HPLC method validations

s The goal of this study was to develop and validate a liquid chromatography (LC) method for the rapid determination of dipyrone (metamizole sodium) in solid and liquid formulations. Samples were extracted with water and filtered prior to LC analysis. The analytical procedure involved reversed phase LC with a Zorbax SB C 18 column (5 m particle size, 4.6 mm × 250 mm i.d.), isoctratic methanol/water (80/20, v/v) mobile phase with UV detection at 254 nm. The flow rate was 1 mL min −1 , injection volume was 100 L and retention time of dipyrone was 3.3 min. The method was validated with respect to linearity, precision and accuracy. Calibration curves were linear in the range 0.5–100 g mL −1 for analyte and the recoveries at three levels (10, 15 and 25 g mL −1) ranged from 93 to 100%. All results for dipyrone were confirmed by liquid chromatography–mass spectrometry (LC–MS) and 1 H nuclear magnetic resonance (NMR) spectroscopy. Due to its simplicity and accuracy, the assay method is suitable for routine analysis of both solid and liquid formulations and seven retail samples were analyzed using the validated method.

A simple, rapid and accurate RP-HPLC method was developed for the determination of doxofylline and photolytic degradation product. The method showed a linear response for concentrations in the range of 1-200 g/ml using acetonitrile: formic acid (90: 10); pH-3.0 as the mobile phase with detection at 274 nm and a flow rate of 1 ml/min and retention time 2.9 min. The method was statistically validated for accuracy, precision, linearity, ruggedness, robustness, forced degradation, solution stability and selectivity. Quantitative and recovery studies of the dosage form were also carried out and analyzed; the % RSD from recovery studies was found to be less than 1. Due to simplicity, rapidity and accuracy of the method, we believe that the method will be useful for routine quality control analysis. The hydrolytic degradation product as well as pathway was characterized by LC-MS/MS.
RESULT AND DISCUSSION:
A sensitive, selective, precise and accurate high
performance liquid chromatographic method of analysis of
doxofylline in both as bulk drug and in formulation was
developed and validated. The mobile phase consisted of
acetonitril: 0.05M formic Acid (90: 10v/v); pH-3.0. The
detection wavelength was 274nm. This system was found to
give the sharp peak for doxofylline (RT-2.9). The method
was validated as per ICH guideline (table 1). Stability
indicating assay method in which hydrolytic stress
condition was used for quantitative estimation of
doxofylline in tablet formulation and identification of
hydrolytic degradation product The separation of drug
from its degradation product were optimized by varying the
ratio &/or nature of organic modifier. Finally method was
developed using same mobile phase composed of
acetonitrile: formic acid (90: 10); pH-3.0, in that both drug
and degradation product showing good elution RT-2.9
(Doxofylline) and RT-3.68 (Hydrolytic degradation
product) and m/e-229 (fig 4). The hydrolytic degradation
product and pure drug were identified by LC-MS/MS in
order to establish hydrolytic degradation pathway (fig 5 and
6).

Objective: To develop and validate a stability-indicating reversed phase high performance liquid chromatography (RP-HPLC) assay for the
determination of phenytoin in bulk and pharmaceutical dosage forms.
Methods: A HPLC instrument incorporating aZorbaxC-18 analytical column (250x4.6 mm, 5μm particles) with a mobile phase comprising
acetonitrile: water in the ratio 50:50 (%v/v) was employed for the determination of phenytoin. The flow rate was set with an isocratic program, the
temperature of the column was maintained at 25 °C and a detection wavelength of 200 nm was employed using an ultraviolet detector. The method
was validated as per The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use
(ICH) guidelines.
Results: Total chromatographic analysis time per sample was 6 min with phenytoin eluting with the reaction time of 4.6±0.2 min. Phenytoin was
exposed to acidic, basic, oxidative, photolytic and thermal stress conditions and the specificity of the assay was confirmed. The calibration plot was
linear (R
≥0.999) over the phenytoin concentration range 5.0-100.0μg/ml. The percentage means recoveries were found to be in the range of 98102%.

The relative standard deviation of precision and robustness were within prescribed limits (<2%). The limit of detection was 0.047 µg/ml
while the limit of quantitation was established as 0.143 µg/ml.
2
Conclusion: A simple, accurate, precise and stability-indicating RP-HPLC assay was successfully developed for the determination of phenytoin in
bulk and dosage forms. Hence, this assay is useful for the analysis of phenytoin in formulations in medicines development and pharmaceutical
manufacturing setting.
Keywords: Phenytoin, RP-HPLC, Stability-indicating assay, Forced degradation, Validation.

Large number of population prefer the Ayurvedic or herbal medicines to treat the various ailments. Herbal medicines are available in three forms as raw plant material or processed plant material or medicinal herbal product but mixtures have several chemical components. Therefore, it is very important to know the specific component having potential for treatment. For qualitative and quantitative analysis of the herbs and herbal drugs, Highperformance thin layer chromatography (HPTLC) is one of the best techniques. Hence, a case study has performed to quantify vasicinone component from Adhatoda zeylanica.

The protein quality of 11 null and 2 tofu soybean genotypes were determined from their total protein content, their amino acid composition, and their glycinin and b-conglycinin contents. There were highly significant differences (P < 0.001) in their total storage proteins, and amino acid contents. Total protein among these genotypes ranged from 33 to 37%, with arginine being the third highest amino acid (7.4-10.9 g/100 g protein) followed by glutamic and aspartic acids. Methionine accounted for only 1.6-2.4 g/ 100 g of protein. All genotypes contained a good balance of essential amino acids (EAA 9 ), ranging from 43.5 to 47.3% of the total protein, limited only in methionine and possibly threonine and valine. Two-dimensional gel electrophoretic (2-DE) reference maps, using narrow range immobilized pH gradient (IPG) strips, revealed unique differences in the proteome, and subunit expression of glycinin and b-conglycinin, among these null genotypes, which can then be correlated with their protein quality. Out of a total of 111 basic (pH 6-11), and 223 acidic (pH 4-7) protein spots separated by 2-DE, 41 soybean storage protein spots were excised, and identified by liquid chromatography on-line with electro spray LCQ DecaXP tandem quadrupole time-of-flight mass spectrometry (LC/MS/MS). These methods will enable accurate evaluation of protein quality in soybeans, based on their protein digestibility-corrected amino acid score, assessment of the genetic variability of soybean genotypes, and serve as very effective tools for assisting plant breeders in their selection of high quality soybean varieties.

