Phase High-performance Liquid Chromatography Method Research Articles

Comparison of Rapid Reversed Phase High-Performance Liquid Chromatography (RP-HPLC) Method with Rapid Reversed Phase Thin Layer Chromatography Method for Detecting Vegetable Oils in Ghee (Clarified Milk Fat)

An efficient, fast, and reliable reversed phase high-performance liquid chromatography based method was developed to detect specific adulterant in premium and pure ghee (clarified butterfat) samples. The method is based on the detection of cholesterol and β-sitosterol as markers in the unsaponifiable matter of pure ghee and adulterated ghee samples, respectively. The reversed phase high-performance liquid chromatography method resolved cholesterol, stigmasterol, campasterol, and β-sitosterol at Rt values of 17.681 ± 0.122, 19.7 ± 0.2, 20.2 ± 0.2, and 22.7 ± 0.2 min., respectively. β-sitosterol served as an indicator for the adulteration in ghee by plant and specific adulterant oil at dose dependent manner (1–5 g/100 g of ghee). Validation of the method revealed that it was fast, economical, highly reliable, and comparable with the reversed-phase thin layer chromatography method with no false negatives.

Matrine and oxymatrine in corroborant plant extracts and fertilizers: HPLC/MS-MS method development and single-laboratory validation

A reversed phase high-performance liquid chromatographic method (HPLC/MS-MS) has been developed and validated for detection of alkaloids matrine and oxymatrine in fertilizer with labeled enhancer plant defense activities. The analytical method was validated statistically. The results show a strong matrix effect, requiring quantification by standard addition method. The regression lines showed r2 > 0.994. Recoveries ranging from 97 to 104% were obtained for the fortification level of 0.01% wt wt−1 and the relative standard deviations ranged from 3 to 4% (n = 10). The limits of detection were below 0.0001% wt wt−1, while the limits of quantification did not exceed 0.0004% wt wt−1. The method is currently applied in ICQRF Laboratory of Catania on fertilized and corroborant plant extract collected in the Italian market in the frame of MIPAAF institutional quality control activity, with the aim to dectect these unpermitted active substances.

Toxin content and toxicological significance in different tissues and development stages of Lepiota brunneoincarnata mushroom

We investigated the quantity and concentration of toxins in different parts and in different growth phases of Lepiota brunneoincarnata mushroom. The amatoxins and phallotoxin levels were measured using the reversed phase high-performance liquid chromatography (RP-HPLC) method. Alpha amanitin (2.38 ± 0.70 mg/g) was followed by beta amanitin (1.97 ± 0.52 mg/g) and gamma amanitin (0.04 ± 0.01 mg/g) in trace amounts; it did not contain any phallotoxin. The cap part is richer in amatoxins than the stipe part. While medium mushrooms were quite rich in amatoxins, less levels of toxin were measured in fully developed mushrooms. The study showed in detail the toxin concentrations of L. brunneoincarnata with regard to different developmental stages and different segments.

Validation of RP-HPLC Method for Simultaneous Quantification of Bicalutamide and Hesperetin in Polycaprolactone-Bicalutamide-Hesperetin-Chitosan Nanoparticles.

Bicalutamide is a non-steroidal anti-androgen drug used for the treatment of androgen-dependent prostate cancer. Hesperetin is a natural bioflavonoid that can be used in combination with bicalutamide to improve efficacy and decrease tolerance. The aim of the present work was to develop and validate a simple, sensitive, rapid reverse phase-high performance liquid chromatographic method for simultaneous estimation of bicalutamide and hesperetin. The validation parameters such as specificity, linearity, precision and accuracy, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2 (R1) guidelines. Chromatographic separation was achieved on Lichrocart(®) CN column (250 × 4 mm, 5 µm, MERCK) with isocratic elution. The retention times and detection wavelength, for hesperetin and bicalutamide were 4.28 min, 288 nm and 5.90 min, 270 nm respectively. The intra-day and inter-day assay precision and accuracy were found to be <2% over linearity of 50-2000 ng/mL with R(2) 0.999. LOD and LOQ, of bicalutamide and hesperetin was 14.70, 44.57 ng/mL and 16.11, 48.84 ng/mL, respectively. The method was successfully applied for encapsulation efficiency and drug release studies from bicalutamide and hesperetin loaded nanoparticles.

