Stability indicating RP-HPLC method for simultaneous determination of atorvastatin and amlodipine from their combination drug products

The study describes development and subsequent validation of a stability indicating reverse-phase HPLC method for the simultaneous estimation of atorvastatin (ATV), and amlodipine (AML) from their combination drug product. The proposed RP-HPLC method utilizes a Lichrospher 100 C18, 5 microm, 250 mm x 4.0 mm i.d. column, at ambient temperature, optimum mobile phase consisted of acetonitrile and 50 mM potassium dihydrogen phosphate buffer (60 : 40, v/v), apparent pH adjusted to 3+/-0.1 with 10% phosphoric acid solution, effluent flow rate monitored at 1.0 ml/min, and UV detection at 254 nm. ATV, AML, and their combination drug product were exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions, and the stressed samples were analyzed by proposed method. The method was applied for the in vitro dissolution of marketed combination drug products. The described method was linear over the range of 1-90 microg/ml and 1-80 microg/ml for ATV and AML, respectively. The mean recoveries were 99.76 and 98.12% for ATV and AML, respectively. The intermediate precision data obtained under different experimental setup, the calculated value of coefficient of variation (CV, %) was found to be less than critical value. The limit of detection for ATV and AML were found to be 0.4 and 0.6 mug/ml, respectively and the limit of quantification was 1.0 microg/ml for both drugs. The average percentage drug release was found to be more than 70% within 30 min for both drugs. Chromatographic peak purity data of ATV and AML indicated no co-eluting peaks with the main peaks of drugs which demonstrated the specificity of assay method for their estimation in presence of degradation products. The proposed method can be useful in the quality control and in vitro dissolution of combination drug products.     

High-performance liquid chromatography quantification of principal antioxidants in black seed (Nigella sativa L.) phytopharmaceuticals

A new, simple, sensitive, rapid, and accurate isocratic RP-HPLC method was developed and validated for simultaneous analysis of the principal antioxidants of Nigella sativa, i.e., thymoquinone (TQ), carvacrol (CR), and its isomer thymol (THY), in different phytopharmaceuticals. The mobile phase was water-methanol (40 + 60, v/v) at a flow rate of 1.5 mL/min. Quantification was achieved with UV detection at 254 nm, based on peak area. The method was validated for linearity, accuracy, precision, selectivity, and robustness. The proposed method is stability-indicating for determination of TQ in the presence of its degradants. The LOD and LOQ (microg/mL) were, respectively, 0.006 and 0.021 for TQ, 0.002 and 0.006 for CR, and 0.027 and 0.090 for THY. The mean recoveries measured at three concentrations were higher than 99%, with RSD <2%. This analytical method is suitable for quality control of the marker substances in this widely used natural protective and curative remedy.     

Development and validation of RP-HPLC method for the determination of stigmasterol in the botanical extract of Ficus deltoidea

A simple, rapid, accurate and precise RP-HPLC method was developed for the determination of stigmasterol in botanical extract of Ficus deltoidea. Separation was achieved with acetonitrile and acetic acid in water (75:25% v/v) in isocratic mode at 210?nm. Single sharp peak of standard stigmasterol was detected at retention time 3.17?min which overlay with the peak of plant extract at 3.14?min. The calibration curve was found to be linear in a concentration range of 2–10?μg/ml with correlation coefficient of 0.998. The LOD and LOQ were found to be 1.50?μg/ml and 4.55?μg/ml respectively. Accuracy and precision was determined with overall recovery of 99.6–100.1% for stigmasterol and RSD values in both intra-day and inter-day repeatability assay lesser than 0.340%, respectively. The robustness study also indicated that there is no influence of minor changes in detecting wavelength and flow rate of mobile phase on the response.     

Development of a simple and stability-indicating RP-HPLC method for determining olanzapine and related impurities generated in the preparative process

A simple and stability-indicating reverse phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the determination of olanzapine (OLN) and related impurities in bulk drugs. Eight impurities were characterized respectively, and particularly a new process impurity from OLN synthesis was structurally confirmed as 1-(5-methylthionphen-2-yl)-1H-benzimidazol-2(3H)-one (Imp-7) by X-ray single crystal diffraction, MS, (1)H NMR, (13)C NMR and HSQC. A mechanism of formation pathway for Imp-7 was proposed. Optimum separation for OLN and eight related impurities was carried out on an Agilent Octyldecyl silica column (TC-C(18), 4.6 mm × 250 mm, 5 μm) using a gradient HPLC method. The method was validated with respect to specificity, linearity, accuracy, precision, LOD and LOQ. Regression analysis showed good correlation (r(2) > 0.9985) between the investigated component concentrations and their peak areas within the test ranges for OLN and eight impurities. The repeatability and intermediate precision, expressed as RSD, were less than 1.74%. The proposed stability-indicating method was suitable for routine quality control and drug analysis of OLN in bulk drugs.     

