Rapid High-performance Liquid Chromatography Research Articles

Development and Validation of the HPLC Method for Simultaneous Quantification of Glucose and Xylose in Sugar Cane Bagasse Extract.

A validated rapid high-performance liquid chromatography (HPLC)-refractive index (RI) method was developed for the identification and quantification of glucose and xylose in hydrolyzed bagasse extract. The separation of compounds was achieved on Eurokat® H column (300 × 8mm, 10μm) at 75°C, using 0.01N sulfuric acid solution as mobile phase and 0.6mL/min as flow rate. The method was validated based on accuracy, precision, linearity, robustness, uncertainty, limit of detection (LOD) and limit of quantification (LOQ). Total chromatographic analysis time per sample was ~11min. Calibration plots were linear over the concentration ranges 11-100μg/100μL for glucose and xylose. The LOD was 0.8ppm and LOQ was 2.5ppm. The high recovery and low relative standard deviation confirm the suitability of the method for determination of glucose and xylose in bagasse extract. The proposed HPLC-RI method was accurate, fast and robust and required less run time due to less analytes retention times and allowed optimal energy consumption owing to lower column oven temperature.

Nanoscale ZIF-8 as an efficient carboplatin carrier for targeted cancer therapy

Cancer is a leading cause of death worldwide and is becoming more prevalent. Carboplatin (CBP) is an anti-cancer medication from the platinum group of substances that are frequently employed in chemotherapy, and it is an active anti-cancer agent. However, the adverse effects of platinum medications, including its lack of selectivity, significant systemic toxicity, and drug resistance, limit its usage. The zeolitic imidazolate framework-8 (ZIF-8) has gained global biomedical interest for its high drug loading capacity and controlled release into target cells. It is synthesized using a non-solvothermal method and has shown the capacity to regulate and enhance the selectivity of cancer cells. A rapid high-performance liquid chromatography equipped with photodiode array detector (HPLC-PDA) method was used to measure the CBP loading capacity on ZIF-8, and the result was found to be 0.066 mg mg−1. ZIF-8 could effectively protect CBP in the in vitro release test in the physiological environment (pH 7.4). When pH 5.5 was reached, ZIF-8 served as a pH-resposive nanocarrier, leading to gradual CPB release. Studies on the in vitro cytotoxicity of CBP showedthat the IC50 of CBP for the lung cancer cell line was significantly lowered upon encapsulation of CBP in nanoparticles.

Analysis of Drug-Alcohol Crime Nexus: A Case Study in Ghana

Background Crimes recorded by law enforcement agencies may be directly or indirectly related to drugs. There is growing concern about the increasing illicit drug use in Ghana, especially among younger people, which is perceived by many as contributing to the increase in criminal activities. The main objective of this research is to explore the role of illicit drugs and alcohol use in relation to crime among prison inmates. The study was carried out at the Kumasi Central Prison to investigate drug use among inmates through empirical interviews and drug tests. A semi-structured interview questionnaire was used to collect qualitative and quantitative demographic data from the prison inmates and the police officers. The inmates were tested for illicit drug use by rapid immune assay test and High performance liquid chromatography. Results The study revealed that the majority of the inmates had used one type of illicit substance or the other, even though only 6% of the inmates tested positive for cannabis. About 40% of respondents were incarcerated as a result of drug related crimes. 85% of persons previously arrested for drug crimes still had their current incarceration resulting from drugs. 82% of the respondents admitted that illicit substance use had both behavioural and psychological effects on the user and influenced crime commission. Conclusions Illicit substance use has a strong correlation to criminal activities. State institutions should prioritise the rehabilitation of drug convicts in order to minimise the reoccurrence of crimes. It is recommended that steps be taken to reduce the continuous traffic of drugs into prisons.

