High-performance Liquid Chromatography System Research Articles

Polycarbonate baby bottles: study of the release of Bisphenol A

Recently, it has been demonstrated that the main Bisphenol A (BPA) release is a degradation process originated by the contact between detergent and polycarbonate (PC) at high temperatures. EU Laws for food contact materials authorize the use of BPA for food contact PC articles (some for the use of young children), and consequently, the degradation process should be taken into account when testing PC articles. In the present study, two types of samples were assayed, baby bottles that have previously undergone some migration assays and new baby bottles of a specific brand in order to test the degradation of not previously used articles. The tests were performed under different conditions and using two types of detergents. To quantify BPA, a high-performance liquid chromatographic system with fluorescence detector was used. The method was validated, and satisfactory results were achieved. Data obtained indicated that the trademark is a substantial factor in the level of BPA release from PC polymer; besides a great dispersion between samples of the same batch independently of the detergent and concentration employed used was found. On the other hand, for both types of samples, a previous food contact (infant formula) as well as the exposure procedure (immersion or article filling) has influenced on the PC degradation.

Rapid Liquid Chromatographic Determination of Itraconazole and its Production Impurities

A simple, rapid ultra-performance liquid chromatography (UPLC) method was developed for the analysis of itraconazole and its associated production impurities. The optimum chromatographic conditions were achieved using an Agilent Zorbax Eclipse XDB C18 column, 1.8 µm (4.6 × 50 mm) installed in a column oven heater utilizing a gradient mobile phase of 0.08M tetrabutylammonium hydrogen sulfate buffer-acetonitrile (80:20, v/v), with ultraviolet detection at 235 nm. An Agilent 1200 RRLC Series was used for the UPLC analysis. UPLC is a technology that greatly reduces analysis time by utilizing columns packed with sub-2 µm particles. The method was validated according to International Conference on Harmonization guidelines with respect to precision, accuracy, linearity, robustness and limits of detection and quantification. All parameters were found to be well within the stated guidelines. The total analysis time was reduced by two-thirds, from over 30 min (the current European Pharmacopeia method) to under 10 min, and the method is applicable for assay and related substance determination. A method utilizing the sub-2 µm column on a conventional high-performance liquid chromatography system was also developed and validated, resulting in substantial time and solvent savings.

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Physico-Chemical Stability of Busulfan in Injectable Solutions in Various Administration Packages

Background and ObjectivesBusulfan is used as part of a conditioning regimen prior to hematopoietic stem cell transplantation for the treatment of certain cancers and immune deficiency syndromes. Due to its instability in aqueous preparations, busulfan for infusion is prepared from a concentrate and has a relatively short shelf life once prepared. The purpose of this study was to identify the most suitable storage container and temperature to maximize the shelf life of busulfan therapeutic infusions prepared from Busilvex®.MethodsBusilvex® 6 mg/mL was diluted to 0.55 mg/mL with 0.9 % NaCl and aliquots dispensed into polypropylene syringes, polyvinyl chloride bags, and glass bottles. Three storage temperatures were evaluated: 2–8 °C, 13–15 °C (thermostatically controlled chamber), and room temperature (20 ± 5 °C). At set time points, samples were analysed for busulfan content, using a high-performance liquid chromatography (HPLC) system with ultraviolet detection. The change in pH and osmolarity on storage was also determined, and solutions were inspected visually for formation of a precipitate or colour change. To determine the contribution of precipitation to loss of busulfan content on storage, samples from one time series were treated with the solvent dimethylacetamide prior to HPLC separation and quantitation of busulfan.ResultsThe results of the active substance content monitoring study over a 48-h period demonstrate that busulfan solution is stable at a 5 % threshold, at 2–8 °C for 16 h in syringes, 14 h in glass bottles, and 6 h in bags. In addition, the period of stability decreases as the temperature increases (4 h at 20 ± 5 °C). The solution is considered to be stable, subject to precipitation liable to be observed regardless of the temperature.ConclusionThe best stability was observed for busulfan solutions placed at 2–8 °C in syringes. This study demonstrated that precipitation, in addition to hydrolysis, has a significant influence on the busulfan content.

