Journal of Pharmaceutical Analysis

Methods for in vitro evaluating antimicrobial activity:A review$

Mounyr Balouiri n, Moulay Sadiki, Saad Koraichi Ibnsouda

2016, 6(2): 71-79. doi: 10.1016/j.jpha.2015.11.005

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Abstract:
In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance. Therefore, a greater attention has been paid to antimicrobial activity screening and evaluating methods. Several bioassays such as disk-diffusion, well diffusion and broth or agar dilution are well known and commonly used, but others such as flow cy-tofluorometric and bioluminescent methods are not widely used because they require specified equip-ment and further evaluation for reproducibility and standardization, even if they can provide rapid re-sults of the antimicrobial agent's effects and a better understanding of their impact on the viability and cell damage inflicted to the tested microorganism. In this review article, an exhaustive list of in vitro antimicrobial susceptibility testing methods and detailed information on their advantages and limita-tions are reported.

The pharmacokinetic study of rutin in rat plasma based on an electrochemically reduced graphene oxide modified sensor$

Pei Zhang a, Yu-Qiang Gou b, Xia Gao a, Rui-Bin Bai a, Wen-Xia Chen a, Bo-Lu Sun a, Fang-Di Hu a, n, Wang-Hong Zhao c

2016, 6(2): 80-86. doi: 10.1016/j.jpha.2015.12.003

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An electrochemical method based on a directly electrochemically reduced graphene oxide (ERGO) film coated on a glassy carbon electrode (GCE) was developed for the rapid and convenient determination of rutin in plasma. ERGO was modified on the surface of GCE by one-step electro-deposition method. Electrochemical behavior of rutin on ERGO/GCE indicated that rutin underwent a surface-controlled quasi-reversible process and the electrochemical parameters such as charge transfer coefficient (α), electron transfer number (n) and electrode reaction standard rate constant (ks) were 0.53, 2 and 3.4 s?1, respectively. The electrochemical sensor for rutin in plasma provided a wide linear response range of 4.70 ? 10 ? 7 ? 1.25 ? 10 ? 5 M with the detection limit (s/n ? 3) of 1.84 ? 10 ? 8 M. The assay was success-fully used to the pharmacokinetic study of rutin. The pharmacokinetic parameters such as elimination rate half-life (t1/2), area under curve (AUC), and plasma clearance (CL) were calculated to be 3.345 7 0.647 min, 5750 7 656.0 mg min/mL, and 5.891 7 0.458 mL/min/kg, respectively. The proposed method utilized a small sample volume of 10μL and had no complicated sample pretreatment (without deproteinization), which was simple, eco-friendly, and time-and cost-efficient for rutin pharmacokinetic studies.

Determination of lercanidipine in human plasma by an improved UPLC-MS/MS method for a bioequivalence study$

Darshan V. Chaudhary, Daxesh P. Patel, Priyanka A. Shah, Jaivik V. Shah, Mallika Sanyal, Pranav S. Shrivastav

2016, 6(2): 87-94. doi: 10.1016/j.jpha.2015.09.001

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An improved and reliable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the determination of lercanidipine in human plasma. Plasma samples with lercanidipine-d3 as an internal standard (IS) were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 100 mL of human plasma. Chromatographic analysis was performed on UPLC BEH C18 (50 mm ? 2.1 mm, 1.7 mm) column under isocratic conditions. Linear calibration curves were obtained over a wide dynamic concentration range of 0.010–20.0 ng/mL. Matrix effect was assessed by post-column infusion, post-extraction spiking and standard-line slope methods. The mean extraction recovery was 4 94%for the analyte and IS. Inter-batch and intra-batch precision (%CV) across five quality controls was o 5.8%. Bioequivalence study was performed with 36 healthy sub-jects after oral administration of 10 mg of lercanidipine and the assay reproducibility was evaluated by reanalysis of 133 incurred samples.