Red clover (Trifolium pratense L.) is an important forage plant that contains the isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds have been studied lately due to their human health benefits. The aim of this study was to develop and validate an HPLC method with simplified sample preparation to quantify daidzein, genistein, formononetin and biochanin A simultaneously in red clover leaves. The validation showed that the method is specific, accurate, precise and robust, not to mention that the sample preparation is easier and faster than those described earlier. The response was linear over a range of 0.01-0.2 lg mL À1 for daidzein, 0.05-0.5 lg mL À1 for genistein, 4-40 lg mL À1 for formononetin and 2-20 lg mL À1 for biochanin A. The range of recoveries was 85.6-101.0%. The RSD for intra-and inter-day precision were <2.54 and <7.22%, respectively. Five populations of red clover, from the National Plant Germplasm System-USDA were analyzed and the content of daidzein, genistein, formononetin and biochanin A ranged from 7. 87-91.31, 51.60-131.30, 6568.33-23461.82, to 2499.55-10337.33 lg g À1 of dried material, respectively.

Major compounds of several commonly used botanicals, including turmeric, have been purported to have antiinflammatory actions. In order to test the anti-inflammatory activity of compounds isolated from rhizomes of Curcuma longa L. (Zingiberaceae), we have established an in vitro test system. HL-60 cells were differentiated and exposed to lipopolysaccharide (LPS) from Escherichia coli (1 mg/ml) in the presence or absence of botanical compounds for 24 h. Supernatants were collected and analyzed for the production of tumor necrosis factor alpha (TNF-a) and prostaglandin E 2 (PGE 2 ) using standard ELISA assays. Water-soluble extracts were not cytotoxic and did not exhibit biological activity. Organic extracts of turmeric were cytotoxic only at concentrations above 50 mg/ml. Crude organic extracts of turmeric were capable of inhibiting LPS-induced TNF-a (IC 50 value ¼ 15.2 mg/ml) and PGE 2 (IC 50 value ¼ 0.92 mg/ml) production. Purified curcumin was more active than either demethoxy-or bisdemethoxycurcumin. Fractions and subfractions of turmeric extracts collected via preparative HPLC had differing biological activity, ranging from no activity to IC 50 values of o1 mg/ml. For some fractions, subfractionation resulted in a loss of activity, indicating interaction of the compounds within the fraction to produce an anti-inflammatory effect. A combination of several of the fractions that contain the turmeric oils was more effective than the curcuminoids at inhibiting PGE 2 . While curcumin inhibited COX-2 expression, turmeric oils had no effect on levels of COX-

Aim: The present work is aimed to develop a simple, rapid, selective, sensitive and validated HPLC method for the determination of fulvestrant in bulk drug.  Method: Fulvestrant was measured using a validated HPLC method with UV detector at 225nm chromatographic peaks were separated on Phenomix, C18 column (250 mm x 4.6 mm x3.5µ) using 50% aetonitrile in channel A and acetonitlile in channel B as mobile phase with gradient elution at a flow rate of 1 ml/min.  Results & Discussions: The chromatograms showed good peak shapes and no interference of solvent peaks. The retention time of fulvestrant was found to be 13.6 min. LOD and LOQ were found to be 0.0226µg/ml (0.001%) and 0.0712µg/ml,(0.004%) respectively. The percentage recovery of fulvestrant was found to be 99.1% - 100.75% which are within the limit. The method was linear over the concentration range of 0.08to 2ìg/ml with coefficient of correlation (r2) 0.99997. Both intraday and inter day accuracy and precision data showed good reproducibility.  Conclusions: The low % RSD values (= 2) indicated that the method was precise and accurate. this method was successfully applied to forced degradation studies and can be adopted for routine estimation of fulvestrant in quality control laboratories.

An analytical method for the simultaneous determination of phenethyl alcohol, methylpropanediol, phenylpropanol, caprylyl glycol, and ethylhexylglycerin, which are used as alternative preservatives in cosmetic products, has been developed. The method is based on liquid chromatography with UV spec-trophotometric detection after chromophoric derivatization with benzoyl chloride and vortex-assisted liquid-liquid semimicroextraction. Different chromatographic parameters, derivatization conditions, and sample preparation variables were studied. Under optimized conditions, the limits of detection values for the analytes ranged from 0.02 to 0.06 mg mL À 1. The method was validated with good recovery values (84-118%) and precision values (3.9-9.5%). It was successfully applied to 10 commercially available cosmetic samples. The good analytical features of the proposed method besides of its environmentally-friendly characteristics, make it useful to carry out the quality control of cosmetic products containing the target compounds as preservative agents.

A stability indicating high performance liquid chromatographic method has been developed for the determination of norfloxacin. Optimum separation was achieved in less than 10 minutes using Phenomenex ODS C 18 (250x 4.6mm packed with 5µ) column. The analyte was resolved by using a mobile phase 20 mmol L -1 ammonium formate and acetonitrile (70:30) pH adjusted to 4.0 with formic acid at flow rate 1 ml/min on a isocratic high performance liquid chromatographic system at a wavelength of 280 nm. The method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy, precision, ruggedness and robustness and can be successfully applied for determination of this drug in commercial tablets. For stress studies the drug was subjected to acid, alkali and neutral hydrolysis, oxidation, dry heat and photolytic degradation. The degradation studies indicated the drug to be susceptible to acid, alkali hydrolysis and oxidative degradation. The analytical conditions and solvent developed provided good resolution within a short analysis time and economic advantages. The proposed method not required highly sophisticated and expensive instrumentation.

Abstract The inulin-type fructans, inulin and fructooligosaccharies (FOS), are part of dietary fibers for production of functional foods with improved health benefits. The increasing application of these components in food technology needs reliable methods for their analysis and control. The aim of the current review is to present and compare the main analytical methods for inulin determination in food products. This study summarizes not only the common applied spectrophotometric and enzymatic methods, but also describes in details chromatographic techniques-HPLC, GC and TLC, IR-FT and real time 1 H NMR spectroscopy. Some of suggested analytical assays, especially spectrophotometric, enzymatic and HPLC-RID methods are applied for routine analysis in analytical practice for monitoring of some process in food technology and control in food labeling. Special attention has been paid to nondestructive IR-FT spectroscopy that reveals the potential to identify the presence of inulin by typical for β-→1 glycosidic bond. Some typical shifts are shown for structure elucidation or hydrolysis of inulin by NMR spectroscopy.

