Photodiode Array Detector Set Research Articles

Liquid chromatographic determination of lumacaftor in the presence of ivacaftor and identification of five novel degradation products using high-performance liquid chromatography ion trap time-of-flight mass spectrometry.

Lumacaftor is a transmembrane conductance regulator potentiator drug, prescribed for the treatment of cystic fibrosis in patients who are homozygous for the F508del mutation. Quantitation of lumacaftor besides its degradation products and ivacaftor was achieved on a fused-core silica particle column packed with pentafluorophenylpropyl stationary phase (Ascentis Express F5, 2.7μm particle size 100mm × 4.6mm; Supelco) using gradient elution (A: 0.1% [v/v] formic acid in water, B: 0.1% [v/v] formic acid in acetonitrile [the mobile phase pH 2.5]). A constant flow rate at 1mL/min was applied, and the detection was realized using a photodiode array detector set at 216nm. The pseudo tablet formulation of the lumacaftor/ivacaftor fixed-dose combination preparation, namely, Orkambi®, was prepared in vitro and used for the analytical performance validation and method application studies. In addition, five novel degradation products, four of which even have no Chemical Abstracts Services registry number, were identified using high-resolution mass spectrometry instrument, and their possible mechanisms of formation were proposed. According to current literature, this paper can be regarded as the most comprehensive liquid chromatographic study on lumacaftor determination, among its counterparts.

Quality by design tool-evaluated stability-indicating ultra-performance liquid chromatography method for the determination of drugs (ritonavir and darunavir) used to treat the human immunodeficiency virus/acquired immunodeficiency syndrome.

Ritonavir and darunavir were examined using a ultra-performance liquid chromatography (UPLC) approach in pharmaceutical dosage forms. The small number of analytical studies that are currently available do not demonstrate the method's stability or nature. The study sought to assess both chemicals using a stability-indicating approach with a relatively short run time. The HSS C18 (100 × 2.1 mm), 2-mm column was used for the chromatographic separation, and isocratic elution was used to achieve this. In the mobile phase, methanol and 0.01 M phosphate buffer (pH 4.0) were included in a 60:40 (v/v) ratio. Throughout the analysis, the flow rate was kept at 0.2 mL min-1 , and a photodiode array detector set to 266 nm was used to find the major components. The proposed method showed a linear response (r2 > 0.999), and the accuracy was between 98.0% and 102.0%. The precision data showed relative standard deviation ≤1.0%. The UPLC method for quantification of ritonavir and darunavir in pharmaceutical dosage forms using a very short run time of under a minute is the subject of the proposed article. To meet current regulatory criteria, the quality by design idea was used in the method performance verification.

Validated HPLC-UV method for quantification of paxalisib, a pan PI3K and mTOR inhibitor in mouse plasma: Application to a pharmacokinetic study in mice.

Paxalisib is a pan-PI3K and mTOR inhibitor, currently entering into Phase II clinical trials as a potential drug to treat glioblastoma patients. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of paxalisib in mouse plasma as per the US Food and Drug Administration regulatory guidelines. From the mouse plasma, paxalisib and the internal standard (IS; filgotinib) were extracted using ethyl acetate as an extraction solvent. The chromatographic separation of paxalisib and the IS was accomplished on a Symmetry C18 (250×4.6mm, 5.0μm) column maintained at 40°C using 10mm ammonium formate and acetonitrile in gradient conditions at a 0.8ml/min flow-rate. The injection volume was 20 μl. The elution was monitored using a photo-diode array detector set at λmax 280 nm. Paxalisib and the IS eluted at 6.5 and 5.9min, respectively with a total run time of 10min. The calibration curve was linear over the range of 111-4,989 ng/ml. Inter- and intraday precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated and the results met the acceptance criteria. The validated HPLC method was extended to assess the pharmacokinetic parameters of paxalisib in mice.