New Analytical Method for the Determination of Metronidazole in Human Plasma: Application to Bioequivalence Study

Purpose: To develop new sensitive, accurate, rapid and reproducible high performance liquid chromatography (HPLC) method to determine metronidazole levels in human plasma and to apply the method in a bioequivalence study. Methods: Metronidazole was extracted from human plasma through one step of protein precipitation by methanol using carbamazepine as internal standard (IS). After centrifugation of the plasma sample, the supernatant layer was separated and injected into HPLC system using Eclipse XDB-phenyl column. The mobile phase consisted of phosphate buffer (pH 4.5): acetonitrile (95:5, v/v). The UV detector was set at 320 nm. The bioavailability of the test metronidazole product (Brand A) was compared to a commercial metronidazole brand as reference product in 24 healthy volunteers who received a single dose equivalent to 500 mg of the test and reference products in a randomized balanced two-way cross-over design separated by two-week wash-out period. Results: Mean standard calibration curves of metronidazole over the concentration range of 0.05 – 30 μg/ml were linear. No significant differences were found based on analysis of variance of the pharmacokinetics parameters required for the assessment of bioequivalence of test and reference formulations. The mean value and 90 %CI of test/reference ratios for the derived parameters were: Cmax, 9.64 vs. 8.38 (0.93 – 1.10), AUC0-24, 124.6 vs.122.3 μg.h/mL (0.973 – 1.051) and AUC0-∞, 140.9 vs. 128.4 h/mL (1.15 – 1.23). Conclusion: The test metronidazole product was bioequivalent to the reference. The method is suitable for bioequivalence and pharmacokinetic studies in humans with a low limit of quantification of 0.05 µg/ml.

Detection and Clinical Significance of Intratumoral EGFR Mutational Heterogeneity in Chinese Patients with Advanced Non-Small Cell Lung Cancer

PurposeThis study evaluated occurrence and potential clinical significance of intratumoral EGFR mutational heterogeneity in Chinese patients with non-small cell lung cancer (NSCLC).Materials and MethodsEighty-five stage IIIa-IV NSCLC patients who had undergone palliative surgical resection were included in this study. Of these, 45 patients carried EGFR mutations (group-M) and 40 patients were wild-type (group-W). Each tumor sample was microdissected to yield 28–34 tumor foci and Intratumoral EGFR mutation were determined using Denaturing High Performance Liquid Chromatography (DHPLC) and Amplification Refractory Mutation System (ARMS). EGFR copy numbers were measured using fluorescence in situ hybridization (FISH).ResultsMicrodissection yielded 1,431 tumor foci from EGFR mutant patients (group-M) and 1,238 foci from wild-type patients (group-W). The EGFR mutant frequencies in group-M were 80.6% (1,154/1,431) and 87.1% (1,247/1,431) using DHPLC and ARMS, respectively. A combination of EGFR-mutated and wild-type cells was detected in 32.9% (28/85) of samples by DHPLC and 28.2% (24/85) by ARMS, supporting the occurrence of intratumoral heterogeneity. Thirty-one patients (36.5%) were identified as EGFR FISH-positive. Patients harboring intratumoral mutational heterogeneity possessed lower EGFR copy numbers than those tumors contained mutant cells alone (16.7% vs. 71.0%, P<0.05). Among 26 patients who had received EGFR-TKIs, the mean EGFR mutation content was higher in patients showing partial response (86.1%) or stable disease (48.7%) compared with patients experiencing progressive disease (6.0%) (P = 0.001). There also showed relationship between progression-free survival (PFS) and different content of EGFR mutation groups (pure wild type EGFR, EGFR mutation with heterogeneity and pure mutated EGFR) (P = 0.001).ConclusionApproximately 30% of patients presented intratumoral EGFR mutational heterogeneity, accompanying with relatively low EGFR copy number. EGFR mutant content was correlated with the response and prognosis of EGFR-TKIs.