The objective was to develop and compare an accurate and reproducible HPLC
method for determination of 5-fluouracil (5-FU) in rabbit and human plasma. A
simple, rapid, accurate, precise and sensitive high pressure liquid chromatography
method has been developed and subsequently validated for determination of 5-FU.
The current method has been developed with simple mobile phase of de-ionized
distilled water, pH adjusted at 3.2 with perchloric acid. An isocratic HPLC system
(Agilent Technologies, 1200 series, USA) was used with variable wavelength
detector and data processing software, ChemStation. Flow rate was 0.8 ml/min and
20 μl sample was injected and eluate was monitored at 260 nm wavelength. The
developed method showed excellent linearity, reproducibility and sensitivity. The
studied method was found robust for analysis of 5-FU in both, rabbit and human
plasma samples. Run time was short (10 min) and simple extraction procedure with
good response even at low drug concentration made this method suitable for
pharmacokinetic and other applications. A rapid, accurate and precise high pressure
liquid chromatography method was developed and validated. It is concluded that
developed method is simple, cost effective, fast and reproducible for the analysis of
drug concentrations, in rabbit and human plasma.

This  study  was  conducted  with  the  aim  to  extract  and  purify  a
polyphenolic compound “Resveratrol” from the skin of black grapes
Vitisvinifera  cultivated  in  Iraq  and  synthesis  new  derivative.
Chemical  investigations  and  tests  for  the  identifications  carried  out
for the qualification of extracted crystals yield include: general tests
for  polyphenoles,  aromatic  unsaturated  compounds,  spectrophotometric  scanning  for  λmax  screening,  High  Performance  Liquid
Chromatography  (HPLC)  comparable  with  standard  and  Fourier
Transform  Infrared  (FTIR)  to  observed  groups  of  compound.
Synthesis  new  derivatives  from  purified  resveratrol  were  done
throughout  series  of  reactions  accompanied  with  FTIR  for  each
derivative.  The  derivative  is  (E)-5-(4-(3,  4,  5-trihydroxybenzoyl)
oxy)  styryl)-1,  3-phenylene bis  (3,  4,  5 tri hydroxyl benzoate). The
IR spectroscopy result confirms the presence of functional groups in
the proposed compounds.

Adecuación al propósito: Es el grado en que la aplicabilidad de un método se ajusta a los criterios o requerimientos acordados entre el analista y el usuario final (cliente) que hace uso de los informes o datos generados a través de un método de ensayo. Es decir es la adaptación de un método para un fin previsto.

In recent years, cleaning validation has achieved a position of increasing in the pharmaceutical industry. FDA regulations, Health Canada Guidelines and the current version of the European Guidelines of Good Manufacturing Practices (CGMP) recognize that cleaning validation of production equipment is the most effective way to ensure quality, efficiency and safety of drugs because it overcame against cross-contamination.
The aim of this work was to establish an effective, robust and reliable strategy for cleaning validation and to verify if the processes of cleaning production equipment within the company GEO-PHARM laboratories, allow to eliminate product residues, degradation products, excipients and cleaning agents, as well as the control of potential microbial contaminants, to ensure that there is no risk of cross-contamination between the active ingredients.
In the pharmaceutical industry, contamination must be avoided. A deep knowledge of the contamination sources and the critical areas of the equipment is essential, for this reason the professional put in place preventive measures, thus means a scrupulous cleaning. The cleaning operation of equipments is an integral part of the manufacturing processes. The implementation of cleaning validation can be broken down into three main stages: definition of a company policy, definition of a methodology and definition of a robust strategy. The company policy is the guideline determined by the analysis of the reasons leading to this validation, the determination of a timetable and the possible means to put in place. The methodology defines the steps to be implemented to perform the validation; it leads to elaborate a validation protocol. The development of an effective, robust and reliable strategy take place on regulatory, normative and experience requirements, based on the grouping principle and the worst case scenario to simplify its realization, increasing the reliability of reasoning, and therefore results.
An analytical method of RP-HPLC-UV reversed phase liquid chromatography and a direct sampling method with swabbing were developed and validated to quantify Diclofenac sodium residues on equipment surfaces after cleaning. Validation studies demonstrated that the method is specific, fast, linear, accurate and sensitive to quantify the acceptable residue limit on surface established at 0.75 μg / cm² and in rinsing water at 2.66 μg / ml. The recovery rate obtained from the stainless steel surfaces was 87%. Stability studies showed that Diclofenac sodium samples were stable within 24 hours under light and ambient temperature.
The cleaning validation report, and the validation of the storage durations of the equipment, the maximum time between completion of processing and equipment cleaning (DHT Dirty Hold Time), and time between equipment cleaning and use (CHT Clean Hold Time) demonstrated that the results were below the acceptable limits, except for certain TOC and conductivity values that were higher than the pre-established limits, which was logical and justifiable because the presence of contaminant below the limit acceptable in rinsing waters increases the conductivity and TOC, that means, the cleaning processes of production equipment were robust and reliable in order to effectively remove chemical, microbiological and particulate contaminants, which prevent the cross-contamination between the active ingredients.

A rapid, specific and sensitive stability indicating reverse phase high performance liquid chromatographic method has been developed and validated for analysis of levosulpiride hydrochloride in both bulk and pharmaceutical dosage form. An isocratic stability indicating reversed-phase liquid chromatographic determination was developed for the quantitative determination of levosulpiride in the pharmaceutical dosage form. A sunfire C-18, 4.5µm column with mobile phase containing methanol-water (10:90, v/v) was used. The flow rate was 1.0 mL min -1 and effluents were monitored at 232 nm. The retention time of Levosulpiride was 5.5 min. Levosulpiride stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation, wet hydrolysis, dry heat degradation and sun light degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values. Stressed samples were assayed using developed LC method. The proposed method was validated with respect to linearity, accuracy, precision and robustness. The method was successfully applied to the estimation of Levosulpiride in tablet dosage forms. The proposed study describes stability indicating LC method for the estimation of Levosulpiride in bulk and their pharmaceutical dosage form. The method is suitable for the routine analysis of Levosulpiride in tablets.

Reliable methods for quantification of synthetic water-soluble colors in complex food matrices are currently not available. The present work describes the development and validation of an improved protocol for the analysis of synthetic food colorants in complex food matrices presenting high protein and/or fat content. The method developed employs an extraction stage, followed by a subsequent sonification, centrifugation and concentration step. A final clean up via SPE on polyamide cartridges was also employed. The isolated colorants were separated and analyzed by an RP-HPLC/DAD system. High and consistent recoveries (min. 81%) and low RSDs (max. 6%) were achieved for all studied colorants. The issue of high fat content matrices was also addressed showing the need for an additional defatting step in the procedure. Overall, the protocol presented shows high precision and accuracy of detection and can provide the basis for future development of similar methods in other complex food matrices.