A novel reverse phase high-performance liquid chromatography (RP-HPLC–UV) method for the estimation of alosetron HCl and its validation

A novel simple, sensitive, and rapid isocratic reverse phase high-performance liquid chromatographic (RP-HPLC) method was developed for the quantitative determination of alosetron HCl, a 5-HT3 antagonist used in the treatment of severe irritable bowel syndrome (IBS) in females. The optimized chromatographic separation was achieved using a stationary phase of Phenomenex® kromasil C-18 (250 mm × 4.6 mm; 5 μm particle size) column, and mobile phase of 0.025 M disodium hydrogen orthophosphate buffer, pH adjusted to 3.0 with orthophosphoric acid, and acetonitrile in the ratio of 65:35 (v/v) with a flow rate of 1 mL min−1. The UV detection was carried out at 217 nm. The developed method provided linear responses within the concentration range 100–2000 ng mL−1, and regression analysis showed a correlation coefficient value (r2) of 0.997. The HPLC method was validated as per International Conference on Harmonization (ICH) guidelines with respect to selectivity, precision, linearity, and robustness. Limit of detec...

Total error-based validation including the experimental design-based robustness evaluation of a stability-indicating method for the simultaneous quantification of hydrochlorothiazide and valsartan in tablet formulations

A gradient reversed phase high-performance liquid chromatography (RP-HPLC) method with ultraviolet (UV) detection to analyze hydrochlorothiazide (HCT) and valsartan (VS) simultaneously in a tablet formulation during forced degradation studies was developed. This method was validated using a novel approach, namely, the accuracy profile or total errors approach. The robustness of the method was evaluated using a Plackett-Burman design for eight factors. The algorithm of Dong was applied to determine the significant factor effects. The validation results showed that the method is precise (RSD: 1.14% for HCT and 0.43% for VS) and accurate (mean recovery: 99.90% for HCT and 99.98% for VS). On the other hand, the results of the robustness study showed that the type of column was the important factor which affects a number of responses, namely, the asymmetry factor (AF), retention time (RT), and resolution (RS). However, the assay results were not affected; therefore, the method can be considered robust. Finally, the method was applied to study the stability of HCT and VS under forced conditions. Significant results were obtained with basic hydrolysis, oxidation, and thermal stress, while the accelerated and acidic conditions did not affect the stability of HCT or VS.

HPLC method for simultaneous determination of salmeterol xinafoate and fluticasone propionate for the quality control of dry powder inhalation products

A simple and accurate reversed phase high-performance liquid chromatography (HPLC) method has been developed for the estimation of uniformity of dosage units, and delivered dose uniformity tests of fluticasone propionate and salmeterol xinafoate in samples obtained during the fine particle mass determination, using C8 Luna (2) 100A 100 × 4.6 mm, 5 micrometer particle size in gradient mode, with mobile phase comprising of buffer solution: 0.6% trifluoroacetic acid solution in water-tetrahydrofurane (8:2 v/v) and acetonitrile-methanol (1:1 v/v) in the ratio of 60:40 v/v. The flow rate was 1.5 mL min−1, and the detection was monitored by ultraviolet (UV) detector at 239 nm and 250 nm. Linearity was observed in the concentration range of 0.025–4.8 μg mL−1 for salmeterol xinafoate and 0.04–32.5 μg mL−1 for fluticasone propionate (R2 > 0.999). The recovery in the range from 98.2% to 102.7% and relative standard deviation (RSD) ≤ 2.2% demonstrated accuracy and high precision of the method. The quantification lim...

(E)-2-Benzylidenebenzocyclanones: part XIII—(E)/(Z)-Isomerization of some cyclic chalcone analogues. Effect of ring size on lipophilicity of geometric isomers

Optimized isocratic reverse phase high-performance liquid chromatography (RP-HPLC) method was developed for separation of the respective (E) and (Z) isomers of the cyclic chalcone analogues (E)-2-(4′-X-benzylidene)-1-indanones, -tetralones, and -benzosuberones. The method has been applied to monitor progress of the light-induced (E)/(Z) isomerization process in the three series. Data indicate formation of equilibrium mixtures of the respective geometric isomers. Comparison of the HPLC retention factors of the respective geometric isomers showed the (Z) isomers less lipophilic in the benzosuberone and the tetralone but more lipophilic in the indanone series. Data provide experimental evidence of opposite effect of ring size and geometric isomerism on lipophilicity of conformationally restricted (cyclic) analogues of chalcone, an open chain pharmacophore.