Evaluation of charged aerosol detector for purity assessment of protein

Commercially available protein reference standard materials are widely used for the quantitation of intact proteins in biopharmaceuticals, food, and consumer products. However, the purity of protein reference standard materials are often assumed to be 100% or they may be assigned an inaccurate value because the methods used to determine protein purity often lack specificity and accuracy. In this study, a high performance liquid chromatography system equipped with a charged aerosol detector (HPLC-CAD) was used for universal response detection to provide a practical, specific, accurate, and robust method for the determination of the purity of protein reference standard materials. This work demonstrates the near uniform CAD responses for six proteins with different molecular weights and different structures. Flow injection analysis (FIA) was used to compare protein responses under various mobile phase and diluent compositions. Similar CAD responses for all six proteins were observed when the mobile phase composition included trifluoroacetic acid (TFA) and acetonitrile. These are typical conditions regularly applied to the separation of proteins by reversed phase (RP) chromatography. The universal response feature of the CAD was employed to determine the purity of Bowman–Birk inhibitor (BBI) reference standard material. This protein is an important ingredient in soybean products and has various therapy applications. Three major components were observed in the commercially available reference standard material by reversed phase gradient HPLC, BBI-Native, BBI-Isoform 1, and BBI-Isoform 2 at relative compositions of 60.0%, 34.2%, and 5.8%, respectively.

Pharmacokinetics of Maraviroc, Raltegravir, Darunavir, and Etravirine in the Semen of HIV-Infected Men

Pharmacokinetics of Maraviroc, Raltegravir, Darunavir, and Etravirine in the Semen of HIV-Infected Men

Solid Phase Micro Tip Extraction Coupled With High Performance Liquid Chromatography for Diclofenac Sodium Analysis in Urine Sample

A solid phase micro tip extraction (SPMTE) has been developed as sample preparation technique for the analysis of diclofenac sodium, a non-steroidal anti-inflammatory drug (NSAID) in human urine sample. The analysis was performed by high performance liquid chromatography (HPLC) system using methanol:water (60:40, v/v) as mobile phase, flow rate of 1.0 mL/min under UV detection wavelength at 282 nm. Extraction conditions such as desorption solvent and extraction time were optimized in this study. The optimized SPMTE coupled with the HPLC method was successfully applied for the determination of diclofenac sodium in urine samples. The best recovery of diclofenac sodium in urine samples is 74.80% with RSD of 3.56% (n=3). The proposed method provides simple and rapid analysis of diclofenac sodium in urine sample.

Identification of the Active Components in Shenmai Injection that Differentially Affect Cyp3a4-Mediated 1′-Hydroxylation and 4-Hydroxylation of Midazolam

Shenmai injection (SMI) is a popular herbal preparation that is widely used for the treatment of atherosclerotic coronary heart disease and viral myocarditis. In our previous study, SMI was shown to differentially affect CYP3A4-mediated 1'-hydroxylation and 4-hydroxylation of midazolam (MDZ). The present study was conducted to identify the active components in SMI responsible for the differential effects on MDZ metabolism, using in vitro incubation systems (rat and human liver microsomes and a recombinant CYP3A4 system) to measure 1'-hydroxylation and 4-hydroxylation of MDZ. First, different fractions of SMI were obtained by gradient elution on an solid phase extraction system and individually tested for their effects on MDZ metabolism. The results demonstrated that lipid-soluble constituents were likely to be the predominant active components of SMI. Second, the possible active components were gradually separated on an high-performance liquid chromatography system under different conditions and individually tested in vitro for their effects on MDZ metabolism. Third, the active component obtained in the above experiment was collected and subjected to structural analysis, and identified as panaxytriol (PXT). Finally, it was validated that PXT had significant differential effects on 1'-hydroxylation and 4-hydroxylation of MDZ in various in vitro systems that were similar to those of SMI. We conclude that PXT is the constituent of SMI responsible for the differential effects on CYP3A4-mediated 1'-hydroxylation and 4-hydroxylation of MDZ.

Distribution of chitin/chitosan-like bioflocculant-producing potential in the genus Citrobacter

Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40-46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66 × 10⁶Da.