Simultaneous estimation of hydroxychavicol and chlorogenic acid from Piper betel L. through RP-HPLC

A RP-HPLC method was developed (λ (max)?=?280) to quantify hydroxychavicol and chlorogenic acid in Piper betel Linn. The method was validated for linearity, limit of detection (LOD?=?3:1σ/S), limit of quantification (LOQ?=?10:1σ/S), precision, accuracy and ruggedness. The response was linear with good correlation between concentration and mean peak area through a coefficient of determinants (r (2)) of 0.9940, y?=?1.98e?+?004x?+?5.19e?+?004 and 0.9945, y?=?2.76e?+?004x?+?1.40e?+?005 with LOD 1.6?μg?mL(-1), 1.0?μg?mL(-1) and LOQ 5.0?μg?mL(-1) and 3.0?μg?mL(-1), respectively, for hydroxychavicol (28.56% w/w) and chlorogenic acid (0.40% w/w). The %RSD of precision and recovery of hydroxychavicol and chlorogenic acid were <2.0%. The proposed method was simple, accurate, specific, precise and reproducible.     

Reversed Phase Chromatographic Analysis of 13 Amino Acids in Honey Samples

A simple method using reversed phase high-performance liquid chromatography (RP-HPLC) was developed for the simultaneous analysis of 13 amino acids. Amino acids were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl) before analysis by RP-HPLC. Experimental parameters affecting the derivatization and chromatographic separation were investigated. Amino acids were derivatized with FMOC-Cl under alkaline condition in 0.1 mol/L borate buffer pH 10.0 at room temperature. The FMOC-amino acid derivatives were separated on an Atlantis C18 column under the gradient elution of 0.05 % trifluoroacetic acid and acetonitrile and UV detection at 265 nm. Linear ranges were 0.2–100.0 μg/mL with the correlation coefficients greater than 0.992. Limits of detection and limits of quantitation were in the range of 0.05–2.0 and 0.2–5.0 µg/L, respectively. The intra-day precision (n = 3) of retention time was less than 1 %, while for the peak area was less than 4 %. The inter-day precision (n = 3 × 3) of retention time was less than 2 % and the peak area was less than 8 %. This method was applied in honey samples and the results showed that proline is the major amino acids in honey samples.

     

A Gradient Reversed Phase High Performance Liquid Chromatography Method for Simultaneous Determination of Hydrochlorothiazide (HCT) and Losartan Potassium (LOS) from Tablets.

ABSTRACT

A new, precise, accurate gradient reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for the simultaneous determination of Hydrochlorothiazide (HCT) and Losartan potassium (LOS) in tablets. The stationary phase was Microbondapak C18 column (10 μ, 300 mm × 3.9 mm ID). A gradient elution with an aqueous methanolic mobile phase (pH=3) was employed for the separation. Detection was carried out at 270 nm using UV detector. The flow rate was 1.0 ml/min and retention times were 7.89 minutes and 15.15 minutes for HCT and LOS, respectively. The linearity was obtained in the concentration range of 0.5 - 200 μg/ml for HCT and 2 - 800 μg/ml for LOS. Mean percentage recoveries were 100.29% and 99.16% for HCT and LOS, respectively.     

Development and validation of a reversed-phase HPLC method for quantitative determination of ginsenosides Rb1, Rd, F2, and compound K during the process of biotransformation of ginsenoside Rb1

A simple and rapid gradient RP-HPLC method for simultaneous separation and determination of related ginsenosides during the process of biotransformation of ginsenoside Rb1 has been developed. As many as four process ginsenosides have been separated and identified on an Eclipse XDB C(18) column (4.6 mm x 150 mm, 5 mum) with gradient elution using water and ACN as a mobile phase. The column was maintained at 30 degrees C and the eluents were monitored with diode array detection at 203 nm. The method was validated in terms of linearity, sensitivity, precision, and accuracy. The correlation coefficients (r) for calibration curves of ginsenosides were in the range of 0.9996-1.0000. The proposed RP-HPLC method was successfully applied to the analysis of fermentation broth and the recoveries of ginsenosides were in the range of 94.4-103.1% with RSD <2.87%. The method could be of use for rapid and routine evaluation of the quantity of ginsenosides during the biotransformation process of ginsenoside Rb1.     