Development and validation of a stability indicating HPLC method for the simultaneous analysis of lopinavir and ritonavir in fixed-dose combination tablets

Abstract Objectives A simple, accurate, precise and rapid stability indicating HPLC method for simultaneous determination of Lopinavir and Ritonavir in combined dosage forms. Methods A validated stability indicating reversed phase high-performance liquid chromatographic method was developed for the quantitative determination of two antiviral drugs viz. lopinavir (LPV) and ritonavir (RTV) on Agilent TC C18 (2) 250 × 4.6 mm, 5 μ column using mobile phase composition of acetonitrile: 0.05 M phosphoric acid (55: 45, v/v) at a flow rate of 1.2 ml/min. Results Quantification was achieved with ultraviolet detection at 240 nm. The retention time obtained for ritonavir was at 4.35 min and for lopinavir was at 6.68 min. The result obtained with the detector response was found to be linear in the concentration range of 8–48 μg/ml for lopinavir and 2–12 μg/ml for ritonavir. This method has been validated and shown to be specific, sensitive, precise, linear, accurate, rugged, robust and fast. LPV and RTV were subjected to different accelerated stress conditions. The degradation products, were well resolved from the pure drug with significantly different retention time values. Conclusion It is concluded that this method can be applied for routine quality control of LPV and RTV in tablet dosage forms as well as in bulk drug. Hence the proposed method is highly sensitive, precise and accurate and it successfully applied for the reliable quantification of active pharmaceutical ingredient (API) content in the commercial formulations of lopinavir and ritonavir.

New reversed phase-high performance liquid chromatographic method for selective separation of yttrium from all rare earth elements employing nitrilotriacetate complexes in anion exchange mode

Separation of Y from other rare earth elements (REE) is difficult because of similarity of its ionic radius to ionic radii of Tb, Dy and Ho. In the new RP-HPLC system with C18 column, tetra-n-butyl ammonium hydroxide (TBAOH) as an ion interaction reagent (IIR), nitrilotriacetic acid (NTA) as a complexing agent at pH=2.8–3.5, and post column derivatization with Arsenazo III, yttrium is eluted in the region of light REE, between Nd and Sm and is base line separated from Nd and Sm and even from promethium. Simple model employing literature data on complex formation of REE with NTA and based on anion exchange mechanism was developed to foresee the order of elution of individual REE. The model correctly predicted that lanthanides up to Tb will be eluted in the order of increasing Atomic Number (At.No.) but all heavier REE will show smaller retention factors than Tb. Concurrent UV/VIS detection at 658nm and the use of radioactive tracers together with γ-ray spectrometric measurements made possible to establish an unique elution order of elution of REE: La, Ce, Pr, Nd, Pm, Y, Sm, Er, Ho, Tm, Yb, Eu, Lu, Dy+Gd, Tb, Sc. The real place of Y however, in this elution series differs from that predicted by the model (Y between Sm and Eu). The method described in this work enables selective separation of Y from La, Ce, Pr, Nd, Pm, Sm and all heavier REE treated as a group.

Development and Validation of Reverse Phase High Performance Chromatography Method for Determination of Olanzapine in Microsample Rat Plasma: Application to Preclinical Pharmacokinetic Study

Purpose: To develop a sensitive and validated reverse phase-high performance liquid chromatographic (RP-HPLC) method for quantification of olanzapine in micro-sample of rat plasma using UV detection.Methods: A single oral dose of olanzapine (7 mg/kg) was given to overnight fasted rats (n = 6). Rat plasma samples containing the drug were extracted by liquid-liquid extraction using a combination of dichloromethane: n-hexane (80:20). A reverse phase chromatographic column C18 hypersil-BDS was used for chromatographic separation with a mobile phase consisting of 50 mM phosphate buffer pH 5.5, acetonitrile and methanol (50:30:20, v/v/v) pumped at a flow rate of 1.2 ml/min. Olanzapine was measured using ultraviolet (UV) detection at 214 nm. The method was validated for precision and accuracy.Results: Separation of compounds of interest was not affected by endogenous interference. Good linearity within the concentration range of 1 - 500 ng/ml in rat plasma was obtained with coefficient of regression (r2) of 0.9986. Liquid-liquid extraction produced comparable recovery to solid phase extraction. Retention time of olanzapine and internal standard (fluoxetine) was 5.0 and 13.4 min, respectively. Lowest limit of quantification (LLOQ) was 1 ng/ml while inter-day and intra-day precision was &lt; 12.5 and 5.1 %, respectively. Accuracy of the method was between 94 and 105 % and the variation of results between two analysts was not significant (p = 0.626). Mean maximum plasma concentration (Cmax) of olanzapine was 412.7 ng/ml, time to attain maximum plasma concentration (tmax) was 1 h and half life (t½) was 2.54 h.Conclusion: The proposed method has been successfully validated for precision and accuracy that are within the limits of U.S. Food and Drug Administration (FDA)’s guidance for bioanalyitcal assay validation. The method was successfully applied to preclinical pharmacokinetic analysis of olanzapine in rats.Keywords: Olanzapine, Antipsychotic, Pharmacokinetics, Rat, Plasma, Bioanalytical assay Olanzapine, Antipsychotic, Pharmacokinetics, Rat, Plasma, Bioanalytical assay