HPLC method for the determination of nifedipine in rat plasma: development, validation, and application to pharmacokinetic drug-herb interaction study

A simple and rapid high-performance liquid chromatography (HPLC) was developed to determine the plasma level of nifedipine in rats after its single administration and combination with herbs. Nifedipine was extracted with acetonitrile to precipitate protein from plasma samples. The separation was implemented on a C18 column with a mobile phase of acetonitrile: water (63:37, v/v). The calibration curve displayed good linearity in the 30–1000 ng/mL range. The lower limit of quantification (LLOQ) was 30 ng/mL. The intraday and interday assay accuracy and precision met the criteria of validation and study sample analysis. The recovery was found to be 101.89%. Stability studies showed that nifedipine was stable after 12 h at room temperature and 21 days at -20 °C. No significant difference was examined between the pharmacokinetic parameters of nifedipine with or without Gynura procumbens leaf extract. The proposed method was helpful for the pharmacokinetic interaction study of nifedipine combined with herbal in rats.

DEVELOPMENT AND VALIDATION OF A NEW RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION AND QUANTIFICATION OF SOFOSBUVIR AND DACLATASVIR

Background: Sofosbuvir (SOF) and daclatasvir (DCV) are usually prescribed in combination for the treatment of hepatitis C worldwide. Objectives: In the present study a novel, simple, rapid, and cost-effective reversed-phase high performance liquid chromatography (HPLC) method was developed, optimised, and validated for simultaneous analysis of sofosbuvir and daclatasvir in human plasma as well as in solid dosage forms. Methodology: The chromatographic analyses were carried out using Shimadzu HPLC gradient system (LC20), while the other HPLC system used for the peak purity and repeated validation studies was Agilent 1260 Infinity fitted with diode array detector. The column used was C18 (150 × 4.6 mm; 5 µm), from Agela Technologies. The mobile phase consisted of acetonitrile and 0.02 M potassium Hexafluorophosphate buffer (42:58, v/v) at pH 2.7, pumped at the rate of 1.3 mL min−1. The limit of detection and limit of quantification were determined using dilution method (RSD/m formula). Results: SOF and DCV combination therapy is significant for the treatment of hepatitis C. Development of this RP-HPLC method for the simultaneous determination and quantification of SOF and DCV is a part of an extensive project of novel formulation development (fixed dose combination tablets of SOF and DCV). This method is successfully applied for raw materials as well as in combination tablets. This method may also be applied for the quantification of sofosbuvir and daclatasvir in blood samples of patients taking these two antivirals for hepatitis C treatment. Conclusion: The optimized method was successfully applied for the determination of sofosbuvir and daclatasvir in single run without any interference. Hence, the developed method is quite appropriate for the simultaneous determination of SOF and DCV in solid dosage form as well as in human plasma for clinical research studies.

Stability Indicating HPLC Method for In-vitro Determination of Pantoprazole Sodium and its Degradation Products in Simulated Gastric and Intestinal Fluids