Validation is one of the important steps in achieving and maintaining the quality of the final product. If each step of production process is validated we can assure that the final product is of the best quality. The study presented here provides the assurance that the manufacturing procedure is suitable for intended purpose and the product consistently meets predetermined specifications and quality attributes, as per specified master formula record. It also provides documented evidence for the operation sequence of manufacturing process and to determine the critical parameters and variables in the process of manufacturing of the tablets.

BAB I PENFAHULUAN Di zaman yang modern seperti sekarang ini, perkembangan Ilmu Pengetahuan dan Teknologi melaju dengan pesatnya. Tuntutan kebutuhan hiduplah yang menjadikan manusia giat mengembangkan ilmu pengetahuan dan teknologi. Berbagai ilmu dikembangkan, mulai dari ilmu sosial hingga ilmu eksak. Seperti salah satu ilmu eksak yaitu ilmu kimia. Dalam praktiknya secara umum ilmu kimia, yaitu ilmu yang mempelajari tentang sususnan, sifat, dan reaksi suatu unsur atau zat dalam berbagai jenis benda material. Dalam ilmu kimia banyak aspek-aspek penelitian yang dilakukan. Seperti halnya, yang paling spesifik yaitu pemisahan molekul-molekul dari senyawa zat nya, atau yang disebut dalam ilmu kimia yaitu Kromatografi. Kromatografi terbagi menjadi berbagai macam lagi. Salah satunya yaitu "Kromatografi Cair Kinerja Tinggi" atau yang biasa di kenal HPLC.

Perindopril erbumine is an antihypertensive, which belongs to the category of angiotensin-converting enzyme inhibitors (ACE inhibitors) that inhibit the conversion of angiotensin I to angiotensin II. The aim of this review is to focus on a comprehensive update of analytical methods for determination of Perindopril and its metabolites in human plasma and in pharmaceutical preparations. It has been described using TLC, RP-HPLC, GC, Mass and UV methods. This review provides detailed information on separation conditions for perindopril alone, perindopril in presence of its degradation products or with its combinations.

A  simple,  robust,  precise  HPLC  method  was  developed  and  the
method  has  been  validated  for  the  estimation  of  valsartan  and
nebivolol  simultaneously  in  its  combined  dosage  forms  as  well  as
individual formulations. Both the compounds are well resolved in a
isocratic  method  by  using  the  mobile  phase  composition  of
acetonitrile: methanol: pH4.0 0.02M Potassium hydrogen phosphate
buffer in the ratio of 50:20:30 v/v at a flow rate of 1.0mL/min using a
C-18 column. The detection was carried out at 210nm.. The retention
time  of  Nebivolol  and  Valsartan  were  2.5  min  and  4.3  min
respectively.  The  developed  method  was  validated  for  specificity,
linearity,  precision,  accuracy,  limit  of  detection  (LOD),  limit  of
quantification (LOQ) and robustness as per ICH guidelines. Linearity
for Nebivolol and Valsartan were found in the range of 48-112 µg/ml
and  3.0-7.0  µg/ml,  respectively.  The  percentage  recoveries  for
Nebivolol  and  Valsartan  ranged  from  98.9-100.8  %  and  98.9-101.1
%,  respectively.  The  proposed  method  could  be  used  for  routine
analysis of Nebivolol and Valsartan in their combined dosage forms

A simple, reliable, reproducible, economical and rapid isocratic RP-HPLC method was developed for the simultaneously separated and quantification of four angiotensin II-receptor antagonists namely Telmisartan (TELM), Losartan (LOSA), Olmesartan (OLME) and Valsartan (VALS) along with thiazide diuretic mostly Hydrochlorothiazide (HCTZ). All the above said drugs were separated by using Welchrom C 18 column having internal diameter of 4.6 X 250 mm with 5 µm particle size as stationary phase with mobile phase consisting a mixture of acetonitrile and phosphate buffer (pH-3.3, 50:50 v/v). The mobile phase was pumped at a flow rate of 1 ml/min. The wave length of the UV detection was done at 238 nm. All the sartans were separated within 6 minutes. The calibration curves were linear (r 2 = 0.9998) in all cases. The relative standard deviation was less than 2% and average recovery was above 99.95%. All four sartans were routinely assayed without interference.

Objectives: Formulation of triplet combined solid dosage film coated
tablet  containing  amlodipine  besylate  (equivalent  to  5  mg
amlodipine), hydrochlorothiazide (12.5 mg) and losartan potassium
(50 mg) for the treatment of severe hypertension. Development and
validated of a simple, fast, precise, selective and accurate HPLC
method  for  the  simultaneous  determination  of  amlodipine  besylate,
hydrochlorothiazide and losartan potassium in the tablets.
Methods:  The  formulation  of  the  tablets  was  carried  out  as  per
standard  protocols. The  various  steps  involve  in  formulation  were
dispensing  of  raw  materials,  sieving,  preparation  of  granulating
solvent,  mixing,  granulation,  drying  (In  FBD),  lubrication,
compression and coating. The separation of these three drugs was
achieved on a Sun Shell C8 column (150 mm x 4.6 mm, 2.6 μm) with
phosphate  buffer-acetonitrile  (70:30%  v/v)  as  mobile  phase  at  1.0
mL/min flow rate and 230 nm detection.
Results  and  Conclusion: The  physical  parameters  of  tablets  were
satisfactory  with  average  weight  deviation  from  3.23  to  3.29%,
friability 0.04%, disintegration time 8.3 minutes, average hardness
85.43N and thickness from 3.92 to 4.01 mm. The assay was found to
be 99.89%, 99.99% and 99.97% of amlodipine, hydrochlorothiazide
and losartan potassium, respectively. The dissolution was found to be
98.8 to 99.70%, 97.85 to 98.95% and 97.98 to 99.99% of amlodipine,
hydrochlorothiazide  and  losartan  potassium,  respectively.  The
uniformity of content was 99.85 to 99.99% and 99.60 to 99.99% of
amlodipine  and  hydrochlorothiazide,  respectively.  The  retention
times  observed  were  to  be  7.338,  2.097  and  10.675  minutes  for
amlodipine  besylate,  hydrochlorothiazide  and  losartan  potassium,
respectively.  The  method  was  statistically  validated  for  linearity,
recovery,  limit  of  detection,  limit  of  quantification,  accuracy, precision,  robustness,  stability  of  drugs  in  pure  form  and  in
presence of matrices and forced degradation study. The method
was successfully applied for analysis of combined dose tablet.