RP-HPLC Method for Determination of Oseltamivir Phosphate in Capsules and Spiked Plasma

A new, sensitive RP-HPLC method was developed for the determination of oseltamivir phosphate in capsules and plasma. The method was based on the reaction of the drug with 4-chloro-7-nitrobenzofurazan in borate buffer solution of pH 8.50. Isocratic chromatography was performed on a C₁₈ column with acetonitrile–10 mM nitric acid (pH 3, 60 + 40, v/v) as the mobile phase with fluorescence detection (λex: 470 nm, λem: 541 nm). Mexiletine hydrochloride was used as an internal standard. Analytical parameters were evaluated. The calibration range was linear from 50.0–750.0 ng/ml. The mean percentage recovery in capsules and plasma were 99.95% and 95.42%, respectively.     

Development and Validation of a High-Performance Liquid Chromatographic Determination of Ceftriaxone Sodium and Its Application to Drug Quality Control

Abstract

A reliable and sensitive RP-HPLC method was developed and validated for the quantitative estimation of ceftriaxone sodium (CFTZ) in pure drug and pharmaceutical dosage forms. The separation of ceftriaxone sodium was achieved on a Waters XTerra RP-18 (5 µm, 250 × 4.6 mm i.d.) column using photodiode array detector at 240 nm. The mobile phase consisted of 0.1 M triethylammoniumacetate–acetonitrile (60:40 v/v) mixture delivered at a flow rate of 1.0 ml/min. Accuracy, evaluated by means of the spike recovery method, was excellent, with percent recovery in the range 99.5–102% with precision in the range 0.3–1.2%.     

Development and validation of a RP-HPLC method for Roflumilast and its degradation products

Roflumilast is a phosphodiesterase type 4 inhibitor that is administered orally as a long-term, in the clinical treatment of chronic obstructive pulmonary disease and asthma. Launched in 2010 for the European market, it currently does not have an official monograph. Here, a reproducible gradient RP-HPLC method was developed and validated for the separation and determination of Roflumilast in the presence of its six major degradation products. Separation was performed on a C₁₈ analytical column (250?×?4.6?mm, 5?µm) with a mobile phase-A of ACN and a phase-B of ammonium acetate buffer (5?mM, pH 4.2) containing triethylamine (0.5% v/v). The most effective RP-HPLC gradient program was determined to be 0/80, 35/10, 36/80, 40/80 (time in minutes/% mobile phase-B). The flow rate was 1.0?ml/min and the column temperature was 25°C. The success of separation of the degradation products with different chemical characteristics was obtained by extending the time of the gradient, changing the proportion of the mobile phases and increasing the velocity of the flow. Two detectors were evaluated for the identification of degradation products and Roflumilast: a diode-arrary detector and a charged aerosol detector. The inability of the charged aerosol detector to dectect one of the six degradation products indicated that the method developed with RP-HPLC and the diode-array detector was more suitable for Roflumilast analysis. The method was validated according to specificity, linearity, LOD, LOQ, accuracy, precision and robustness.     

Stress Studies and Identification of Degradation Products of Cephalexin Using LC–PDA and LC–MS/MS

A stability indicating RP-HPLC method for cephalexin has been developed and validated to identify and characterize potential degradation products. Drug was subjected to hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stresses as per ICH guidelines Q1A (R2) and Q1B. Chromatographic separation was achieved on C8 column with mixture of ammonium acetate buffer pH 4.5 and acetonitrile in gradient mode as a mobile phase with PDA detection. Specificity of the method was established by peak purity studies. Method was validated as per ICH guideline Q2 (R1) for accuracy, precision, linearity, sensitivity, and robustness. Kinetics for each degradation condition was studied with respect to order of reaction and rate constant. Method was found to comply with acceptance criteria of validation parameters with respect to specificity (peak purity greater than 0.999) linearity (r ² greater than 0.99), accuracy (% recovery in the range of 98–102%), and precision (% RSD not more than 2). A total of six degradation products were generated in different stress conditions; these were identified and structures were proposed using LC–MS/MS. Cephalexin undergoes degradation in almost all the conditions. The developed stability indicating method is suitable for analysis of stability samples as it adequately separates all degradation products. Degradation products generated in photolytic and oxidative conditions are reported for the first time.     