Extended stability study of an extemporaneously analgesic solution of clonidine, ropivacaine and fentanyl

The aim of this work was to perform an extended stability study for the analgesic containing fentanyl, clonidine and ropivacaine in physiological saline solution 0.9% at different infusion sites, such as infusion bags, epidural infusion sets and syringes. The extended stability was assessed by an HPLC system equipped with a photodiode array detector set at 210 nm. The separation was conducted on a C18 column maintained at 40˚C and using an isocratic mobile phase consisted of buffer solution-methanol-acetonitrile (45:45:10, v/v/v). The presence of particulate matter and the pH of each solution were also investigated. Twenty-four hours after the preparation, the formation of one suspected product was observed and for all drugs, in 24 hours it was observed the concentration decrease in different sets (PVC infusion bags, syringes and epidural infusion administration sets). The pH values of each solution varied no more than 5% during the study and no particle was observed. Conclusion: The extended stability study was applied to the analgesic solution and promoted the detection of an unexpected peak in 24 hours. Based on it, further stability studies are necessary to determine the extended stability data.

Quality by Design Approach to Develop Stability Indicating Method to Quantify Related Substances and Degradation Products of Sacubitril by High Performance Liquid Chromatography.

Sacubitril (SBT) is a neprilysin inhibitor, approved by food and drug administration (FDA) in 2015, under the FDA's priority review process. In this work, we report the validated stability indicating method of SBT by employing quality by design (QbD) principles related to analytical method development, capable in separation of 11 impurities. Chromatographic separation was performed on an ascentis phenyl hexyl column using 10mM KH2PO4 as a mobile phase-A and the pH adjusted to 2.1. Methanol: acetonitrile (70:30v/v) solvent mixture was employed as the mobile phase-B in a gradient mode of elution at a flow rate 0.8mL/min at 30°C. The column effluents were monitored by a photo diode array detector set at a wavelength of maximum absorption 254nm noted for all the impurities and furthermore for SBT. This method was remarked to be accurate in the range from 92 to 116%, precise with relative standard deviation 0.9% for SBT (0.8mg/mL) and 1.0 to 2.1% for its related impurities (0.0005mg/mL) also linear with correlation coefficient r≥0.9989. The limits of quantification for all impurities were 0.05% with respect to sample concentration 0.8mg/mL. The developed method revealed a good method operable design range for the experimental chromatographic conditions. Forced degradation of SBT carried under acidic, basic and oxidative stressed conditions manifested that the method is stability indicating.

A stability-indicating UPLC method for the determination of curcumin diethyl disuccinate, an ester prodrug of curcumin, in raw materials

A stability-indicating reversed-phase ultra-performance liquid chromatographic (UPLC) method for quantitative analysis of curcumin diethyl disuccinate (CDD) in raw materials was developed for applications in product development and quality control. Chromatographic separation was performed using the Waters ACQUITY UPLC® H-Class system consisting of an ACQUITY UPLC® BEH C18 (1.7 μm, 2.1 × 50 mm) column and a photodiode array detector set at a wavelength of 400 nm. The mobile phase consisting of 2%v/v acetic acid in water and acetonitrile was delivered at a flow rate of 0.3 mL/min under gradient elution program. The method was validated according to the ICH Q2(R1) guideline for the validation of analytical procedures. The method was found to be linear over the concentration range of 8–12 μg/mL with the coefficient of determination >0.995. The accuracy of the method established as %recovery ranged from 98.3 – 100.8%. The precision of the method expressed as %CV was found to be <1%. The coelution of degradation products from six stress test conditions was not observed. The method was robust under the variation of chromatographic parameters. The method was successfully applied in the determination of CDD content in raw materials.

A Novel HPLC Method for Simultaneous Determination of Methyl, Ethyl, n-propyl, Isopropyl, n-butyl, Isobutyl and Benzyl Paraben in Pharmaceuticals and Cosmetics.

The alkyl esters of p-hydroxybenzoic acid at the C-4 position, "the parabens," including methyl, ethyl, propyl, and butyl, are widely used as antimicrobial preservatives in foods, cosmetics, and pharmaceuticals. Official regulations on the use of these compounds make their analysis essential for the estimation of their exposure. On this basis, the presented study was realized to develop a simple, selective and cheap high-performance liquid chromatographic method for the quantitative determination of methylparaben, ethylparaben (EP), n-propyl paraben (NPP), isopropyl paraben (IPP), n-butyl paraben (NBP), isobutyl paraben (IBP) and benzyl paraben (BP) in pharmaceuticals and cosmetic products. The chromatographic separation of the analytes was achieved under flow rate gradient elution conditions using a C18-bonded core-shell silica particle column (2.6 μm particle size, 150 × 3.0 mm from Phenomenex Co.). The samples were injected into the system as aliquots of 1.0 μL, and the compounds were detected by using a photodiode array detector set at 254 nm wavelength. With this technique, seven paraben derivatives can be determined in the concentration range of 250-2000 ng/mL. The recovery of the method is in the range of 99.95-13.84%, and the RSD is at a maximum value of 3.95%. The proposed method was fully validated and successfully applied to different pharmaceutical and cosmetic samples (n=16), including syrups, suspensions, oral sprays, gels, etc. At least one paraben derivative was detected in six samples and was determined quantitatively. The maximum amount of a paraben derivative found in the analyzed samples was 321.7 ng/mL, which was MP. To the best of our knowledge, this is the first LC method, which is applicable both on pharmaceutical and cosmetic samples.