Pharmaceutical industry has been emerging rapidly for the last decade by focusing on product Quality, Safety, and Efficacy. Pharmaceutical firms increased the number of product development by using scientific tools such as QbD (Quality by Design) and PAT (Process Analytical Technology). ICH guidelines Q8 to Q11 have discussed QbD implementation in API synthetic process and formulation development. ICH Q11 guidelines clearly discussed QbD approach for API synthesis with examples. Generic companies are implementing QbD approach in formulation development and even it is mandatory for USFDA perspective. As of now there is no specific requirements for AQbD (Analytical Quality by Design) and PAT in analytical development from all regulatory agencies. In this review, authors have discussed the implementation of QbD and AQbD simultaneously for API synthetic process and analytical methods development. AQbD key tools are identification of ATP (Analytical Target Profile), CQA (Critical Quality Attributes) with risk assessment, Method Optimization and Development with DoE, MODR (method operable design region), Control Strategy, AQbD Method Validation, and Continuous Method Monitoring (CMM). Simultaneous implementation of QbD activities in synthetic and analytical development will provide the highest quality product by minimizing the risks and even it is very good input for PAT approach.

Context: Drug solubility is one of the most important physicochemical properties for the development of new pharmaceutical products, which, in our study, is a new synthetic molecule, which has been shown to have a potential neuroprotective effect in rat.

Aims: To evaluate the equilibrium solubility of JM-20 within the physiological pH range and aqueous chemical stability.

Methods: It was performed at pH 1.2, 4.5 and 6.8 at 37 ± 0.5°C for 27 h by the method of the saturated flasks. HPLC and FTIR were used for the study of aqueous stability and to evaluate the molecular structure of the non-ionized fraction by DSC/TGA.

Results: Concentrations calculated at pH 1.2 were higher than at pH 4.5 and pH 6.8. A retention time greater than pH 4.5 and 6.8 after 24 h was detected in the aqueous stability study at pH 1.2, in addition to other peaks in these chromatograms. Analyses by FT-IR and DSC/TGA demonstrated the absence of polymorphism in the dissociated fraction of saturated solutions.

Conclusions: The JM-20 presented at 6 hours its maximum steady state of solubility, being inferior to pH 4.5 and 6.8. It is corroborated by the qualitative analysis by HPLC, that the compound is chemically unstable from 24 h to pH 4.5 and 6.8. By FT-IR and DSC/TG it was elucidated that the fractions of the molecule not dissociated, did not undergo structural changes in it, and it is inferred that there is no polymorphism.

Background: In Algeria, important quantities of secondary date variety (Phoenix dac-tylifera L.) are generated in each campaign; their chemical composition is similar to that of commercial dates. The present work aims to valorize this common date variety (Degla-Beida) which is often poorly exploited. Methods: In this context, we tried to prepare syrup from the secondary date variety and evaluate the effect of conventional extraction (CE) or water bath extraction (WBE) and alternative extraction (mi-crowaves assisted extraction (MAE) and ultrasound-assisted extraction (UAE)) on its total sugar content (TSC), using response surface methodology (RSM). Then, the analysis of individual sugars was performed by high performance liquid chromatography (HPLC). Results: Maximum predicted TSC recoveries under the optimized conditions for MAE, UAE and CE were 233.248 ± 3.594 g/l, 202.889 ± 5.797g/land 233.535 ± 5.412g/l, respectively, which were close to the experimental values: 233.796 ± 1.898 g/l; 202.037 ± 3.401 g/land 234.380 ± 2.425 g/l. HPLC analysis revealed high similarity in the sugar composition of date juices obtained by MAE (60.11% sucrose, 16.64% glucose and 23.25% fructose) and CE (50.78% sucrose, 20.67% glucose and 28.55% fructose), although a large difference was detected for that obtained by UAE (0.00% sucrose, 46.94% glucose and 53.06% fructose). Conclusion: Microwave-assisted extraction was the best method for the preparation of date syrup with an optimal recovery of total sugar content. However, ultrasound-assisted extraction was the best one for the preparation of date syrup with high content of reducing sugars.

ABSTRACT
An accurate, sensitive, precise and stability-indicating reversed phase high performance liquid chromatographic (RP-HPLC) method for the analysis of clopidogrel bisulfate was developed and validated. The chromatographic conditions comprised of a reversible phase C18 column (250 × 4.6 mm, 5 μm) with a mobile phase consisting of a mixture of 50 mM potassium di-hydrogen phosphate and acetonitrile in the ratio of 75:25 at pH 3.0 adjusted with phosphoric acid. The flow rate was 1ml/min, the detection was carried out at 247 nm and the retention time of clopidogrel was 6.5 min. Clopidogrel was subjected to acid and alkali hydrolysis, oxidation and photodegradation. The method was validated for accuracy, precision and robustness. The results indicate that the drugs are susceptible to degradation in different conditions. All the peaks of degraded products were resolved from the active pharmaceutical ingredient with significant different retention times. As the method could effectively separate the drug from its degradation products, it could be employed as a stability-indicating method.

A reproducible and selective method for the simultaneous determination of cyclophosphamide (CP) and ifosfamide (IF) in plasma has been developed and validated using isocratic elution. The assay is performed by HPLC-UV, with a C18 column (5μm, 150 x 4 mm) and detection in 195 nm. The mobile phase was constituted by phosphate buffer 10 mM pH 6.0: acetonitrile (77.25:22.75), with a flow of 1 mL/min. SPE was used for sample clean-up in a range from 3 (LOQ) to 540 μM. The repeatability coefficients of variation (CV) ranged from 0.5 to 7.8% and the intermediate precision CVs varied from 0.9 to 5.7%. Extraction efficacy and accuracy varied from 94 to 115%. The simple method may permit the determination of cyclophosphamide and ifosfamide in plasma, simultaneously, to pharmacokinetics and bioequivalence studies.

