortification): a known quantity of analyte added to a sample

• Tests whether the response to the spike is the same as expected
based on known calibration curve
• Response is different from expectations indicates problems
(contamination or loss)

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 12
Example: Spike Recovery (1 of 3)
Let C stand for concentration. One definition of spike recovery is

C spiked sample − Cunspiked sample

% recovery =  100
C added

An unknown was found to contain 10.0 μg of analyte per liter. A spike

of 5.0 μg/L was added to a replicate portion of unknown. Analysis of
the spiked sample gave a concentration of 14.6 μg/L. Find the percent
recovery of the spike.

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 13
Example: Spike Recovery (2 of 3)
Solution: The percent of the spike found by analysis is

14.6 g/L − 10.0 g/L

% recovery =  100 = 92%
5.0 g/L

If the acceptable recovery is specified to be in the range of 96 to 104%,

then 92% is unacceptable. Something in your method or techniques
needs improvement.

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 14
Example: Spike Recovery (3 of 3)
Test Yourself: Find percent recovery if the fortified sample gave a
concentration of 15.3 μg/L.

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 15
Dealing with Large Numbers of Samples
Perform periodic calibration checks.
Method drift can be due to changes such as room temperature or
spoilage of reagents/standards.
Calibration check solutions should be different from ones used to
prepare the original calibration curve.
Performance test samples (quality control samples or blind samples)
help to eliminate bias introduced by an analyst who knows the
concentration of the calibration check sample.
Standard operating procedures (SOP) outline steps to be taken for the
procedure. Serve as control experiments to detect problems in the lab.
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 16
Assessment
Assessment is the process of
(1) Collecting data to show that analytical procedures are operating
within specific limits
(2) Verifying that final results meet use objectives
Documentation is critical. Standard protocols provide direction for what
must be documented and how documentation is to be done.
Control charts can be used to monitor performance.
If final results meet the use objectives, the method is fit for purpose.
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 17
Box 5-2 Control Charts
Laboratory measuring lanthanum (La) in uranium as part of
international nuclear non-proliferation monitoring
• Control chart: visual representation of
confidence intervals for a Gaussian
distribution
• Warns when a property strays dangerously
far from an intended target value
• For a Gaussian distribution, 95.5% of all
observations are within ±2; 99.7% within
±3
• ±2 = warning lines and ±3 = action
lines
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 18
Table 5-2 Agencies that develop standard methods
(1 of 2)
Government agencies such as the U.S. Environmental Protection
Agency (EPA) set requirements for quality assurance and publish
standard methods for certified analysis.
American Public Health Association (APHA)
• 400 methods for analysis of water, water supplies, and wastewater
AOAC International (formerly Association of Official Analytical Chemists)
• 3 000 standardized chemistry and microbiological methods to ensure safety of food, beverages, dietary
supplements and similar products, and purity of their ingredients
ASTM International (formerly American Society for Testing and Methodology)
• 5 400 test methods for 90 industrial sectors, including oil and gas, mining, pulp and paper, industrial
chemicals, agriculture, and energy
National Institute for Occupational Safety and Health (NIOSH)
• Methods for industrial hygiene monitoring

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 19
Table 5-2 Agencies that develop standard methods
(2 of 2)
Occupational Safety and Health Administration (OSHA)
• Methods for sampling and analysis of contaminants in workplace air, on workplace surfaces, and in the
blood and urine of workers who are occupationally exposed

U.S. Environmental Protection Agency (EPA)

• 1 600 methods for drinking water, air pollution, water pollution, hazardous waste, pesticides, and
radiochemistry

U.S. Food and Drug Administration (FDA)

• Methods for allergens, additives, supplements, pesticides/herbicides, drug residues, toxic elements,
bacteria, and microbiologics in food and cosmetics
• Regulations and guidelines for the validation of analytical methods and procedures for drugs and biologics

U.S. Pharmacopeia (USP)

• Standards for medicines, food ingredients, dietary supplement products, and ingredients
• Other similar agencies are the British Pharmacopeia and European Pharmacopeia
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 20
Agencies that develop standard methods in VN
• Bộ Y Tế (Ministry of Health)
• Bộ TNMT (MONRE)
• Ex. Tiêu chuẩn Việt Nam TCVN 6180:1996 (ISO 7890/3 : 1988 (E)) về chất
lượng nước - Xác định nitrat - Phương pháp trắc phổ dùng axit
sunfosalixylic do Bộ Khoa học Công nghệ và Môi trường ban hành
• Bộ Xây Dựng (Ministry of Construction)
• Bộ KH và CN (Ministry of Science and Technology)

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 21
Section 5-2
Method Validation

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 22
Method Validation
Method validation: the process of proving that an analytical method is
acceptable for intended purpose

• Method selectivity • Range

• Linearity • Limit of detection
• Accuracy • Limit of quantitation
• Precision • Robustness

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 23
Selectivity
Selectivity: extent to which an analytical method can distinguish analyte
from everything else in the sample
Figure 5-1

• Method is specific if it selects for

only one analyte (no interferences).
• Chromatogram separates drug
imidacloprid (peak 7) from other
potential impurities/degradation
peaks.