Stability Indicating Method to Analyze Benidipine and Chlorthalidone Using HPLC Technique: Establishment, Validation and Application to Tablets

Background : The combination of chlorthalidone and benidipine was used to manage hypertension. The mixture of chlorthalidone and benidipine in tablet dosage form has not been previously determined by any method. A stability indicating HPLC method was developed for the simultaneous determination of benidipine and chlorthalidone in bulk and tablets. Methods: Chromatographic separation was accomplished in a reverse phase system using an isocratic elution with a mobile phase composed of methanol-0.1M dipotassium hydrogen phosphate buffer (40:60, v/v), at 1 ml/min flow rate. The photodiode array (PDA) detector set at 260 nm was used to detect and quantify benidipine and chlorthalidone. Benidipine and chlorthalidone tablet samples were subjected to degradation under acid, neutral, alkali, thermal, photo and oxidative. The proposed method was effectively adapted to quantify benidipine and chlorthalidone in the combined tablet formulation. Results: The elution times for benidipine and chlorthalidone were approximately 4.573 min and 6.422 min, respectively. The method was validated within a concentration range of 2 - 6 μg/ml (R2 = 0.9997) for benidipine and 6.25 - 18.75 μg/ml (R2 = 0.9998) for chlorthalidone. Adequate results were obtained for precision (RSD% = 0.106% for benidipine and RSD% = 0.031% for chlorthalidone) and accuracy (99.95 - 100.25 % mean recovery for benidipine and 99.60 - 99.63% mean recovery for chlorthalidone). Robustness has also been found to be acceptable. During the degradation study, interference was not noticed in the analysis of studied drugs. Conclusion: The findings demonstrated that the method could be useful for determination of the selected drug combination in routine analysis.

A Simple and Sensitive High Performance Liquid Chromatographic Method for Estimation of Transitmycin in Plasma

A high performance liquid chromatographic method for estimation of Transitmycin (Tr) in human plasma was developed. The analyte was extracted using solid phase cartridges and the analysis of the eluent was carried out using Atlantis T3 column (150 cm × 4.6 mm ID) and photo diode array detector set at wavelength of 214 nm. The assay was specific for Tr and linear from 0.5 µg/mL to 20.0 µg/mL. The relative standard deviations for intra- and inter-day assays were less than 10%. The method yielded a recovery for Tr that ranged from 94% to 107% and could be used in toxicology and pharmacokinetic studies.

Development and Validation of a Stability indicating Related Substances of Baricitinib by RP-HPLC and its Degradation

Reverse phase high performance liquid chromatography method, for estimation of related substances or chromatographic impurities of Barcitinib was developed and validated. Baricitinib was developed by separating its degradation products on a X-Terra RP18 (150x4.6mm, 5.0 µm) column using 0.1% Tri ethyl amine in water adjusted pH-2.5 with OPA and Acetonitrile in simple gradient at a flow rate 1.0 ml/min. The column effluents were monitored by a photodiode array detector set at 224nm. The method was validated in terms of specificity, linearity, accuracy, precision, detection limit, quantification limit and robustness. Forced degradation of Baricitinib was carried out under acidic, basic, peroxide, reduction, thermal, photo and hydrolysis conditions. The proposed method is validated as per ICH Q2 (R1) guidelines. The proposed method is simple as selected chromatographic conditions are not so difficult to apply in routine analysis for testing the chromatographic impurity of baricitinib.