Enzyme-based online monitoring and measurement of antioxidant activity using an optical oxygen sensor coupled to an HPLC system

It is estimated that up to 50% of the adult population take antioxidant products on a daily basis to promote their health status. Strangely, despite the well-recognized importance of antioxidants, currently there is no international standard index for labeling owing to the lack of standardized methods for antioxidant measurement in complex products. Here, an online high-performance liquid chromatography (HPLC)-based method to detect and measure the total antioxidant capacity of antioxidant samples is presented. In this approach, complex samples containing antioxidants are separated by the HPLC system, which is further coupled to an antioxidant measuring system consisting of an optical oxygen sensor, laccase, and tetramethoxy azobismethylene quinone (TMAMQ). The antioxidants, separated via HPLC, reduce TMAMQ to syringaldazine, which is then reoxidized by laccase while simultaneously consuming O(2). The amount of consumed oxygen is directly proportional to the concentration of antioxidants and is measured by the optical oxygen sensor. The sensor is fabricated by coating a glass capillary with an oxygen-sensitive thin layer made of platinum(II) meso-tetra(4-fluorophenyl)tetrabenzoporphyrin and polystyrene, which makes real-time analysis possible (t(90) = 1.1 s in solution). Four selected antioxidants (3 mM), namely, catechin, ferulic acid, naringenin (used as a control), and Trolox, representing flavonol, hydrocinnamic acid, flavanone, and vitamin E, respectively, were injected into the online antioxidant monitoring system, separated, and then mixed with the TMAMQ/laccase solution, which resulted in oxygen consumption. This study shows that, with the use of such a system, the antioxidant activity of individual antioxidant molecules in a sample and their contribution to the total antioxidant activity of the sample can be correctly assigned.

Development and Validation of a Simple, Rapid and Stability-Indicating High Performance Liquid Chromatography Method for Quantification of Norfloxacin in a Pharmaceutical Product

A stability-indication high performance liquid chromatographic method has been developed for the determination of norfloxacin in tablet dosage forms. Optimum separation was achieved in less than 7 minutes using Eclipse Plus Zorbax C18 Agilent, 150 mm×4.6 mm i.d., 5 μm particle size column. The analyte was resolved by using a mobile phase 5% acetic acid aqueous solution and methanol (80:20, v/v) at a flow rate 1.0 ml/min on an isocratic high performance liquid chromatographic system at a wavelength of 277 nm. Linearity, system suitability, precision, sensitivity, selectivity, specific, and robustness were established by International Conference Harmonization guidelines. For stress studies the drug was subjected to photolysis, oxidation, acid, alkaline and neutral conditions. The analytical conditions and the solvent developed provided good resolution within a short analysis time and economic advantages. The proposed method not required sophisticated and expensive instrumentation.

Mixed Extractant-Dispersive Liquid Phase Microextraction Based on Solidification of Floating Organic Drop for Determination of S -phenylmercapturic Acid in Urine

A method of dispersive liquid phase microextraction based on solidification of floating organic drop(SFO-DLLME) with mixed extractant by high performance liquid chromatography(HPLC) for the determination of S-phenylmercapturic acid(SPMA) in urine was established.Detailed research on the conditions of microextraction was evaluated in the experiment.An aliquot of 20 mL of urine sample was adjusted to pH 2.0 and 7.2 g NaCl was added to increase the ionic strength.Then,100 μL mixed solvent(1-dodecanol/chloroform,1.8 ∶ 1,V/V) as extractant and 100 μL methanol as dispersive agent were injected into sample solution.The solution was shaken to disperse the extractant.The extraction solvent could be floated on the surface of the solution with the help of centrifugation and solidified at -20 ℃.Then the solid organic phase was transferred to EP tube and melted at room temperature.The supernatant of mixed extraction solvent was injected into HPLC system for.The analytical method showed good linearity in the range of 0.025-1.25 mg/L SPMA.The detection limit was 0.019 mg/L.The intra-day and inter-day precisions(relative standard deviation,RSD) of the method were 2.2%-5.4% and 2.5%-6.1%,respectively.The method was applied for determination of SPMA in human urine with satisfactory results.The spiked recoveries ranged from 86.8%-99.9%.The method was simple,fast and suitable for batch analysis of SPMA in urine samples.

