Journal of Pharmaceutical Analysis

Determination of cilostazol and its active metabolite 3,4-dehydro cilostazol from small plasma volume by UPLC-MS/MS

Nejal M. Bhatt, Vijay D. Chavada, Daxesh P. Patel, Primal Sharma, Mallika Sanyal, Pranav S. Shrivastav

2015, (1): 1-11. doi: 10.1016/j.jpha.2014.08.001

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Abstract:
A simple, rapid and sensitive ultra performance liquid chromatography-tandem mass cilostazol and its pharmacologically active metabolite 3,4-dehydro cilostazol in human plasma using deuterated analogs as internal standards (ISs). Plasma samples were prepared using solid phase extraction 18 (50 mm ? 2.1 mm, 1.7 mm) column. The method was established over a concentration range of 0.5–1000 ng/mL for cilostazol and 0.5–mL for 3,4-dehydro cilostazol. Intra-and inter-batch precision (%CV) and accuracy for the analytes were found within 0.93–1.88 and 98.8–101.7% for cilostazol and 0.91–2.79 and 98.0–102.7% for the metabolite respectively. The assay recovery was within 95–97% for both the analytes and internal standards. The method was successfully applied to support a bioequivalence study of 100 mg cilostazol in 30 healthy subjects.

Development of an LC - MS/MS method for determination of 2-oxo-clopidogrel in human plasma

Yu-Han Li, Min Song, Tai-Jun Hang

2015, (1): 12-17. doi: 10.1016/j.jpha.2014.07.004

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Abstract:
A sensitive and selective liquid chromatography–tandem mass spectrometric (LC ? MS/MS) method was established to determine 2-oxo-clopidogrel, a crucial intermediate metabolite in human plasma. A chromatographic separation was performed on a Sapphire C18 column following a liquid–liquid extraction sample preparation with methyl t-butyl ether. Detection was carried out on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) with an electrospray ionization (ESI) mode. The method was validated in terms of specificity, accuracy, precision and limit of quantification. The calibration curves ranged from 0.50 to 50.0 ng/mL with good linearity. The stability was fully validated with addition of 1,4-dithio-DL-threitol (DTT) into the plasma sample prior to and in the preparation procedure. The validated method was proved to be suitable for use in pharmacokinetic study after single oral administration of 75 mg clopidogrel tablets in human subjects, which could make contribution to intensive study of the clinical drug–drug interactions of clopidogrel and individual treatment.

Development and validation of microbial bioassay for quantification of Levofloxacin in pharmaceutical preparations

Nishant A. Dafale, Uttam P. Semwal, Piyush K. Agarwal, Pradeep Sharma, G.N. Singh

2015, (1): 18-26. doi: 10.1016/j.jpha.2014.07.007

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Abstract:
The aim of this study was to develop and validate a simple, sensitive, precise and cost-effective one-level agar diffusion (5t1) bioassay for estimation of potency and bioactivity of Levofloxacin in pharmaceutical preparation which has not yet been reported in any pharmacopoeia. Among 16 microbial strains, Bacillus pumilus ATCC-14884 was selected as the most significant strain against Levofloxacin. Bioassay was optimized by investigating several factors such as buffer pH, inoculums concentration and reference standard concentration. Identification of Levofloxacin in commercial sample Levoflox tablet was done by FTIR spectroscopy. Mean potency recovery value for Levofloxacin in Levoflox tablet was estimated as 100.90%. A validated bioassay method showed linearity (r2 ? 0.988), precision (Interday RSD ? 1.05%, between analyst RSD ? 1.02%) and accuracy (101.23%, RSD ? 0.72%). Bioassay was correlated with HPLC using same sample and estimated potencies were 100.90%and 99.37%, respectively. Results show that bioassay is a suitable method for estimation of potency and bioactivity of Levofloxacin pharmaceutical preparations.

Analytical quality-by-design approach for sample treatment of BSA-containing solutions

Lien Taevernier, Evelien Wynendaele, Matthias D’Hondt, Bart De Spiegeleer

2015, (1): 27-32. doi: 10.1016/j.jpha.2014.06.001

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Abstract:
The sample preparation of samples containing bovine serum albumin (BSA), e.g., as used in transdermal Franz diffusion cell (FDC) solutions, was evaluated using an analytical quality-by-design (QbD) approach. Traditional precipitation of BSA by adding an equal volume of organic solvent, often successfully used with conventional HPLC-PDA, was found insufficiently robust when novel fused-core HPLC and/or UPLC-MS methods were used. In this study, three factors (acetonitrile (%), formic acid (%) and boiling time (min)) were included in the experimental design to determine an optimal and more suitable sample treatment of BSA-containing FDC solutions. Using a QbD and Derringer desirability (D) approach, combining BSA loss, dilution factor and variability, we constructed an optimal working space with the edge of failure defined as Do0.9. The design space is modelled and is confirmed to have an ACN range of 8373%and FA content of 170.25%.

LC, MSn and LC-MS/MS studies for the characterization of degradation products of amlodipine

Ravi N. Tiwari, Nishit Shah, Vikas Bhalani, Anand Mahajan

2015, (1): 33-42. doi: 10.1016/j.jpha.2014.07.005

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Abstract:
In the present study, comprehensive stress testing of amlodipine (AM) was carried out according to International Conference on Harmonization (ICH) Q1A(R2) guideline. AM was subjected to acidic, neutral and alkaline hydrolysis, oxidation, photolysis and thermal stress conditions. The drug showed instability in acidic and alkaline conditions, while it remained stable to neutral, oxidative, light and thermal stress. A total of nine degradation products (DPs) were formed from AM, which could be separated by the developed gradient LC method on a C18 column. The products formed under various stress conditions were investigated by LC–MS/MS analysis. The previously developed LC method was suitably modified for LC–MS/MS studies by replacing phosphate buffer with ammonium acetate buffer of the same concentration (pH 5.0). A complete fragmentation pathway of the drug was first established to characterize all the degradation products using LC–MS/MS and multi-stage mass (MSn) fragmentation studies. The obtained mass values were used to study elemental compositions, and the total information helped with the identification of DPs, along with its degradation pathway.