SIMULTANEOUS QUANTIFICATION OF PENTAZOCINE AND NALOXONE BY STABILITY INDICATING REVERSE-PHASE-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD

Objective: The objective of the study was to develope a stability indicating high-performance liquid chromatographic (HPLC) method for simultaneous assay of pentazocine and naloxone in bulk and tablets.&#x0D; Methods: Pentazocine and naloxone were analyzed on Dionex C18 column using 0.1M K2HPO4 buffer (pH 4.0) and methanol (60:40, v/v) as the mobile phase. The concentration of pentazocine and naloxone was quantified by photodiode array detector set at 248 nm. The method was validated in compliance with ICH rules. Pentazocine and naloxone tablet formulation was subjected to forced degradation such as acid, neutral and alkali hydrolysis, oxidation, photo, and thermal degradation.&#x0D; Results: The method was linear, with R2=0.9999 in the concentration range 100–300 μg/ml for pentazocine and R2=0.9995 in the concentration range 1–3 μg/ml for naloxone. The level of detection and quantification was 0.097 μg/ml and 0.322 μg/ml for pentazocine and 0.0073 μg/ml and 0.0243 μg/ml for naloxone, respectively. The degraded products are resolved well from pentazocine and naloxone with significantly different retention time values. From validation results, it was proved that the method is selective, precise, robust, and accurate for the estimation of pentazocine and naloxone simultaneously.&#x0D; Conclusion: The developed stability-indicating HPLC method can be applied for quantitative determination of pentazocine and naloxone in tablets.

EFFECT OF DIFFERENT ANTICOAGULANTS IN DETERMINING METFORMIN HYDROCHLORIDE LEVELS IN HUMAN PLASMA USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: This study aimed to develop and validate an analytical method and to determine the effect of different anticoagulants for analyzingmetformin HCl level in human plasma.Methods: Analysis was performed with high-performance liquid chromatography method using a photodiode array detector set at 234 nm, a C18SunFire™ column (5 μm, 250 mm×4.6 mm), a temperature of 40°C, a mobile phase comprising 10 mM sodium dodecyl sulfate and 10 mM phosphatebuffer in water-acetonitrile (60:40 v/v) at pH 7.0, with a flow rate of 1.0 mL/min, and atorvastatin calcium as the internal standard. Plasmaextraction was performed through protein precipitation using human plasma (300 μL) and acetonitrile (600 μL; 1:2 v/v). The method was linear at aconcentration range of 20.0–5000.0 ng/mL, with r&gt;0.9998.Results: The stability and recovery of metformin HCl in human plasma were not different between citrate with ethylenediaminetetraacetic acid(EDTA) and heparin with EDTA as anticoagulants. However, a significant difference in the peak area response ratio (p&lt;0.05) was observed betweenthe three anticoagulants.Conclusion: The validation results for the three types of plasma anticoagulants met validation requirements based on the European MedicinesAgency bioanalytical guidelines of 2011 for accuracy and precision, selectivity, calibration curve linearity, dilution integrity, carry-over, and stability.

INCURRED SAMPLE STABILITY OF METFORMIN HYDROCHLORIDE IN PLASMA OF SIX HEALTHY SUBJECTS USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: This study aimed to measure metformin levels and evaluates the incurred stability in plasma of six healthy subjects after administration850 mg metformin hydrochloride tablet using high-performance liquid chromatography.Methods: Samples were collected from six healthy subjects who were administered 850 mg metformin hydrochloride tablet. Blood was collected fromthe subjects at up to 12-time points throughout a 12 h period. Sample stability was analyzed on days 7, 14, and 30. The analysis was conducted usingC-18 columns, a column temperature of 40°C, a mobile phase consisting of acetonitrile-phosphate buffer (pH 7.0; 40:60 v/v), a flow rate of 1 mL/min,the photodiode array detector set at a wavelength of 234 nm, and calcium atorvastatin as the internal standard.Results: All metformin HCl samples collected on days 7, 14, and 30 from subjects 1 to 6 met stability requirement based on the European MedicinesAgency Bioanalytical Guideline (2011), which state that the percentage difference value not &gt;20%. In addition, the developed method for analyzingmetformin HCl in plasma was linear in the concentration range of 20.0–5000.0 ng/mL (r=0.9999).Conclusion: The incurred sample of metformin hydrochloride in plasma of six healthy subjects was stable until 30 days after drug administration.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING REVERSE-PHASE HIGHPERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS QUANTIFICATION OF SAQUINAVIR, RITONAVIR, AND AMPRENAVIR