Simultaneous determination of linagliptin and metformin by reverse phase-high performance liquid chromatography method: An application in quantitative analysis of pharmaceutical dosage forms.

To enhance patient compliance toward treatment in diseases like diabetes, usually a combination of drugs is prescribed. Therefore, an anti-diabetic fixed-dose combination of 2.5 mg of linagliptin 500 mg of metformin was taken for simultaneous estimation of both the drugs by reverse phase-high performance liquid chromatography (RP-HPLC) method. The present study aimed to develop a simple and sensitive RP-HPLC method for the simultaneous determination of linagliptin and metformin in pharmaceutical dosage forms. The chromatographic separation was designed and evaluated by using linagliptin and metformin working standard and sample solutions in the linearity range. Chromatographic separation was performed on a C18 column using a mobile phase of 70:30 (v/v) mixture of methanol and 0.05 M potassium dihydrogen orthophosphate (pH adjusted to 4.6 with orthophosphoric acid) delivered at a flow rate of 0.6 mL/min and UV detection at 267 nm. Linagliptin and metformin shown linearity in the range of 2-12 μg/mL and 400–2400 μg/mL respectively with correlation co-efficient of 0.9996 and 0.9989. The resultant findings analyzed for standard deviation (SD) and relative standard deviation to validate the developed method. The retention time of linagliptin and metformin was found to be 6.3 and 4.6 min and separation was complete in <10 min. The method was validated for linearity, accuracy and precision were found to be acceptable over the linearity range of the linagliptin and metformin. The method was found suitable for the routine quantitative analysis of linagliptin and metformin in pharmaceutical dosage forms.

RP-HPLC Estimation of Imipramine Hydrochloride and Diazepam in Tablets.

A simple and rapid reversed phase-high performance liquid chromatographic method was developed for simultaneous determination of imipramine hydrochloride and diazepam in pharmaceutical formulations. The elution was done in isocratic mode utilizing a mobile phase consisting of methanol:water:0.1M sodium acetate (30:50:20 v/v/v) on Chromosil C18 column with a flow rate of 1.0 ml/min and with detection at 243 nm. The measured retention time was 3.33±0.02 min for imipramine hydrochloride and 4.64±0.02 min for diazepam. Linearity was measured in the range 25-150 μg/ml for imipramine hydrochloride (r(2)=0.999) and in the range 5-30 μg/ml for diazepam (r(2)=0.9994), respectively. The limits of detection and quantitation were 0.03 and 0.1 μg/ml for imipramine hydrochloride and 0.02 and 0.07 μg/ml for diazepam. Satisfactory validation was also obtained from recovery (100.95-101.52% for imipramine hydrochloride and 99.47-100.33% for diazepam) studies, intraday and interday precision (<2%) and robustness results. The reported method was the first study of these drugs in combination and could be employed for routine quantitative determination of imipramine hydrochloride and diazepam in tablets.

A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts.