Background: A high-performance liquid chromatography (HPLC) method was developed for the determination of Pantoprazole Sodium (PPZ) in the presence of its degradation products. The degradation of PPZ was studied in simulated intestinal fluid (SIF) and simulated gastric fluids (SGF) in various temperature conditions. Aim: This study aimed to establish a simple, sensitive, and rapid RP HPLC method for in-vitro determination of Pantoprazole Sodium and its degradation products in simulated gastric and intestinal fluids. Objective: Pantoprazole is acid labile drug. In order to determine pantoprazole in various oral dosage forms, the stability-indicating assay of PPZ was performed in phosphate buffer (pH 6.8) representing simulated intestinal fluid (SIF) and in 0.1 molars (M) Hydrochloric acid (HCl) as simulated gastric fluid (SGF) at two different temperature conditions, i.e., 25°C and 0°C, respectively. Method: Pantoprazole sodium was obtained from the Akums laboratory in Haridwar. The analysis was performed by high-performance liquid chromatography (HPLC), Shimadzu, equipped with two LC-10 AD VP solvent-delivery modules, a SPD-10A UV–-visible detector, and a manual injector valve with 20 μL sample loop. Phenomenex ODS analytical column (150 mm × 4.6 mm i.d., 5 μm particles) was done under reversed-phase partition chromatographic conditions. The mobile phase was phosphate buffer and acetonitrile (ACN) of pH 7.4, respectively, optimized in a 70:30 (v/v) ratio followed by filtration through a 0.45 μm membrane filter and degassed by ultrasonicator before use. The mobile phase was delivered at the flow rate of 2 mL/min. The various parameters, such as linearity, accuracy and precision of the analytical method, were studied. Result: The standard curve of PPZ was linear (R2>0.99) over the concentration range of 5-30 μg/mL, and the relative standard deviation (RSD) values for intra-day and inter-day variations were in the range of 1.0-1.8%. The range of RSD was within ±2. Conclusion: The stability of PPZ in aqueous solution was pH dependent. The rate of degradation increases with decreasing pH. The pH stability of pantoprazole was studied at the above-mentioned temperature conditions. The PPZ peaks were analyzed by comparing them with fresh samples and were stable in SIF solution after 24 hours elapsed time at pH 6.8. The obtained degraded peaks in SGF (pH 1) were successfully separated from the PPZ.

A Simple and Rapid High-Performance Liquid Chromatography Method for Preparation and Content Detection of the Mainly Numbing Taste Substances of Zanthoxylum bungeanum Maxim.

As the characteristic numbing taste substances, hydroxyl-α-sanshool (HAS) and hydroxyl-β-sanshool (HBS) were considered vital indicators to evaluate the quality of Zanthoxylum bungeanum Maxim. However, it is very difficult to obtain their high-purity monomers individually, as the only difference between HAS and HBS is that C-6 cis-trans isomerism. In our study, a simple and rapid Ag +-HPLC method was developed to pure the standard chemicals of Z. bungeanum with numbing taste, and 1H NMR and 13C NMR were employed to determine the purity and structure. Moreover, an HPLC method was established to determine the content of numbing taste components of 16 varieties of Z. bungeanum from different regions. The analytical methods were validated for accuracy, precision, and linearity, respectively. The validated method was accurate (spiked recoveries 0.94-1.10), precise in terms of peak area (intra-day RSDs <1.25% and inter-day RSDs <1.61%), and linear (r2 ≥ 0.999). It was found that there were significant differences in the content of HAS and HBS among different types of Z. bungeanum, with HAS content ranging from 60.06 ± 1.14 to 164.13 ± 3.28mg/g and HBS ranging from 7.81 ± 0.36 to 21.11 ± 0.75mg/g. The RSDs of HAS range were 1.73-3.80% and that of HBS range 2.03-4.73% (RSDs ≤5%), which indicated that the measurements of HAS and HBS were reliable.

Simultaneously measure the concentrations of busulfan and phenytoin in human plasma using an HPLC-MS/MS method: Application to the TDM for children underwent hematological stem cell transplantation

AbstractIn this work, a simple and rapid high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to carry out the simultaneous measurement of busulfan (BU) and phenytoin (PHT) in the plasma of children. In this method, plasma sample could be prepared by one-step protein precipitation using 1 mL of methanol/water (1:1, v/v). After centrifugation (14,500 rpm, 5 min, 4 °C), 10 μL of the supernatant was injected into a Hypersil Gold C18 column (150 × 2.1 mm, 5 μm, Thermo Fisher Scientific) for separation by gradient elution. Quantification was carried out using multiple reactions monitoring (MRM) under positive scan mode. In the method verification, the calibration curves of BU and PHT showed satisfactory linearity (r &gt; 0.99) at the concentration ranging from 0.02 to 20 μg mL−1. The accuracy and precision were tested at four concentration levels (including the LLOQ level) with the relative error (RE) ranging from −0.80% to 11.45% and coefficient of variation (CV) between 0.93% and 7.74%. There was no pronounced matrix effect to interfere with the quantitative analysis. Compared to determine BU and PHT using two individual methods, less pre-treatment process, labor and blood sample volume are required in this proposed method. Finally, this method was successfully applied to the therapeutic drug monitoring of BU and PHT for children underwent hematological stem cell transplantation.