Objective: The objective of the study was to develop and validate a sensitive, precise, and accurate stability indicating reverse-phase (RP) high-performance liquid chromatography method for the quantification of saquinavir, ritonavir, and amprenavir simultaneously.Methods: The determination of saquinavir, ritonavir, and amprenavir in their mixtures was done using a mobile phase consisted of 0.1M phosphate buffer (pH 3.5) and methanol (70:30, v/v). The method is based on the simultaneous separation of studied drugs in a RP Inertsil ODS C18 (4.6 mm×100 mm, 5 μm) column at ambient temperature. Detection and quantitation were achieved with photodiode array detector set at 260 nm.Results: Saquinavir, ritonavir, and amprenavir showed linearity over a concentration range of 40–200 μg/ml (R2-0.9994), 20–100 μg/ml (R2-0.9992), and 30–150 μg/ml (R2-0.9990), respectively. The limit of quantification was 0.64 μg/ml, 0.57 μg/ml, and 0.53 μg/ml for saquinavir, ritonavir, and amprenavir, respectively. The accuracies for the three drugs were in the range of 99.40–100.53% (saquinavir), 99.45-100.47% (ritonavir), and 100.03–100.53% (amprenavir). The percentage relative standard deviations for the studied drugs were 0.785–0.848% (saquinavir), 0.338–0.499% (ritonavir), and 0.336–0.775% (amprenavir). No peaks were observed at the retention time of saquinavir, ritonavir, and amprenavir in placebo blank, mobile phase blank and stress degraded samples which suggested that the proposed was selective and specific.Conclusion: The method was found to be suitable for the regular analysis of saquinavir, ritonavir, and amprenavir simultaneously in the presence of their stress degradation products.Objective: The objective of the study was to develop and validate a sensitive, precise, and accurate stability indicating reverse-phase (RP) high-performance liquid chromatography method for the quantification of saquinavir, ritonavir, and amprenavir simultaneously. Methods: The determination of saquinavir, ritonavir, and amprenavir in their mixtures was done using a mobile phase consisted of 0.1M phosphate buffer (pH 3.5) and methanol (70:30, v/v). The method is based on the simultaneous separation of studied drugs in a RP Inertsil ODS C18 (4.6 mm×100 mm, 5 μm) column at ambient temperature. Detection and quantitation were achieved with photodiode array detector set at 260 nm. Results: Saquinavir, ritonavir, and amprenavir showed linearity over a concentration range of 40–200 μg/ml (R2-0.9994), 20–100 μg/ml (R2-0.9992), and 30–150 μg/ml (R2-0.9990), respectively. The limit of quantification was 0.64 μg/ml, 0.57 μg/ml, and 0.53 μg/ml for saquinavir, ritonavir, and amprenavir, respectively. The accuracies for the three drugs were in the range of 99.40–100.53% (saquinavir), 99.45-100.47% (ritonavir), and 100.03–100.53% (amprenavir). The percentage relative standard deviations for the studied drugs were 0.785–0.848% (saquinavir), 0.338–0.499% (ritonavir), and 0.336–0.775% (amprenavir). No peaks were observed at the retention time of saquinavir, ritonavir, and amprenavir in placebo blank, mobile phase blank and stress degraded samples which suggested that the proposed was selective and specific. Conclusion: The method was found to be suitable for the regular analysis of saquinavir, ritonavir, and amprenavir simultaneously in the presence of their stress degradation products.

Midazolam Hydroxylation Assay as a Marker for Endogenous CYP3A4‐mediated Metabolism: Metabolic Interaction with Natural Health Supplements