Direct injection HILIC-MS/MS analysis of darunavir in rat plasma applying supported liquid extraction

Bokka Ramesh, Nemali Manjula, Sistla Ramakrishna, Potturi Sita Devi

2015, (1): 43-50. doi: 10.1016/j.jpha.2014.05.001

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Abstract:
A novel bioanalytical method was developed and validated for the quantitative determination of darunavir (DRV) in rat plasma by employing hydrophilic interaction chromatography and tandem mass spectrometry (HILIC–MS/MS) with supported liquid extraction (SLE). Irbesartan (IRB) was used as an internal standard (IS). The analyte in rat plasma (200 mL) was isolated through SLE using ethyl acetate as the eluting solvent. The chromatographic separation was achieved on Luna-HILIC (250 mm4.6 mm, 5 μm) column with a mobile phase of 0.1% of formic acid in water:acetonitrile (5: 95, v/v), at a constant flow rate of 1.0 mL/min. The MS/MS ion transitions for DRV (548.1-392.0) and IS (429.2-207.1) were monitored on an ion trap mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The lower limit of quantitation (LLOQ) was 0.2 ng/mL and quantitation range was 0.2–5000 ng/mL. The method was validated for its selectivity, sensitivity, carryover, linearity, precision, accuracy, recovery, matrix effect and stability. The method was successfully applied to pharmacokinetic study in rats.

Protective effect of Genistein against N-nitrosodiethylamine (NDEA)-induced hepatotoxicity in Swiss albino rats

Fahad Ali, Rahul, Falaq Naz, Smita Jyoti, Yasir Hasan Siddique

2015, (1): 51-57. doi: 10.1016/j.jpha.2014.07.003

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Abstract:
In the present study, we studied the effect of Genistein against the hepatotoxicity induced by N-nitrosodiethylamine (NDEA). NDEA is present in almost all kinds of food stuff and has been reported to be a hepatocarcinogen. The male rats were exposed to NDEA (0.1 mg/mL) dissolved in drinking water separately and along with 25, 50, 100 mg/mL of Genistein for 21 days. The activities of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were measured in blood serum. Lipid peroxidation, protein carbonyl content, micronucleus frequency and DNA damage (Comet assay) were performed on rat hepatocytes. The results of the study reveal that the treatment of NDEA along with Genistein showed a significant dose-dependent decrease in the levels of blood serum enzymes i.e., SGOT, SGPT, ALP and LDH (Po0.05). The HE staining of histological sections of the liver also revealed a protective effect of Genistein. A significant dose-dependent reduction in the lipid peroxidation and protein carbonyl content was observed in rats exposed to NDEA (0.1 mg/mL) along with Genistein (Po0.05). The results obtained for the comet assay in rat hepatocytes showed a significant dose-dependent decrease in the mean tail length (Po0.05). Thus the present study supports the hepatoprotective role of Genistein.

A newfangled study using risk silhouette and uncertainty approximation for quantification of acyclovir in diverse formulation

Karan Mittal, Riddhish Patadia, Chintan Vora, Rajashree C. Mashru

2015, (1): 58-69. doi: 10.1016/j.jpha.2014.05.005

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Abstract:
Risk assessment and uncertainty approximation are two major and important parameters that need to be adopted for the development of pharmaceutical process to ensure reliable results. Additionally, there is a need to switch from the traditional method validation checklist to provide a high level of assurance of method reliability to measure quality attribute of a drug product. In the present work, evaluation of risk profile, combined standard uncertainty and expanded uncertainty in the analysis of acyclovir were studied. Uncertainty was calculated using cause-effect approach, and to make it more accurately applicable a method was validated in our laboratory as per the ICH guidelines. While assessing the results of validation, the calibration model was justified by the lack of fit and Levene’s test. Risk profile represents the future applications of this method. In uncertainty the major contribution is due to sample concentration and mass. This work demonstrates the application of theoretical concepts of calibration model tests, relative bias, risk profile and uncertainty in routine methods used for analysis in pharmaceutical field.

Capillary electrophoresis to determine entrapment efficiency of a nanostructured lipid carrier loaded with piroxicam

Jessica Otarola, Adriana Guillermina Lista, Beatriz Fernández Band, Mariano Garrido

2015, (1): 70-73. doi: 10.1016/j.jpha.2014.05.003

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Abstract:
A simple and fast capillary electrophoresis method has been developed to determine the amount of piroxicam loaded in a drug delivery system based on nanostructured lipid carriers (NLCs). The entrapment efficiency of the nanostructured lipid carrier was estimated by measuring the concentration of drug not entrapped in a suspension of NLC. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was studied; these parameters included the pH of the electrophoretic buffer solution and the applied voltage. The piroxicam peak was obtained with a satisfactory resolution. The separation was carried out using a running buffer composed of 50 mM ammonium acetate and 13.75 mM ammonia at pH 9. The optimal voltage was 20 kV and the cartridge temperature was 20 1C. The corresponding calibration curve was linear over the range of 2.7–5.4 mg/mL of NLC suspension. The reproducibility of migration time and peak area were investigated, and the obtained RSD% values (n ? 5) were 0.99 and 2.13, respectively.

2015 Vol. 5, No. 1