Highly selective and sensitive high performance liquid chromatography method for determination of fumonisins from feed samples with MIL-101 based dispersed micro-solid-phase extraction and precolumn fluorescence derivatization

A selective and rapid high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for determining fumonisins was established and validated, which based on dispersed microsolid-phase extraction (D-μ-SPE) and precolumn derivatization. The MIL-101 was prepared as sorbents to selectively enrich two fumonisins from feed samples. The obtained extracts were labeled by a novel derivatization reagent 2,5-dioxopyrrolidin-1-yl 4-(1-methyl-1H-phenanthro[9,10-d]imidazol-2-yl)benzoate (MPIBA-Su). There was no fluorescence from fumonisins itself, while their derivatives exhibited considerable fluorescence with a maximum excitation at λex 255 nm and a maximum emission at λem 457 nm. Key factors affecting extraction efficiency and derivatization efficiency were fully optimized. Excellent linearity was achieved with correlation coefficients > 0.9935 under optimal conditions. The detection limits (LODs) were between 2.4 and 3.5 ng·mL−1, and the quantitation limits (LOQs) were between 8.1 and 11.7 ng·mL−1. With recoveries in the range of 83.8% − 94.3%, the established method has successfully been applied to the quantitative analysis of fumonisins in feed samples.

Determination of ATP-related compounds by HPLC to study the effect of cell-free supernatants of Lactiplantibacillus plantarum on the shelf life of sliced dry-cured ham

This research aimed to study the evolution of ATP-related compounds in dry-cured ham and to assess their suitability as chemical markers of food contamination. A rapid high-performance liquid chromatography (HPLC) method was developed for the analysis of five ATP-related compounds namely, ATP, ADP, AMP, hypoxanthine, and Ino in sliced dry-cured ham. Moreover, a positive correlation between ATP content and microbial load was found in ham during storage. In addition, the method developed herein was employed to quantify ATP-related compounds in sliced dry-cured ham wrapped in a novel antibacterial packaging film containing the cell-free supernatant (CFS) of a probiotic Lactiplantibacillus plantarum strain. The contents of ATP-related compounds in ham samples wrapped in the antibacterial packaging film were lower than in control samples during the 7 days of storage (181 vs 269 mg/kg on day 2; 247 vs 286 mg/kg on day 5; 272 vs 297 mg/kg on day 7). Thus, the evaluated CFS could be considered in packaging material applications for the preservation of sliced ham. This study presents an effective method to assess the quality of sliced ham, which might also contribute to the adoption of active packaging in the food industry.

Rapid high performance liquid chromatography method for erlotinib quantification in vitro: Application to study the effect of resveratrol on metabolism and cellular uptake of erlotinib.

Erlotinib is a selective epidermal growth factor receptor inhibitor that is used for the treatment of non-small cell lung cancer and pancreatic cancer. Its metabolism is mainly mediated by cytochrome P450 3A (CYP 3A). Resveratrol, a natural compound found in many plants and supplements, is known to inhibit CYP3A enzyme, therefore, it may act as an inhibitor for the metabolism of erlotinib. Development of a rapid high performance liquid chromatography with photodiode array detection (HPLC-PDA) method for the quantification of erlotinib in liver microsomes and cancer cells and its application to study resveratrol effect on metabolism and cellular uptake of erlotinib. HPLC-PDA was used to develop an efficient bioanalytical method with a 2.5-min runtime preceded by a simple protein precipitation step. The method was validated according to the European Medicines Agency guidelines. Erlotinib metabolic stability and resveratrol effect on erlotinib metabolite formation were evaluated in rat liver microsomes. Furthermore, the method was used to measure the intracellular concentrations of erlotinib in cancer colorectal cells and investigating resveratrol effect on the cellular uptake of erlotinib. A rapid HPLC-PDA method was developed and validated for the first time to address potential drug interaction of erlotinib with resveratrol. Resveratrol was a strong inhibitor of erlotinib metabolism in vitro with IC50 = 4.03 μM. Resveratrol, however, had no effect on erlotinib cellular uptake after 1h incubation in human colorectal cancer cells. The study suggests that resveratrol may produce a potential herb-drug interaction with erlotinib at the metabolism level and should be investigated in patients in the clinic.