A simple, fast, precise, specific, highly sensitive, an accurate and reproducible isocratic reversed phase HPLC method has been developed for the separation of four sartans and validated for quantitation of Olmesartan in pure and in pharmaceutical formulation. One of the prominent aims for the method development is to achieve constant, reproducible separation. The effects of mobile phase composition, buffers, pH, and acetonitrile concentrations were investigated on the separation of sartans (Valsartan, Telmisartan, Olmesartan and Irbesartan) were assayed without any interference. The chromatographic separation was achieved on Welchrom C18 Column (4.6 X 250 mm, 5 µm) as stationary phase with the mobile phase comprising of phosphate buffer pH-3.3 and acetonitrile in the portion of 50:50 v/v. Isocratic elution at a flow rate of 1ml/min was employed. The detection was performed with Shimadzu SPD-20A prominence UV-Vis detector set at 232 nm. Total run time was 8 minutes and the elution window of two minutes. The validated method was successfully applied to the commercially available Olmesartan pharmaceutical dosage form yielding very good and reproducible results. During method validation parameters such as linearity, precision, specificity, robustness and ruggedness were evaluated from spiked tablet samples according to ICH guidelines, which remained within acceptable limits. The proposed method can also be extended for the determination of other three sartans or their combinations with hydrochlorothiazide. This method provides a fast simple method with good retention, excellent peak shape and high resolution.

For the first time simple, selective, sensitive, rapid isocratic RP-HPLC method was developed for the separation and quantitative development Telmisartan (TEL), Losartan (LOS), Olmesartan (OLM) and Irbesartan (IRB) are angiotensin II-receptor antagonists which are used in the treatment of hypertension alone or in combination with other drugs mainly Hydrochlorothiazide (HCTZ). The most important advantage of developed method was that the five separate drugs can be determined on a single chromatographic system without modifications in detection wavelength and mobile phase by RP-HPLC. RP-HPLC method was developed by using Welchrom C 18 column (4.6 X 250 mm, 5 µm) as stationary phase with the mobile phase comprising of phosphate buffer pH-3.3 and acetonitrile in the portion of 50:50 v/v. Isocratic elution at a flow rate of 1ml/min was employed. The detection was performed with Shimadzu SPD-20A prominence UV-Vis detector set at 232 nm. Total run time was 8 minutes and the elution window of only two minutes. The proposed method was statistically validated in terms of linearity, precision, accuracy, specificity, robustness and ruggedness. The optimized method proved to be specific, robust and accurate for the quality control of four above said angiotensin-II receptor blockers alone or in presence of HCTZ with TEL, HCTZ with LOS, HCTZ with OML and HCTZ with IRB in bulk drug and pharmaceutical formulations.

Red clover is one of the most utilized forage in agriculture and contains many of the isoflavones known for their human health benefits. The objectives of this study were: i) to quantify, using HPLC analysis, isoflavones in 77 accessions from the USDA core collection and a Brazilian line; ii) to verify possible relationships depending on their origin, improvement status or maturity type and; iii) to verify the seasonal variation. The isoflavone mean contents were 29.27 µg g -1 of dry material for daidzein, 163.69 µg g -1 for genistein, 11353.29 µg g -1 for formononetin and 6568.8 µg g -1 for biochanin A. Clustering was mainly influenced by the total amount of isoflavones and partially due to maturity type, improvement status and geographic origin. The seasonal evaluation demonstrated an increase of concentration during winter, and decrease during spring. These results highlighted accessions that can be used to develop new varieties with low or high isoflavones concentration.

In the present study total twelve impurities of Bisoprolol (BISO) and Hydrochlorothiazide
(HCTZ) were separated simultaneously in a single HPLC method. Out of these 12 impurities,
five are potential degradants, which are validated as per ICH guidelines. As the two active drug
substances BISO and HCTZ have different solubilities and polarities, the most critical
parameters in resolving the components from each other are pH, temperature, and solvents. The
method is precise (RSD<1.0%), accurate, linear (r2>0.999), robust, and stability indicating in the
range of LOQ to 150%. The HPLC method is then migrated to UPLC to further reduce the, run
time, solvent consumption, and increase the sample through put.

A new rapid, precise and sensitive reverse phase high performance
liquid chromatographic (RP-HPLC) method has been developed and
validated  for  the  estimation  of  Perindopril  and  Amlodipine
simultaneously  in  combined  dosage  form.  The  two  components
Perindopril  and  Amlodipine  were  well  resolved  on  an  isocratic
method,  C18  column,  utilizing  a  mobile  phase  composition  of
acetonitrile:  methanol:  a  mixed  buffer  of  0.02M  Potassium
dihydrogen  phosphate  buffer  and  0.02M  Sodium  dihydrogen
Phosphate buffer with 1mL Tri ethyl amine (40:20:40), v/v, pH 5.0)
at  a  flow  rate  of  1.0  mL/min  with  UV  detection  at  226  nm.  The
retention time of Perindopril and Amlodipine were 2.9 min and 4.9
min  respectively.  The  developed  method  was  validated  for
specificity,  linearity,  precision,  accuracy,  limit  of  detection  (LOD),
limit of quantification (LOQ) and robustness as per ICH guidelines.
Linearity for Perindopril and Amlodipine were found in the range of
48-112 µg/ml and 60.0-140.0 µg/ml, respectively. The percentage
recoveries for Perindopril and Amlodipine ranged from 98.9-100.4 %
and 98.9-100.7 %, respectively. The proposed method could be used
for routine analysis of Perindopril and Amlodipine in their combined
dosage forms.

The goal of the present investigation was carried out to develop a validated analytical RP-HPLC method for simultaneous separation and estimation of Lamivudine and Zidovudine for pharmaceutical formulations. The chromatographic separation was accomplished on Welchrom RP-C 18 Column (250 mm X 4.6 mm; 5µm), Shimadzu LC-20AT Prominence Liquid Chromatograph and with a mixture of 10 mM Phosphate buffer (pH 3.6): acetonitrile (50:50, v/v). The flow rate was fixed at 1.2 mL/minute and the analysis was performed using Shimadzu SPD-20A Prominence UV-Visible detector at 241 nm. The Lamivudine and Zidovudine were separated within three minutes. The retention time for Lamivudine and Zidovudine was found to be 2.393 minutes and 2.7 minutes respectively. The calibration plots were linear over the concentration range of 2-10 µg/mL for Lamivudine (r 2 =0.9995) and 4-20 µg/mL for Zidovudine (r 2 =0.9991). There was no interference due to commonly used excipients. The % RSD for both the drugs were calculated and showed less than 2% which obviously indicates that the present method was said to be highly precise. Regarding accuracy of the developed method the %RSD were also found less than 2% which shows the method is completely accurate. The method was very sensitive with regard to limit of detection and limit of quantitation for Lamivudine and Zidovudine were found to be 0.181 µg/mL, 0.576 µg/mL and 0.551 µg/mL, 1.747µg/mL respectively. The mean assay values for Lamivudine and Zidovudine were arrived at 98.88% and 99.70 % respectively. The developed RP HPLC method was found to be simple, rapid, sensitive, highly precise and accurate and can be successfully employed for simultaneous estimation of Lamivudine and Zidovudine in pharmaceutical formulations.