Background:Orthosiphon stamineus Benth. (Lamiaceae) is a traditional medicinal plant which has been used in treating various ailments such as kidney diseases, bladder inflammation, arthritis and diabetes. The leaves contain high concentration of phenolic compounds, thus, rosmarinic acid (RA), 3’-hydroxy-5, 6, 7, 4’-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) were chosen as a marker compounds for standardization of various O. stamineus leaf extracts.Objective:The aim was to develop and validate a new high-performance liquid chromatography (HPLC) method for quantification of 4 marker compounds (RA, TMF, SIN, EUP) in various O. stamineus leaf extracts.Materials and Methods:The method was developed and validated using RP-HPLC-diode-array detection at 320 nm for accuracy, precision and limits of detection and was applied for quantification of it markers in five different extracts prepared in solvents with increasing polarity, using a gradient mobile phase 0.1% formic acid: Acetonitrile at a flow rate of 1 ml/min on reverse phase acclaim polar advantage II C18 column (3 μm, 3 × 150 mm) with 18 min separation time.Results:The developed method provided satisfactory precision, and the accuracy of this method was in the range of 90.2% to 105.5%. All of 4 compounds showed good linearity at R2 > 0.999.Conclusion:The developed method is a simple, cost effective with shorter run time (18 min) in comparison to previous methods (30 min) and utilization of environmental-friendly solvents system. Therefore, this method has the potential to replace currently used methods in the routine standardization work of O. stamineus extracts, raw materials and its commercial products.

Analysis of L-citrulline and L-arginine in Ficus deltoidea leaf extracts by reverse phase high performance liquid chromatography.

Background:Ficus deltoidea (FD) is one of the native plants widely distributed in several countries in Southeast Asia. Previous studies have shown that FD leaf possess antinociceptive, wound healing and antioxidant properties. These beneficial effects have been attributed to the presence of primary and secondary metabolites such as polyphenols, amino acids and flavonoids.Objective:The aim was to develop a reverse phase high-performance liquid chromatography method with ultraviolet detection that involves precolumn derivatisation with O-phthaladehyde for simultaneous analysis of two amino acids L-citrulline and L-arginine in FD leaf extracts.Materials and Methods:An isocratic elution program consisting of methanol: acetonitrile: Water at 45:45:10 v/v (solvent A) and 0.1 M phosphate buffer pH 7.5 (solvent B) at A: B v/v ratio of 80:20 on Zorbax Eclipse C18 SB-Aq column (250 × 4.6 mm, 5 μm) were used. The flow rate was set at 1 ml/min and detection was carried out at 338 nm with 30 min separation time.Results:Good linearity for L-citrulline and L-arginine was obtained in the range 0.1-1000 μg/ml at R2 ≥ 0.998. The limit of detection and limit of quantification values for both L-citrulline and L-arginine were 1 and 5 μg/ml, respectively. The average of recoveries was in the range 94.94-101.95%, with relative standard deviation (%RSD) less than 3%. Intra- and inter-day precision was in the range 96.36-102.43% with RSD less than 2%.Conclusion:All validation parameters of the developed method indicate the method is reliable and efficient for simultaneous determination of L-citrulline and L-arginine for routine analysis of FD.

A novel validated RP-HPLC-DAD method for the estimation of Eluxadoline in bulk and pharmaceutical dosage form

The present study describes about novel approach to develop and validate a rapid, accurate, precise, simple, efficient and reproducible isocratic Reversed Phase-High Performance Liquid Chromatographic (RP-HPLC-DAD) method for the estimation of Eluxadoline in bulk and pharmaceutical dosage form. Eluxadoline were separated using Hypersil C18 column (250mm×4.6 mm, 5μm particle size), Waters Alliance e2695 HPLC system with 2998 PDA detector and the mobile phase contained a mixture of Acetonitrile and Water (70: 30, v/v). The flow rate was set to 1ml/min with the responses measured at 260nm. The retention time of Eluxadoline was found to be 3.481min. Linearity was established for Eluxadoline in the range of 10–125μg/ml with correlation coefficient (r2=0.999). The percentage recoveries were between 99.92% to 100.07%. Validation parameters such as specificity, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ) were evaluated for the method according to the International Conference on Harmonization (ICH) Q2 R1 guidelines. The developed method was successfully applied for the quantification and hyphenated instrumental analysis.

Development of an HPLC Method for Absolute Quantification and QAMS of Flavonoids Components in Psoralea corylifolia L.