Determination of torasemide in human plasma and its bioequivalence study by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry$

Lin Zhang, Rulin Wang, Yuan Tian, Zunjian Zhang

2016, 6(2): 95-102. doi: 10.1016/j.jpha.2015.11.002

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A sensitive and selective method using high-performance liquid chromatography coupled with elec-trospray ionization tandem mass spectrometry (HPLC–ESI–MS) to determine the concentration of tor-asemide in human plasma samples was developed and validated. Tolbutamide was chosen as the internal standard (IS). The chromatography was performed on a Gl Sciences Inertsil ODS-3 column (100 mm ? 2.1 mm i.d., 5.0 mm) within 5 min, using methanol with 10 mM ammonium formate (60:40, v/v) as mobile phase at a flow rate of 0.2 mL/min. The targeted compound was detected in negative io-nization at m/z 347.00 for torasemide and 269.00 for IS. The linearity range of this method was found to be within the concentration range of 1–2500 ng/mL (r?0.9984) for torasemide in human plasma. The accuracy of this measurement was between 94.05%and 103.86%. The extracted recovery efficiency was from 84.20% to 86.47% at three concentration levels. This method was also successfully applied in pharmacokinetics and bioequivalence studies in Chinese volunteers.

Optimized high performance liquid chromatography-ultraviolet detection method using core-shell particles for the therapeutic monitoring of methotrexate$

Milagros Montemurro, María M. De Zan n, Juan C. Robles

2016, 6(2): 103-111. doi: 10.1016/j.jpha.2015.12.001

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Methotrexate (MTX) is an antineoplastic drug, and due to its high toxicity, the therapeutic drug mon-itoring is strictly conducted in the clinical practice. The chemometric optimization and validation of a high performance liquid chromatography (HPLC) method using core–shell particles is presented for the determination of MTX in plasma during therapeutic monitoring. Experimental design and response surface methodology (RSM) were applied for the optimization of the chromatographic system and the analyte extraction step. A Poroshell 120 EC-C18 (3.0 mm ? 75 mm, 2.7μm) column was used to obtain a fast and efficient separation in a complete run time of 4 min. The optimum conditions for the chroma-tographic system resulted in a mobile phase consisting of acetic acid/sodium acetate buffer solution (85.0 mM, pH?4.00) and 11.2%of acetonitrile at a flow rate of 0.4 mL/min. Selectivity, linearity, accuracy and precision were demonstrated in a range of 0.10–6.0 mM of MTX. The application of the optimized method required only 150 mL of patient plasma and a low consumption of solvent to provide rapid re-sults.

Liquid chromatography-tandem mass spectrometry method for simultaneous determination of valproic acid and its ene-metabolites in epilepsy patient plasma$

Huan Lu, Chong Su, Lei Yin, Liqiang Gu, Jingkai Gu, Xiaohui Chen

2016, 6(2): 112-116. doi: 10.1016/j.jpha.2015.11.006

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A simple and high throughput method was developed and validated for simultaneous determination of valproic acid and its two toxicant ene-metabolites, 2-enevalproic acid and 4-enevalproic acid in epilepsy patient plasma using liquid chromatography–tandem mass spectrometry. Probenecid was used as in-ternal standard and solid-phase extraction was selected for sample preparation. A chromatographic separation was performed on an Agilent Poroshell SB-C18 column (50 mm ? 4.6 mm i.d., 2.7μm) by an optimized gradient elution at a flow rate of 0.9 mL/min. The total run time was 7 min. Electrospray ionization was used in negative ion mode by multiple reaction monitoring of the precursor-to-product ion transitions at m/z 143.0-143.0 for valproic acid, m/z 140.9-140.9 for 2-enevalproic acid and 4-enevalproic acid for their poor fragments, and m/z 283.9-239.9 for probenecid. The results showed good linearity of valproic acid, 2-enevalproic acid and 4-enevalproic acid in their respective linear ranges. The correlation coefficients were more than 0.998. The intra- and inter-day precision of the assay was less than 11.0%and the accuracy ranged from 2%to 12%. This analytical method was successfully applied to assay plasma concentrations of valproic acid and its two ene-metabolites in epilepsy patient plasma and used for therapeutic drug monitoring.