BackgroundEndogenous substances (endobiotics) such as vitamin D3 and estrogen are metabolized by cytochrome P450 3A4 (CYP3A4). However, measurement of endobiotic metabolites is often technology‐limited and expensive. Because of structural similarity of midazolam with vitamin D3, midazolam metabolic profile could present valuable information in identifying potential interaction of drugs or natural health supplements (NHS) with endogenous substances. Polyphenols (quercetin, raspberry ketone, gallic acid, rutin), furanocoumarin (imperatorin) and terpenoids (ursolic acid) are the biologically active human dietary constituents from grapes, berry fruits, pomegranate, and olive oil, but are also widely ingested as over‐the‐counter natural health supplements (NHS) in United States. NHS have the ability to inhibit CYP3A4‐mediated metabolism, possibly causing the plasma concentrations of the midazolam as well as vitamin D3 to increase. The objective of the present study was to develop a simple and effective metabolism assay to measure CYP3A4‐mediated midazolam hydroxylation and to screen certain NHS (polyphenols, furanocoumarin, terpenoids) for their potential to cause CYP3A4 related herb‐endobiotic interactions in humans.MethodsThe hydroxylation of midazolam to 1′‐hydroxymidazolam assay was developed and optimized. Reaction mixtures containing potassium phosphate buffer (pH 7.4), recombinant CYP3A4 protein (30 pmol/ml), NADPH‐regenerating system, and midazolam (5 uM or 2 uM) were incubated for 15 min. Substrate, metabolites and internal standard were analyzed with a reverse phase assay by a Shimadzu Prominence high‐performance liquid chromatography (HPLC) coupled to a photodiode array detector set at 220 nm using a C18 column. SigmaPlot enzyme kinetics module (version 13; Systat Software, Inc., San Jose, CA) and GraphPad Prism software (GraphPad Software Inc., San Diego, CA) were used to determine the kinetics parameters and inhibitory constants, respectively.ResultsThe Km and Vmax values for rCYP3A4‐mediated 1′‐hydroxymidazolam formation were 1.9±0.3 uM and 75.4±3.7 pmol/min/mg, respectively. Imperatorin inhibited 1′‐hydroxymidazolam formation to undetectable levels at both 2 uM and 5 uM concentrations of midazolam, whereas quercetin inhibited the formation by 31–44%. The IC50 values of imperatorin for 1′‐hydroxymidazolam and 4‐hydroxymidazolam were 7.7 uM and 2.6 uM, respectively. Ketoconazole, a known potent inhibitor of CYP3A4, yielded IC50 concentrations of 0.16–0.20 uM for midazolam hydroxylation.ConclusionsA midazolam hydroxylation assay was developed to screen potential metabolic interactions. Imperatorin was identified as an inhibitor of rCYP3A4. Quercetin showed low rCYP3A4 inhibition potential. This research further shows that NHS studied here can potentially inhibit one of the most ubiquitous enzymes responsible for inactivation of endobiotics and may provide additional health benefits. Vitamin D3 is metabolized by CYP3A4 and midazolam could potentially be used as a marker for interaction of commonly used NHS with vitamin D3. The cost‐effective HPLC method developed in this study provides a useful tool in predicting NHS‐endobiotic interactions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DETERMINATION OF PINAVERIUM BROMIDE IN PHARMACEUTICAL DOSAGE FORMS BY A VALIDATED STABILITY-INDICATING LC METHOD

A reversed-phase liquid chromatography (LC) method is validated for the determination of pinaverium bromide (PB) in pharmaceutical dosage forms. The LC method is carried out on a reversed-phase monolithic C18 column (100 x 4.6 mm i.d.), maintained at 45 ºC. The mobile phase consisted of acetonitrile and a solution of triethylamine 0.3% pH 5.0 (50:50; v/v), run at a flow rate of 2.0 mL/min, with photodiode array detector set at 213 nm. The chromatographic separation is obtained with PB retention time of 3.4 min, and it is linear in the range of 5-100 μg/mL (r2 = 0.9991). The limits of detection and quantitation are 1.41 and 4.70 μg/mL, respectively. The specificity and stability-indicating capability of the method are proven through degradation studies, which also showed that there is no interference of the formulation excipients, showing that peak is free from any coeluting peak. The method showed adequate precision, with a relative standard deviation values lower than 1.38%. Excellent values of accuracy were also obtained, with a mean value of 100.68%. Experimental design is used during validation to calculate and prove method robustness. The proposed LC method is applied for the analysis of the PB pharmaceutical dosage forms, contributing to improve the quality control and to assure the therapeutic efficacy.

Fast and parallel determination of PCB 77 and PCB 180 in plasma using ultra performance liquid chromatography with diode array detection: A pharmacokinetic study in Swiss albino mouse.