Pharmacokinetics and Tissue Distribution Study of Daphnoretin in Ethanol Extract from the Roots of Wikstroemia Indica in Rats by a Validated UPLC-MS/MS Method

Background: Daphnoretin, as a known bicoumarin compound that contained various pharmacological activities, was isolated from Wikstroemia indica C.A. Mey (RWI). Objective: The study aims to investigate the pharmacokinetic characteristics of daphnoretin from RWI ethanol extracts in rat plasma and to determine daphnetin in rat plasma and various tissues by a rapid, reliable and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method. Methods: The UPLC-MS/MS method was established. Daphnoretin and IS (buspirone) were chromatographed on an agilent Zorbax XDB-C18 column (2.1 mm × 50 mm, 3.5 μm), and Gradient elution of acetonitrile-0.15% formic acid in aqueous solution. Quantification was performed using electrospray ionization in positive ion multiple reaction monitoring mode of the transitions m/z 353.1→179.1 for daphnoretin and m/z 386.3→122.3 for IS. Results: Good linearity between 5-10000 ng/mL for cyperidin in plasma and tissue samples (r ≥ 0.99) was resulted. The accuracies of plasma and tissue homogenates ranged from-3.31% to 9.00%, and the precision was less than 5.78%. After that, the validated method was successfully applied to the pharmacokinetics and tissue distribution study of daphnoretin after oral administration of ethanol extract from the roots of RWI to rats. Conclusion: Daphnoretin was well absorbed in the systemic circulation after oral administration and was widely distributed in tissues, with the highest concentration in lung tissue. This study is beneficial to the development and utilization of RWI and provides a reasonable reference for its clinical administration.

Development and Validation of a RP-HPLC Method for the determination of Venlafaxine hydrochloride in bulk and Pharmaceutical Dosage Forms

A simple, accurate, precise and rapid reverse phase high performance liquid chromatography method was developed and validated for the analysis of venlafaxine hydrochloride in bulk and marketed pharmaceutical dosage form. The chromatographic separation was carried out using a Phenomenex C18, (250mm × 4.6mm × 5µ) column. The optimum mobile phase consisted of ammonium acetate buffer, methanol (60:40) with pH adjusted to 5. The mobile phase was delivered isocratically at a flow rate of 1mL/min with UV detection at 227nm. Venlafaxine hydrochloride was eluted at 2 minutes. The method was validated for linearity, precision, accuracy, robustness and recovery. The method was linear in the concentration range of 15-165µg/ml with correlation coefficient 0.9997. limit of detection and limit and quantification were found to be 185ng/ml and 563ng/ml respectively.

HPLC-UV Method Development and Validation to Monitor Difluprednate Synthesis.

During the synthesis of active pharmaceutical ingredients (APIs) there is a need for the development and validation of a simple and rapid high performance liquid chromatography (HPLC) method for the determination and quantification of the synthesized product and related by-products. An HPLC method gives a better understanding of how a synthesis is proceeding. A rapid and easy to use HPLC-UV (ultraviolet) method for the determination of difluprednate and monitoring of impurities generated during synthesis was developed and validated. A Shimadzu VP Series HPLC equipped with a LabSolutions software and UV detector set at 240nm was used for analysis. The mobile phase consisted of phosphate buffer (pH6) and acetonitrile 50:50 (v/v) and was eluted at a flow rate of 1.2mL/min. Separation took place on a reversed-phase Kinetex C18 column (150×4.60mm; 5μm i.d.). Column temperature was set at 40°C. The developed method was found to have good linearity and acceptable accuracy and precision. The developed method may be effectively applied to determine products and by-products formed during synthetic reactions of steroids and to calculate the yield of the products obtained during each step of the synthesis.