Determination of tryptophan in raw materials, rat brain and human plasma by RP-HPLC technique

This paper describes tryptophan (TRP) estimation in raw human plasma and rat brain by reversed-phase high-performance liquid chromatography (RP-HPLC). Estimation was carried out on a Purospher STAR C18 column using water-acetonitrile (90:10 v/v, at pH 2.7) mixture at a rate of 1.5 mL/min as mobile phase. Eluents were monitored at 273 nm by an ultraviolet detector. The method was linear (R(2) > 0.999), precise (intra-day and inter-day precision <2%) in the range of 0.25-20 μg/mL. The detection and quantification limits were 0.0144 μg/mL and 0.0437 μg/mL, respectively. In human plasma, Day 1 and Day 2 precision were 0.054-2.29% and 1.66-3.7%; whereas precisions in rat brain were 1.23-2.3% and 0.677-4.2%, respectively. The method was applied to study TRP level in human smokers and in arthritic rat brain. An efficient RP-HPLC method was developed for TRP determination that worked for clinical and research purposes.     

Simultaneous RP-HPLC Determination of Six Platycosides: Application to an Enzymatic Preparation Study

Platycosides, main pharmacological effective compounds, are known to have several biological activities, including anti-obesity, anti-cancer and anti-diabetes. Although enzymatic preparation of platycosides was considered as effective method to obtain them, few analytical methods have been reported on process control. In the present study, we developed an application of reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of six platycosides during the process of enzymatic preparation of deapio-platycodin D (dPD) and platycodin D (PD). The method employed a Hypersil ODS2 analytical column (250 × 4.6 mm I.D., 5 μm) coupled with UV detector at 210 nm with flow rate of 1.0 mg mL⁻¹. A step gradient of acetonitrile–water (v/v) was applied, leading to a sample analysis of 60 min. The method was validated in terms of linearity, sensitivity, precision and accuracy. The correlation coefficients (R ²) for calibration curves of platycosides were in the range of 0.9995–1.0 when the linearity range was from 0.85 to 10.2 mg mL⁻¹. The proposed RP-HPLC method was successfully applied to the analysis of enzymatic preparation study and the recoveries of platycosides were in the range of 96.22–102.56% with RSD <3.3%. The method could be of use for rapid and routine evaluation of the quantity of platycosides during the enzymatic preparation process.

     

Stability-Indication LC Determination of Entacapone in Tablets

A rapid and sensitive RP-HPLC method with UV detection at 305 nm for routine quality control of entacapone in tablets was developed. The procedure was validated by specificity, robustness, linearity, accuracy, repeatability and intermediate precision. Experimental design was used during validation to calculate method robustness. The method employs an Ace RP-18 (250 × 4.6 mm i.d., particle size 5 μm), with a mobile phase consisting of water pH 3.0: acetonitrile (65:35, v/v). Entacapone solutions were exposed to direct UV radiation (254 nm), alkaline hydrolysis, acid hydrolysis and effect of oxidation by hydrogen peroxide to evaluate method stability-indication and peak purity tool was utilized to verify the peak purity. The results confirm that the method is highly suitable for its intended purpose.     

Simultaneous RP-HPLC Determination of Six Platycosides: Application to an Enzymatic Preparation Study

Platycosides, main pharmacological effective compounds, are known to have several biological activities, including anti-obesity, anti-cancer and anti-diabetes. Although enzymatic preparation of platycosides was considered as effective method to obtain them, few analytical methods have been reported on process control. In the present study, we developed an application of reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of six platycosides during the process of enzymatic preparation of deapio-platycodin D (dPD) and platycodin D (PD). The method employed a Hypersil ODS2 analytical column (250 × 4.6 mm I.D., 5 ??m) coupled with UV detector at 210 nm with flow rate of 1.0 mg mL^?1. A step gradient of acetonitrile?Cwater (v/v) was applied, leading to a sample analysis of 60 min. The method was validated in terms of linearity, sensitivity, precision and accuracy. The correlation coefficients (R ²) for calibration curves of platycosides were in the range of 0.9995?C1.0 when the linearity range was from 0.85 to 10.2 mg mL^?1. The proposed RP-HPLC method was successfully applied to the analysis of enzymatic preparation study and the recoveries of platycosides were in the range of 96.22?C102.56% with RSD <3.3%. The method could be of use for rapid and routine evaluation of the quantity of platycosides during the enzymatic preparation process.     