The seeds of Psoralea corylifolia L. (Fabaceae) are a commonly used medicinal herb in eastern Asia with many beneficial effects in clinical therapies. In this study, a simple, sensitive, precise, and specific reverse phase high-performance liquid chromatography (HPLC) method was established for quantification of 9 flavonoids in P. corylifolia, including isobavachin, neobavaisoflavone, bavachin, corylin, bavachalcone, bavachinin, isobavachalcone, corylifol A, and 4′-O-methylbavachalcone. Based on this method, a quantitative analysis of multicomponents by single marker (QAMS) was carried out, and the relative correction factors (RCFs) were calculated for determining the contents of other flavonoids. The accuracy of QAMS method was verified by comparing with the results of external standard method, as well as the feasibility and adaptability of the method applied on quality control of P. corylifolia. The 9 compounds were baseline separated in 60 min with a good linearity of regression coefficient (R 2) over 0.9991. The accuracies of QAMS were between 92.89% and 109.5%. The RSD values of f in different injection volume were between 2.3% and 3.6%. The results obtained from QAMS suggested that it was a convenient and accurate method to determine multicomponents especially when some authentic standard substances were unavailable. It can be used to control the quality of P. corylifolia.

High-performance liquid chromatography for analytical and small-scale preparative separation of (R,S)-mexiletine using (S)-(—)-(N)-trifluoroacetyl-prolyl chloride and (1S)-(—)-camphanic chloride and recovery of native enantiomer by detagging

A reversed phase high-performance liquid chromatographic method has been established for enantioseparation of (R, S)-mexiletine. Two volatile and thermally stable acyl chlorides, viz., (S)-(—)-(N)-trifluoro acetyl prolyl chloride and (1S)-(—)-camphanic chloride, were used as chiral derivatizing reagents. Binary composition of aqueous trifluoroacetic acid (0.1%)-acetonitrile as mobile phase was successful with ultraviolet (UV) detection at 210 nm. The method was optimized and validated for accuracy, precision, and limit of detection. The limit of detection was found to be 45 ng mL−1 and 80 ng mL−1 for the two types of diastereomers. Besides, kinetic resolution was achieved, and the experimental conditions optimized for this purpose provided diastereomeric excess up to 74% for (R)-isomer. On achieving a resolution value greater than 2, the optimized method for analytical enantioseparation was scaled-up to smallscale preparative level, and the native (R)-mexiletine was recovered by acid hydrolysis of the diastereomer.

A Study of Method Development, Validation, and Forced Degradation for Simultaneous Quantification of Paracetamol and Ibuprofen in Pharmaceutical Dosage Form by RP-HPLC Method.

A rapid and stability-indicating reversed phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous quantification of paracetamol and ibuprofen in their combined dosage form especially to get some more advantages over other methods already developed for this combination. The method was validated according to United States Pharmacopeia (USP) guideline with respect to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity, and system suitability. Forced degradation study was validated according to International Conference on Harmonisation (ICH). For this, an isocratic condition of mobile phase comprising phosphate buffer (pH 6.8) and acetonitrile in a ratio of 65:35, v/v at a flow rate of 0.7 mL/minute over RP C18 (octadecylsilane (ODS), 150 × 4.6 mm, 5 μm, Phenomenex Inc.) column at ambient temperature was maintained. The method showed excellent linear response with correlation coefficient (R2) values of 0.999 and 1.0 for paracetamol and ibuprofen respectively, which were within the limit of correlation coefficient (R2 > 0.995). The percent recoveries for two drugs were found within the acceptance limit of (97.0–103.0%). Intra-and inter-day precision studies of the new method were less than the maximum allowable limit percentage of relative standard deviation (%RSD) ≤ 2.0. Forced degradation of the drug product was carried out as per the ICH guidelines with a view to establishing the stability-indicating property of this method and providing useful information about the degradation pathways, degradation products, and how the quality of a drug substance and drug product changes with time under the influence of various stressing conditions. The degradation of ibuprofen was within the limit (5–20%, according to the guideline of ICH), while paracetamol showed <20% degradation in oxidation and basic condition.

Determination of Catechin and Epicatechin Content in Chocolates by High-Performance Liquid Chromatography

A simple and sensitive reversed phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of catechin and epicatechin in cocoa powder and chocolates. The separation was achieved on a reversed phase C 18 column (TARGA) 5 μm by gradient elution with a flow rate of 1.0 mL/minute with an operating temperature of 30°C and detection with a UV-Visible detector was at 280 nm. The method was validated for linearity, precision, intra- and interday precision, and accuracy. The developed method is successfully applied for the determination of catechin and epicatechin content in chocolates. The Godiva brand chocolate contains high concentration of epicatechin.