A Validated RP-HPLC Method Development and Validation for The Simultaneous Estimation of Nebivolol and Valsartan in Bulk Drug and Pharmaceutical Dosage Form

A novel, simple, accurate and rapid Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed and validated for the determination of Nebivolol and Valsartan in bulk and tablet dosage form. Gradient elution chromatography has been developed on C18 column (5µm, 4.6mm* 250mm) with a mobile phase consist of pH 4 acetonitrile,water and Methanol (25:35:40) with a flow rate of 1ml/min at detection of 270 nm wavelength. The retention time for Nebivolol and Valsartan was found to be 4.59 and 10.2 respectively. Chromatography parameters were validated as per ICH guidelines and the method can be applicable for routine quantitative analysis of drugs in combined dosage form.

Determination of Apigenin in Cosmetics Containing Chamomile by High-Performance Liquid Chromatography with Ultraviolet Detection (HPLC-UV)

A simple, rapid, and precise high performance liquid chromatography (HPLC) method was developed to determine the flavonoid apigenin in cosmetic products containing chamomile extracts. A C18 column was used as stationary phase and 70:30 ethanol:water was used as mobile phase with a 1 mL/min flow rate and isocratic elution. The temperature was stabilized at 25 °C during the separation. The injection volume was 50 μL. The apigenin peak was eluted at 4.15 ± 0.4 min. The method was validated according to International Conference on Harmonisation (ICH) criteria in terms of linearity, limit of detection, limit of quantitation, selectivity, sensitivity, robustness, accuracy, and precision. The limits of detection and quantitation were 0.060 and 0.2 μg/mL respectively. The linear range was from 0.2 to 20 μg/mL. The relative standard deviation values for intraday and interday analyses were less than 1.64%. The developed procedure was applied to the analysis of various cosmetic products. It is expected that the method is suitable for routine analysis, quality control and standardization of cosmetic products for apigenin.

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Haloperidol and Trihexyphenidyl Hydrochloride in Tablet Dosage Form

Haloperidol is a common antipsychotic drug used to treat schizophrenia, and Trihexyphenidyl hydrochloride is an antiparkinsonian medication. The combination formulation is used to treat schizophrenia as well as depression. A simple, accurate, and precise assay as well as a rapid stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Haloperidol (HAL) and Trihexyphenidyl hydrochloride (THP) from their combination drug product have been developed and validated. The proposed method is based on reversed-phase separation of the two drugs using a Zorbax Eclipse XDB C18 column (150 mm x 4.6 mm, 5 um) kept at 30°C. The optimal mobile phase was composed of 0.5 mL triethylamine 0.05 M phosphate buffer having pH 3.75: acetonitrile: (65:35 v/v), a flow rate of 1.2 mL.min-1, and PDA detection at 210 nm. The method was validated in accordance with ICH standards. It was found to be precise and reproducible. Linearity was achieved in the concentration ranges of 6-60 ug mL-1 for HAL and 8-80 ug mL-1 for THP, with correlation coefficients of 0.999 and 1.000, respectively. The mean percent recovery of triplicate samples at each level for both drugs was 99.10-100.7% for Haloperidol and 98.7-100.6% for Trihexyphenidyl hydrochloride. The proposed method can be used to successfully test quality control and stability samples from bulk manufacturing and pharmaceutical dosage forms.