Determination of Pongamol and Karanjin in Karanja Oil By Reverse Phase Hplc

ABSTRACT

Pongamol and karanjin, naturally present in karanja (Pongamia glabra) oil possess potential UV A and UV B sunscreen activities, respectively. A reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for direct estimation of these constituents of karanja oil. This method of analysis requires no sample preparation of active ingredients. The analysis uses methanol, water and acetic acid in the ratio of 85: 13.5: 1.5 as mobile phase at a flow rate of 0.5 ml/ min on a Kromasil 100 C18 column, ex Tracer (250mm X46mm, particle size 5 microns) and dual wavelength detection at 350 and 300 nm. 2-ethylhexyl-p-methoxy cinnamate (Parsol MCX) was used as the internal standard. The identical UV spectrum of the chromatographic region (upslope, apex and downslope) confirmed that the analysis was free from substrate interference. The detection limits for pongamol and karanjin were found to be 0.1 μg/ml.     

Simultaneous Estimation of Escitalopram and Clonazepam in Tablet Dosage Forms Using HPLC-DAD Method and Optimization of Chromatographic Conditions by Box-Behnken Design

The study aimed to develop a new reverse-phase high-performance liquid chromatography (RP-HPLC) method with diode array detection (DAD) detection for simultaneous estimation of escitalopram (EST) and clonazepam (CZP) in tablet dosage forms with a quality by design (QbD) approach. The chromatographic conditions were optimized by Box-Behnken design (BBD) and developed method was validated for the linearity, system suitability, accuracy, precision, robustness, sensitivity, and solution stability according to International Council for Harmonization (ICH) guidelines. EST and CZP standard drugs peaks were separated at retention times of 2.668 and 5.046 min by C-18 column with dimension of 4.6 × 100 mm length and particle size packing 2.5 µm. The mobile phase was methanol: 0.1% orthophosphoric acid (OPA) (25:75, v/v), with a flow rate of 0.7 mL/min at temperature of 26 °C. The sample volume injected was 20 µL and peaks were detected at 239 nm. Using the standard calibration curve, the % assay of marketed tablet was founded 98.89 and 98.76 for EST and CZP, respectively. The proposed RP-HPLC method was able to detect EST and CZP in the presence of their degradation products, indicating the stability-indicating property of the developed RP-HPLC method. The validation parameter’s results in terms of linearity, system suitability, accuracy, precision, robustness, sensitivity, and solution stability were in an acceptable range as per the ICH guidelines. The newly developed RP-HPLC method with QbD application is simple, accurate, time-saving, and economic.     

A Rapid Method for Determination of Progesterone By Reversed-Phase Liquid Chromatography from Aqueous Media

ABSTRACT

A simple reversed-phase liquid chromatographic method to assay progesterone in aqueous receiving medium, following in vitro skin permeation studies is presented. Progesterone was analysed using 5 μm LichroCART® RP-18 column (125 × 4 mm i.d.), after extraction with chloroform. The mobile phase used was methanol:water (70:30) at a flow rate of 1 ml/min. Detection was carried out at 254 nm at room temperature. The method showed a mean recovery of 99% for progesterone, and intra-assay and inter-assay coefficients of variation were below 2%. The proposed method was validated for linearity, specificity, precision and accuracy and showed to be useful for analysis of progesterone in in vitro skin permeation studies.     

Development and validation of a new RP-HPLC method for determination of quercetin in green tea

Green tea (Camellia sinensis) contains quercetin as a bioactive compound. Quercetin has anti-inflammatory and anticancer effects. The aim of this paper was to develop and validate an RP-HPLC method for determination of quercetin in green tea simpler and faster than other available methods. RP-HPLC analysis was performed by isocratic elution with a flow rate of 1.0 mL/min. Pure methanol was used as a mobile phase, while the quantification was effected at 370 nm. The separation was performed at 35°C using a C₁₈ column (4.6 × 250 mm, 5 μm). The results showed that the peak area response is linear within the concentration range of 10–70 μg/mL (r = 0.9986). The values of LOD and LOQ were 1.2 and 4 μg/mL, respectively. For the intra-day and intra-instrument reproducibility, RSD were in the range of 0.05–0.84% and 0.89–1.55%, respectively. The results of accuracy for the different concentrations of quercetin (40, 50 and 60 μg/mL) were 101.3, 98.4, and 98.2%. The developed and validated method was successfully applied to the determination of quercetin in green tea extract.