For the first time the author bring forward newly developed RP-HPLC method for the simultaneous separation and determination of Atenolol (ATN), Metoprolol succinate (MET), Hydrochlorothiazide (HZT) and Amlodipine besylate (AML) individually and in combination with ATN with AML in tablet formulations which is quite distinct from that of other methods in vogue till today. The most important advantage of developed method was that the 4 separate drugs and combinations can be determined on a single chromatographic system without modifications in detection wavelength and mobile phase composition by RP-HPLC. The chromatographic separation of the selected drugs was carried out on Welchrom C 18 column consisting of 250 mm X 4.6 mm, 5 µm particle size utilizing mixture of 10 mM phosphate buffer (pH 3.0): Acetonitrile in the ratio of 50:50, v/v as mobile phase at the flow rate of 1mL/min with detection wave length at 235 nm by using UV spectrophotometric detector with total run time of 6 minutes. The retention times for ATN, MET, HZT, and AML were found to be 2.240, 2.813, 3.543, 3.753 and Combination of ATN and AML were arrived at 2.240 and 3.753 minutes respectively. All calibration curves for four drugs showed linearity over a concentration range of 2-10 µg/mL and for Amlokind-AT tablet 5-25 µg/mL for ATN and 1-5 µg/mL for AML. The corresponding correlation coefficient were also calculated from the linear regression analysis and found to be above 0.9995 in all cases. For intra and inter-day precision the % RSD values for each drug were evaluated and found RSD percentages of all above specified drugs were found less than 2 %. The results relating to limit of detection (LOD) and limit of quantitation (LOQ) for ATN, MET, HZT, AML and (ATN and AML combination) were found to be 0.3177 µg/mL and 0.9627 µg/mL; 0.1840 µg/mL and 0.5578 µg/mL; 0.1266 µg/mL and 0.3836 µg/mL; 0.2251 µg/mL and 0.6823 µg/mL; and for combination of drugs are 0.4941 μg/mL and 1.4973 μg/mL; 0.1181 μg/mL and 0.3580 μg/mL, respectively. The method was validated for its recovery, intra-and interday precision, selectivity and specificity. Therefore it is concluded that these methods are successfully feasible for the application of routine analysis within the said four antihypertensive drugs and binary combination of ATN and AML. Please cite this article in press as P. Ravisankar et al. Rapid Separation and Quantification of Certain Anti-Hypertensive Agents In Their Dosage Forms By Reversed Phase-High Perpromance Liquid Chromatography. INTRODUCTION The four different drugs namely ATN, MET, HZT and AML [1] are anti-hypertensive drugs mainly aimed to treat prevention of high blood pressure, stroke and myocardial infarction. After going through relevant literature survey on the drugs showed that there are some reported analytical methods for the estimation of these drugs individually and with other binary combination of anti-hypertensive agents in their dosage forms based on RP-HPLC [2-7], HPTLC [8], UV-Vis spectroscopy [9], LC-MS [10,11]. According to the available present knowledge, no unique single RP-HPLC method was available for the estimation of ATN, MET, HZT and AML individually and in combination with ATN with AML. The present proposed method would help in assay of drugs in a single run that reduces the time of analysis and does not need any separate method for each drug. After thoroughly evaluating the existing methods in operation the author had attempted to develop an alternative simple, rapid, reliable, precise, accurate and cost-effective, reproducible RP-HPLC method for the separation and quantification of four above said anti-hypertensive agents and also develop a validated analytical method for simultaneous quantitative determination of ATN and AML in combination tablet dosage form. The structural formulas of ATN, MET, HZT and AML are shown in Figure 1 to Figure 4.

A simple, accurate, precise, economic, robust and rugged UV spectrophotometric method has been developed for the simultaneous estimation of Atenolol and Ivabradine HCl in synthetic mixture. Combination of Atenolol and Ivabradine HCl has not any analytical method developed yet, so this UV method is novel for combined synthetic dosage of Atenolol and Ivabradine HCl (10:1). The absorbance maxima of Atenolol and Ivabradine HCl were found to be 276.00 nm and 286.50 nm respectively. Beer law obeyed the concentration range of 20-100 µg/ml and 2-10 µg/ml for Ivabradine HCl. The result of analysis was validated statistically and by the recovery studies. The %RSD value of all validation parameter less than 2. The proposed method can be effectively applied for the estimation of these two drugs.

The present work describes a simple, sensitive, accurate and rapid reverse phase high Performance liquid chromatographic method for simultaneous estimation and validation of Atenolol (ATN) and Hydrochlorothiazide (HCTZ) in tablet dosage form. The chromatographic separation was accomplished on Welchrom RP-C 18 Column (250 mm X 4.6 mm; 5µm), Shimadzu LC-20AT Prominence Liquid Chromatograph and with a mixture of 10 mM Phosphate buffer (pH 3.0): acetonitrile (50:50, v/v). The flow rate was fixed at 1.0 mL/minute and the entire separation was carried out at an ambient temperature for the HPLC system, the analysis was performed using Shimadzu SPD-20A Prominence UV-Visible detector at 235 nm. The anti-hypertensive agents, ATN and HCTZ were separated within 4 minutes. ATN and HCTZ showed retention times of 2.257 min and 3.367 min respectively. The calibration plots were linear over the concentration range of 5-25 µg/mL for Atenolol (r 2 = 0.9999) and 2.5-12.5 µg/mL for Hydrochlorothiazide (r 2 = 0.9999). The method was very sensitive with regard to LOD and LOQ for Atenolol and Hydrochlorthiazide were found to be 0.0296 µg/mL and 0.6028 µg/mL; and 0.0899 µg/mL and 1.8269 µg/mL respectively. The method was statistically validated for its linearity, precision, accuracy, specificity, Robustness and ruggedness. The optimized method proved to be specific, robust and accurate for the quality control of ATN and HCTZ in bulk drug and pharmaceutical formulations.