A rapid and sensitive method for the determination of malondialdehyde as its hydralazine derivative in human plasma using high performance liquid chromatography

Malondialdehyde (MDA) is a presumptive biomarker for lipid peroxidation in plasma, live organisms, and cultured cells. This study presents an accurate and reproducible method to measure MDA as its hydralazine (HDZ) derivative in plasma using high-performance liquid chromatography (HPLC). A reference curve was prepared by mixing a 250 μL volume of each concentration of standard MDA (0.05–10.0 nmol mL−1) with 25 μL HDZ 5 mM solution in 2 M hydrochloric acid and the mixture was incubated for 60 min at room temperature in the dark. An aliquot of 50 μL of this reaction mixture was injected into the HPLC system. This method was also used for the determination of MDA in phosphate buffer and plasma. The standard addition method was used to determine MDA in plasma. In order to compare this method with another method for the determination of MDA, a HPLC method was applied using derivatization of MDA with 2,4-dinitrophenylhydrazine (DNPH). Results showed that this method was valid, linear, simple, highly sensitive and reproducible for MDA determination in plasma. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.03 and 0.1 nmol mL−1, respectively. This method has a shorter run time, and a lower LOQ in comparison with other HPLC methods described for MDA determination. Also, it can serve as a cost-beneficial method for determination of MDA in plasma as a biomarker for lipid peroxidation.

Pharmacokinetic studies of meloxicam after its intravenous administration in local goat (Capra hircus) of Assam

Aim: The aim of the present study was to determine the meloxicam concentrations in the plasma of goats at different time intervals after its administration by IV route and data were used to determine the different pharmacokinetic parameters and the bioavailability of meloxicam in goats. Materials and Methods: The present study was conducted on 12 to 18 months old, five adult male local goats (Capra hircus) of Assam, with body weight ranging between 10 to 18 kg. All goats were clinically sound and healthy and reared at Goat Research station, Burnihat, Assam. A single dose of meloxicam was administered intravenously into the jugular vein of goats at the rate of 1 mg/kg body weight. Blood samples were analyzed with the help of high performance liquid chromatography (HPLC) system. Results:After single intravenous administration of meloxicam at the dose rate of 1mg/kg, the mean peak plasma level of the drug was 6.538±0.1189 μg/ml at 2 min. The mean elimination half life (t ) and mean distribution half life (t ) were 1/2â 1/2a 484.5±85.1202 min and 28.68±1.4206 min respectively. The mean values of AUC and AUMC were 2635.272±94.2389 μg.min/ml and 1620332±92456.46 μg.min2/ml respectively. The mean values of volume of distribution (Vd) and mean residence time (MRT) were 0.276±0.0103 L/kg and 612.948±14.5771 min respectively. Conclusion: After calculating various pharmacokinetic determinants, dosage regimens were computed for meloxicam in goats, based on minimum effective therapeutic plasma concentration of 0.7 μg/ml.To maintain the minimum effective concentration of 0.7 μg/ml in plasma for 25 hours following iv administration of meloxicam,an initial loading dose of 1.1 mg/kg body weight followed by a maintenance dose of 1mg/kg body weight/day is recommended.

Analysis of metformin, glimepiride and pioglitazone in human serum and its application to pharmacokinetics

A robust method, for the chromatographic separation of three antidiabetic drugs viz metformin, pioglitazone and glimepiride using an isocratic reversed phase high-performance liquid chromatographic (HPLC) system having ultraviolet detection at 254 nm is presented in this paper. The method was developed in human serum and dosage formulation with high-quality chromatographic separation between the drug peaks by using a stainless steel analytical column Nucleosil, C18 (10 micron, 25 × 0.46 cm). The system was operated at room temperature using a mobile phase consisting of acetonitrile, phosphate buffer (pH 4.3) in the ratio of 60 : 40 v/v at a flow rate of 1 mL min−1. The parametric statistics, i.e., correlation coefficient of 0.999 was assessed for all the drugs having linearity over the tested concentration range (10 to 10 000 ng mL−1) in human serum. The accuracy and the relative standard deviations of samples for six replicate measurements were not less than 97% and greater than 2%, respectively. The proposed method was validated for selectivity, linearity, accuracy, and precision according to the International Conference on Harmonization (ICH). The method is applicable for the quality control of the mentioned drugs in raw material, bulk drug, and pharmaceutical formulations as well as in human serum.