A simple, sensitive and reproducible ultra-performance liquid chromatography (UPLC) method has been developed and validated for simultaneous estimation of polychlorinated biphenyl (PCB) 77 and PCB 180 in mouse plasma. The sample preparation was performed by simple liquid-liquid extraction technique. The analytes were chromatographed on a Waters Acquity H class UPLC system using isocratic mobile phase conditions at a flow rate of 0.3 mL/min and Acquity UPLC BEH shield RP18 column maintained at 35°C. Quantification was performed on a photodiode array detector set at 215 nm and PCB 101 was used as internal standard (IS). PCB 77, PCB 180, and IS retention times were 2.6, 4.7 and 2.8 min, respectively, and the total run time was 6 min. The method was validated for specificity, selectivity, recovery, linearity, accuracy, precision and sample stability. The calibration curve was linear over the concentration range 10-3000 ng/mL for PCB 77 and PCB 180. Intra- and inter-day precisions for PCBs 77 and 180 were found to be good with CV <4.64%, and the accuracy ranged from 98.90 to 102.33% in mouse plasma. The validated UPLC method was successfully applied to the pharmacokinetic study of PCBs 77 and 180 in mouse plasma.

Stress testing of linaclotide: Development of a validated stability-indicating RP-HPLC method

Linaclotide, a first-in-class guanylate cyclase-C agonist, was recently approved by US Food and Drug Administration (FDA) as a promising pharmacotherapy for the management of constipation-predominant irritable bowel syndrome (IBS). In this communication, we present a novel stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of linaclotide along with its degradation products. During the International Conference on Harmonization (ICH) prescribed stress study, linaclotide was found susceptible to degrade under hydrolytic (acid and base) and oxidative (peroxide) conditions. The separation of the degradants from the analyte was achieved on a Zorbax Eclipse XDB C8 Column (250 mm × 4.6 mm, 5 μm) using 0.01 N potassium dihydrogen orthophosphate buffer and acetonitrile (80:20 v/v) as mobile phase at a flow rate of 1.00 mL min−1 at column temperature of 40 °C. The detection of the column effluents was realized on a photodiode array detector set at 220 nm. Under the above optimal condition, the method was validated with respect to specificity, linearity, range, precision, robustness, and sensitivity in compliance to the regulatory requirements.

Development and Validation of Stability Indicating HPLC Method for Gefitinib and Its Related Compounds and Characterisation of Degradation Impurities

A validated stability indicating RP-HPLC was developed for the estimation of Gefitinib related compounds as well as degradants on Inertsil C8 (250 × 4.6 mm, 5 μ) column using 50 mM aqueous ammonium acetate: acetonitrile as the mobile phase in a gradient mode of elution at a flow rate of 1.0 mL/min at 50°C. The column effluents were monitored by a photo diode array detector set at 300 nm. The method was validated in terms of accuracy, precision and linearity as per the ICH guidelines. The limits of quantification of Gefitinib and impurities were obtained in the range of 0.015-0.05%. The forced degradation of Gefitinib was carried out under acidic, basic, thermal, reduction and oxidation conditions. The degradation products were characterized by MS-MS and 1 H NMR spectroscopy. The method was successfully applied to quantify the related substances and degradation products of Gefitinib in bulk drugs. The recoveries of Gefitinib and impurities were well within the range.

Evaluation of recombinant human parathyroid hormone by CZE method and its correlation with in vitro bioassay and LC methods

A stability-indicating capillary zone electrophoresis (CZE) method was validated to assess the content/potency of the recombinant human parathyroid hormone (rhPTH 1-34), using ranitidine as internal standard (IS). A fused-silica capillary, (i.d. of 50µm; effective length of 40cm) was used at 25°C; the applied voltage was 20kV. The background electrolyte solution consisted of 50mmolL−1 sodium dihydrogen phosphate solution at pH 3.0. Injections were performed using a pressure mode at 50 mbar for 45s, with detection by photodiode array (PDA) detector set at 200nm. Separation was obtained with a migration time of 5.3min, and was linear over the concentration range of 0.25–250µgmL−1 (r2 =0.9992). Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.28% with bias lower than 0.85%. Analyses of the same batches showed mean differences of the estimated content/potencies of 0.61%, 1.31% higher and 0.86% lower as compared to the validated reversed-phase and size exclusion liquid chromatography methods, and to the UMR-106 cell culture bioassay, respectively, with non-significant differences (p>0.05). Degraded forms were also subjected to the in vitro cytotoxicity test. The results obtained showed the capabilities of each one of the methods, and constitute an alternative strategy to monitor stability, improve the quality control and ensure the batch-to-batch consistency of bulk and finished biotechnology-derived medicine.