O crescimento significativo na produção e consumo de sucos demanda métodos de
controle, capazes de mensurar a proporção da fruta e de açúcares, em cada tipo de
produto. Pelos direitos básicos dos consumidores, informações sobre a composição e
riscos, devem estar claras e adequadas nas embalagens. Apesar disto, frequentemente
o brasileiro é confundido, á começar pela nomenclatura dos sucos. Pela legislação,
os sucos - integral e concentrado devem conter 100% da fruta sem qualquer adição.
Já, o néctar, bebida pronta para o consumo, deve ser elaborado com um mínimo de
50% da fruta, adicionado de água, sacarose, acidulantes e conservantes. Portanto,
este estudo objetivou verificar se as quantidades de açúcares totais dos sucos de uva
- concentrado e néctar são comparáveis e concordantes, quando determinadas por
3 técnicas/métodos distintos – refratometria, espectrofotometria e cromatografia,
verificando a sua aplicabilidade. A refratometria (RF), a mais aplicada no controle
industrial, emprega a leitura direta do oBrix em refratômetro. A espectrofotometria
mede glicose em 490 nm pelo método convencional fenol-sulfúrico (ESP-FS).
Enquanto, a cromatografia líquida de alta eficiência determina os teores de frutose,
glicose e sacarose empregando detector por índice de refração (HPLC-RID). Para
fins de comparação direta, a concentração dos açúcares totais por HPLC-RID foi
obtida pela soma dos teores individuais dos carboidratos. A quantidade de açúcares
totais média empregando RF, ESP-FS e HPLC-RID foi considerada semelhante
(Tukey p<0,05) para o néctar (14,85±0,44 g 100 mL-1) e para o suco concentrado
de uva (61,74±2,60 g 100 mL-1), excetuando o concentrado analisado por ESP-FS
(80,47±8,6 g 100 mL-1). A menor variação dos teores foi observada por RF (0,5-3,0%),
seguida por HPLC-RID (3,4-6,3%), com ESP-FS (8,4-17,2%) apresentando a maior
dispersão. Desta forma, a RF provou ser adequada para a determinação rotineira
dos açúcares totais, considerando sua precisão, praticidade e simplicidade de uso.
Entretanto, o HPLC-RID apresenta um diferencial adicional, além de fornecer o teor
dos açúcares totais. Baseado no perfil cromatográfico possibilita distinguir entre
sucos - integral ou concentrado, elaborados só com a fruta, pelas proporções de
frutose e glicose encontradas naturalmente na própria uva, de produtos adicionado
de sacarose, como o néctar. Este método consiste, portanto, em uma ferramenta
alternativa que poderia ser utilizada em caso de suspeita de fraudes, para atestar a
autenticidade e o cumprimento da legislação.

Coffee as high-price commodity is quite vulnerable to adulteration by cheaper roasted grains. Frauds are conventionally
detected by microscopy; however, this technique is limited semi-quantitative assays that require training
and skilled analysts. In this regard, carbohydrates as majormacronutrients of grains can be used as chemical
markers to qualitatively and quantitatively assess coffee authenticity. Although, some tamper's studies have
already been reported, this paper approaches on new analytical resources for detection of ground roasted coffee
adulteration, applying roasted soybean and wheat as sources of fraud. The characterization of the pure roasted
coffee beans and of adulterations profiles was taken by total carbohydrates validated method based on highperformance
anion-exchange chromatography with pulsed amperometric detection. The influence of each
matrix was evaluated employing the simplex-centroid design for experiments with mixtures thus relating the
mixing ratio with each monosaccharide by response surface. The proposed models were effective in recognition
and prediction of different mixture proportions, thereby allowing the distinction of genuine coffee by principal
component analysis and linear discriminant analysis. Predominantly, pure roasted coffee presented higher levels
of galactose and mannose. Glucose can be considered as a marker for wheat adulteration and fructose for
soybean, respectively. These results correspond to polysaccharides of pure raw grains, confirming this approach
as a feasible analytical tool for detection of adulterants in ground roasted coffee.

Parkia speciosa seeds are a common ingredient in Malay cuisine with traditional interest because of its medicinal importance and content of health-promoting phytochemicals. This study evaluated the phytochemical constituents and biological activities (antioxidant and antibacterial activities) of Parkia speciosa Hassk seeds collected from three different regions of Malaysia (Perak, Negeri Sembilan and Johor). Phytochemical constituents (total flavonoid and total phenolic) were measured using the spectrophotometric method, and individual flavonoids and phenolic acids were identified using ultra-high-performance liquid chromatography. Ferric reducing antioxidant potential (FRAP) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay we used in order to evaluation of antioxidant activities. Disc diffusion method was employed for the evaluation of antibacterial activity of extracts against Gram-positive and Gram-negative bacterial strains.

Results
The primary screening of phytochemicals showed that P. speciosa seeds contain alkaloids, terpenoids, flavonoids, and phenolics. Samples collected from Perak contained the highest levels of the phytochemical constituents, with highest DPPH and FRAP activity followed by Negeri sembilan and Johor. From the identified compounds, quercetin and gallic acid were identified as the most abundant compounds. Seeds collected from the Perak location exhibited potent antibacterial activity, against both Gram-positive and Gram-negative bacteria strains. Staphylococcus aureus and Bacillus subtilis were recorded as the bacterial strains most sensitive to P. speciosa seed extracts. Correlation analysis showed that flavonoid compounds are responsible for the antioxidant activities of the P. speciosa seeds studied, while antibacterial activity showed a high correlation with the levels of gallic acid.

Conclusions
Parkia speciosa seed grown in Perak exhibit the highest concentrations of phytochemicals, as well as the highest biological activity. It may also be recommended for the food industry to use seeds from this area for their products, which are going to compete in the expanding functional food markets.

The present study depicts the development of a validated RP-HPLC method for the determination of the Levetiracetam in pharmaceutical tablet dosage form. RP-HPLC method was developed by utilizing Welchrom C18 Column (250 x 4.6 mm, 5µ), Shimadzu LC-20AT Prominence Liquid Chromatograph. The mobile phase composed of 10 mM Phosphate buffer: acetonitrile (50:50 v/v) (pH-3.0, adjusted with triethylamine). The flow rate was set to 1.0 ml/min with the responses measured at 243.2 nm using Shimadzu SPD-20A Prominence UV-Visible detector. The retention time of Levetiracetam was found to be 3.030 minutes. Linearity was established for Levetiracetam in the range of 10-50 µg/ml with correlation coefficient 0.9998. The LOD and the LOQ for Levetiracetam found to be 0.468 μg/ml and 1.421 μg/ml respectively. The amount of Levetiracetam present in the formulation was found to be 99.56 %. None of the excipients interfered with the analyte of interest. Considering all the results of validation parameters simplicity of the method and the cost effectiveness of the overall procedure, it is possible to conclude that the developed method can be suitable for the regular quality control determination of Levetiracetam in bulk as well as pharmaceutical dosage form.