Evaluation of a low cell constant conductance detector for detection of charged species in high-performance liquid chromatography

The present study evaluates high performance liquid chromatography (HPLC) detection based on a commercial conductance detector with a low cell constant of 0.005 cm−1 and a volume of 2 μL and a photodiode array UV-vis detector typically used in HPLC. When detecting a static NaCl solution, the conductance detector yields a limit of detection (LOD, 3 × noise) for NaCl of 39 parts per trillion. When flowing methanol through both conductance and UV-vis detectors and injecting benzoic acid/methanol, the signal-to-noise (S/N) ratio of the conductance detector is 8-fold higher than that of the UV-vis at its optimal wavelength. Using an HPLC with a C-18 column, flowing a 75 : 25 water : methanol solution, and using an acetylsalicylic acid (ASA)/water solution, the conductance detector yielded an ∼18-fold higher S/N ratio. It was found that HPLC system noise reduces the S/N ratio of the conductance detectors. The conductance detector detected non-chromophoric species generated by atmospheric CO2 as well as by decomposition of ASA. Conductance chromatograms yielded ASA peak heights and areas that varied linearly with the ASA concentration from 0.5 ppm to 10 ppm with linear correlation coefficients exceeding 0.999. In view of the sensitivity of conductance detection, its potential application as a sensitive tool for cleaning assessments for pharmaceutical equipment was confirmed by dispersing 50 μg of ASA on a 2′′ × 2′′ stainless steel sheet, swabbing the surface, dissolving the collected material in water and injecting the solution into the HPLC. The results in this study demonstrate that low cell constant conductance detection can be remarkably sensitive to ionized/charged species and thereby has potential to serve as an analytical tool for this important class of molecules.

Method Development and Validation for Simultaneous Estimation of Ethinyl Estradiol and Drospirenone and Forced Degradation Behavior by HPLC in Combined Dosage Form

A simple, accurate, rapid and precise isocratic High performance liquid chromatographic (HPLC) method was developed and validated for the determination of ethinyl estradiol and drospirenone in tablet formulation. The method employs Waters HPLC system on Thermo Hypersil BDS C18 Column (4.6×250 mm and 5 μm) and flow rate of 1.0 ml/min with a load of 15 μl. Acetonitrile and ammonium acetate buffer was used as mobile phase in the composition of 30:70. The detection was carried out at 258 nm. Linearity ranges for ethinyl estradiol and drospirenone were 0.06- 0.18 μg/ml, 6-18 μg/ml respectively. Retention Time of ethinyl estradiol and drospirenone were found to be 1.4 min, 5.3 min respectively. Percent Recovery study values of ethinyl estradiol and drospirenone were found to be within 97-103%. The combination product is exposed to acid/base, hydrolytic, photolytic and peroxide stress conditions and the stressed samples were analyzed. This developed method was successfully utilized for the quantitative estimation of ethinyl estradiol and drospirenone in pharmaceutical dosage forms. This method was validated for accuracy, precision, linearity and Robustness as per ICH guidelines.

High Performance Liquid Chromatography Coupled with Pre-column Derivatization for Determination of Oxidized Glutathione Level in Rats Exposed to Paraquat.

Glutathione (GSH) is one of the most important antioxidants that plays an essential role in detoxification of reactive oxygen species (ROS) which oxidizes to glutathione disulfide (GSSG). Paraquat (PQ), awidely used herbicide, causes pulmonary injury with the productionof ROS. Excessive ROS accumulation as a consequence of PQ exposure are frequently targeted by GSH thereby oxidative stress leads to depletion of cellular GSH by transforming of GSH to glutathione disulfide (GSSG). A precise method of measuring of GSSG concentration in plasma as indicator of oxidative stress is needed. Some analytical techniques such as high-performance liquid chromatography (HPLC), gas chromatography and capillary electrophoresis have been used for determination of GSSG concentration. In the present study, a new HPLC method with fluorescence detection based on derivatization of the amine group of glutathione with 9-fluorenylmethyl chloroformate (FMOC-Cl) was developed. Male Wistar albino rats exposed to different doses of PQ (20-60 mg/kg) and control group were used and after protein precipitation, their plasma was subjected to derivatization with FMOC in the presence of borate buffer. The derivatized samples were injected to HPLC system with C18 column, mobile phase consisting of methanol and phosphate buffer, λem= 315 nm, λex= 260 nm. Among all experimental groups, the rats which received 60 mg/kg PQ, showed a significant increase in the amount of oxidized glutathione (GSSG) compared to the control group. In this study, the applied derivatization and HPLC method made it possible to measure small amounts of glutathione in plasma using a precise and sensitive technique.