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 24
 Linearity
Linearity: measures how well a calibration curve follows straight line
➔ response is proportional to quantity of analyte
A common (but superficial) measure of linearity is the
square of the correlation coefficient, R2.
 ( x − x )( y
− y )
2

R = 2 i i

( xi − x )2 ( yi − y)2
• A value of R2 > 0.995–0.999 is a good fit for many
purposes.
• Plotting a graph shows the true nature of the calibration.

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 25
• Another criterion for linearity is that the y-intercept of the
calibration curve (after the response of the blank has been
subtracted from each standard) should be close to 0.
• An acceptable degree of “closeness to 0” might be 2% of the
response for the target value of analyte

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 26
Residual Plots
Residual plots emphasize the difference between calibration data and
the least-squares line.
Figure 5-2
Residual
di = yi – y
= yi – (mxi + b)

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 27
Example: Linearity (1 of 3)
Samples from 2 to 3 mg/L quinine are to be analyzed using fluorescence
spectroscopy. Standards from 1 to 10 mg/L were measured in triplicate to yield the
calibration curve in Figure 5-2a. Least-squares yields y = 92.4x + 72.9 with R2 =
0.996. Is the calibration linear? Figure 5-2a

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 28
Example: Linearity (2 of 3)
Solution: R2 is greater than 0.995, but R2 alone should not be used to judge linearity. The intercept is
21% of the 3-mg/L standard, which is greater than the 2% criterion. Inspection of the calibration
plot in Figure 5-2a reveals that low standards are below the line, middle standards are above the
line, and high standards are below the line. The residual plot in Figure 5-2b clearly indicates
curvature. Figure 5-2b

X 2 3
Y 257.7 350.1
% of
intercept 28.3 20.8

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 29
Example: Linearity (3 of 3)
Solution: Fitting the calibration data to a quadratic function yields y = −2.25x2 +
117.1x + 23.4, with R2 = 0.999 8. Residuals for the quadratic fit are randomly
scattered about 0 and reduced to one-third of those in Figure 5-2b. The intercept is
still >2% of the 3-mg/L standard.

Reviewing the procedure revealed that fluorescence was zeroed using distilled
water rather than the 0.05 M sulfuric acid used to prepare standards. Sulfuric acid
may have given the fluorescence responsible for the positive intercept. In this case,
deviation of the intercept from 0 cannot be used to assess the fit. Based on these
considerations, the quadratic fit is appropriate. X 2 3
Y 248.6 354.45
% of
intercept 9.4 6.6
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 30
Accuracy: “Nearness to the Truth”
Accuracy can be demonstrated by:

• Analyzing a certified reference material in a matrix similar to that

of the unknown
• Comparing results from two or more different analytical methods
• Analyzing a blank sample spiked with known addition of analyte
• Standard additions of analyte to the unknowns

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 31
Example: Testing Accuracy (1 of 3)
A specification for determining ~3 mg/L quinine by fluorescence in the preceding
example is a spike recovery of 100 ± 2%. Using the quadratic fit for the 1- to 10-
mg/L calibration, we can estimate that blank solutions spiked to 1.50 and 4.50 mg/L
quinine would yield signals of 194.0 and 504.8, respectively.
Figure 5-2a
Is the method fit for purpose if the linear
calibration in Figure 5-2a is used?

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 32
Example: Testing Accuracy (2 of 3)
Solution: To be fit for purpose, the percent recovery for spiked blanks must be within
98 to 102% of the concentration added. For 1 to 10 mg/L quinine, the linear least-
squares equation is y = 92.4x + 72.9. The quinine concentration corresponding to a
signal of 194.0 is y − 72.9 194.0 − 72.9
x = = = 1.31 mg/L
92.4 92.4
The spike recovery for the 1.50-mg/L spike is
C spiked sample − Cunspiked blank 1.31 − 0
% recovery =  100 =  100 = 87.3%
C added 1.50

For the 4.50-mg/L spike, the calculated concentration is 4.67 mg/L and its recovery
is 103.8%. Spike recoveries differ from 100% by more than ±.2%. The method lacks
the accuracy necessary to be fit for purpose.
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 33
Example: Testing Accuracy (3 of 3)
Test Yourself: If a blank spiked to 3.00 mg/L quinine gives a signal of
354.4, what is the predicted quinine concentration and percent
recovery?

Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 34