Simple and Rapid High-Performance Liquid Chromatography Method for Simultaneous Determination of Picloram and 2,4-D in Pesticide Formulations

Picloram and 2,4-D are systemic herbicides used to control a wide range of broad-leaved weeds and post-emergence annual and perennial broad-leaved weeds, respectively. The use of plant protection products containing Picloram and 2,4-D necessitates the development of analytical methods, useful for the laboratories focused on control, for monitoring. In this study, we designed and validated an analytical method for the rapid determination of picloram and 2,4-D by HPLC-DAD. The method involves the extraction of the substances by sonication of the sample with methanol, followed by dilution in acetonitrile, and direct injection on a liquid chromatography system, based on the use of a Gemini C18 column. We used an isocratic mobile elution consisting of acetonitrile and water acidified 1% with H3PO4 (50:50, v/v). We validated the proposed method, which demonstrated linearity within the concentration range of 0.01–0.028 mg/mL for picloram and 2,4-D, with a correlation coefficient (R2) of 0.9993 for picloram and 0.9999 for 2,4-D. We considered precision, repeatability and selectivity in the validation. The repeatability of the method expressed as percent of relative standard deviation (%RSD) was lower than 1% for both substances. The proposed method is suitable for the simultaneous determination of picloram and 2,4-D in pesticide formulations.

Method Development and Validation for the estimation of Amlodipine and Perindopril in Bulk and Pharmaceutical Dosage Form

Literature survey reveals that few spectrophotometric, high performance liquid chromatographic (HPLC)and High performance thin layer chromatographic (HPTLC)methods were reported for the estimation of Amlodipine and perindopril in bulk and combined pharmaceutical dosage forms. But there is no any stability indicating high performance liquid chromatography study is reported for amlodipine. So the present study was aimed to development of simple, accurate, rapid and economical HPLC stability indicating assay method were established for the determination of Amlodipine and Peridopril in bulk and tablet dosage form. A High-performance liquid chromatograph WATERS, software: Empower 2,2695 separation module, 996 PDA detector, using Phenomenex Luna C18 (4.6mm×250mm) 5µm or equivalent column, with mobile phase composition of Methanol: Phosphate Buffer pH3.0 (70:30v/v) was used. The flow rate of 1.0ml min-1 and effluent was detected at 230nm. The retention time of Amlodipine and Perindopril was found to be 1.870min and 2.499minutes respectively. Linearity was observed over concentration range of 10-50µg ml-1 for Amlodipine and 16-80µg ml-1 for Perindopril respectively. The accuracy of the proposed method was determined by recovery studies and the Amlodipine was found to be 99.1% and Perindopril was found to be 98.8% respectively. The proposed method is applicable to routine analysis of Amlodipine and Perindopril in bulk and pharmaceutical formulations. The proposed method was validated for various ICH parameters like linearity, limit of detection, limits of quantification, accuracy, precision, range and specificity.

Bioanalytical RP-HPLC method development and validation for estimation of azelnidipine and Olmesartan medoxomil in human plasma

An accurate, rapid and simple reversed-phase high performance liquid chromatography (RP-HPLC) bioanalytical method was developed for estimation of Azelnidipine (AZP) and Olmesartan medoxomil (OLM)in human plasma. The AZP, OLM and internal standard (IS) were extracted by liquid-liquid extraction technique. Chromatographic separation was accomplished using BDS Hypersil C18, 250 mm X 4.6 mm, 5µ analytical column. The mobile phase consisted of Acetonitrile: Water, pH adjusted with ortho-phosphoric acid in the ratio 60:40 with flow rate of 1ml/min. Detection and quantification were performed by UV detector at 256 nm. The retention times were found to be 10.05±0.02, 4.3±0.02 and 6.9±0.02 for AZP, OLM and IS respectively. The linearity range was found to be 0.5 to 12 µg /ml and 1 to 15 µg /ml for AZP and OLM respectively.The method was validated as per CDER guideline, was found to be suitable for analysis in biological fluid.