Study and ICH validation of a reverse-phase liquid chromatographic method for the quantification of the intact monoclonal antibody cetuximab$

Antonio Martínez-Ortega, Agustín Herrera, Antonio Salmerón-García, José Cabeza, Luis Cuadros-Rodríguez, Natalia Navas

2016, 6(2): 117-124. doi: 10.1016/j.jpha.2015.11.007

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Cetuximab (CTX) is a potent chimeric mouse/human monoclonal antibody (mAb) approved worldwide for treatment of metastatic colorectal cancer. Among the various biological and physical analyses per-formed for full study on this biopharmaceutic, the determination of the concentration preparations throughout manufacturing and subsequent handling in hospital is particularly relevant. In the present work, the study and validation of a method for quantifying intact CTX by reverse-phase high-perfor-mance liquid chromatography with diode array detection ((RP)HPLC/DAD) is presented. With that end, we checked the performance of a chromatographic method for quantifying CTX and conducted a study to validate the method as stability-indicating in accordance with the International Conference on Harmo-nization guidelines (ICH) for biotechnological drugs; therefore, we evaluated linearity, accuracy, preci-sion, detection and quantification limits, robustness and system suitability. The specificity of the method and the robustness of the mAb formulation against external stress factors were estimated by compre-hensive chromatographic analysis by subjecting CTX to several informative stress conditions. As de-monstrated, the method is rapid, accurate, and reproducible for CTX quantification. It was also suc-cessfully used to quantify CTX in a long-term stability study performed under hospital conditions.

Free radical scavenging potential and HPTLC analysis of Indigofera tinctoria linn (Fabaceae)$

Sakthivel Srinivasan, Wankupar Wankhar, Sheeladevi Rathinasamy, Ravindran Rajan n

2016, 6(2): 125-131. doi: 10.1016/j.jpha.2015.04.003

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The objective of this study was to evaluate the free radical scavenging potential and high performance thin layer chromatography (HPTLC) fingerprinting of Indigofera tinctoria (I. tinctoria). Phytochemical analysis was carried out using standard methods, and free radical scavenging activity of the plant was determined using 2,2-diphenyl-1-picrylhydrazy (DPPH), nitric oxide (NO) and superoxide anion ( O?2 ) radical scavenging capacities. HPTLC plate was kept in CAMAG TLC Scanner 3 and the Rf values at fin-gerprint data were recorded by WINCATS software. Aqueous extract of I. tinctoria reliably showed the total phenolics (267.2 7 2.42 mg/g), flavonoids (75.43 7 3.36 mg/g) and antioxidants (349.11 7 8.04 mg/g). The extract was found to have DPPH (52.08%), NO (23.12%) and O?2 (26.79%) scavenging activities at the concentration of 250μg/mL and the results were statistically significant compared with ascorbic acid standard (p o 0.05). HPTLC results confirmed that the extract contained several potential active com-ponents such as phenols, flavonoids, saponins and terpenoids as the slides revealed multi-colored bands of varying intensities. This study confirmed that the plant had multipotential antioxidant and free ra-dicals scavenging activities.

Comparison of reversed-phase enantioselective HPLC methods for determining the enantiomeric purity of (S)-omeprazole in the presence of its related substances$

Bruno Gallinella, Rosella Ferretti, Leo Zanitti, Isabella Sestili, Antonina Mosca, Roberto Cirilli n

2016, 6(2): 132-136. doi: 10.1016/j.jpha.2015.11.001

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A simple analytical high-performance liquid chromatography (HPLC) method was applied for the en-antiomeric excess determination of esomeprazole ((S)-OME), the enantiopure active ingredient con-tained in drug products, in the presence of its potential organic impurities A-E. The enantioselective separation was accomplished on the immobilized-type Chiralpak ID-3 chiral stationary phase (CSP) under reversed-phase conditions. The results were evaluated and compared with those obtained by the official enantioselective method of European Pharmacopoeia used as the reference for checking the enantiomeric excess of (S)-OME. It has been established that the use of the Chiralpak ID-3 CSP allows the determination of the enantiomeric purity of (S)-OME without any interference coming from its chiral and achiral related substances. The analytical procedure of the drug regulatory agencies based on the AGP CSP suffered instead from poor specificity due to overlap of the peaks pertinent to the achiral impurity A and the chiral impurity (R)-OME (impurity F).

Journal of Pharmaceutical Analysis

2016, 6(2): 137-138.

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2016 Vol. 6, No. 2