human plasma

A simple, rapid and sensitive liquid chromatography / electro spray ionization tandem mass spectrometry (LC–ESI–MS/MS) assay method has been proposed for the determination of apremilast in human plasma samples using apremilast d5 as internal standard (IS). Analyte and the IS were extracted from the 200 µL of human plasma via simple solid–phase extraction. The Chromatographic separation was obtained on a C18 column operating at a flow rate of 1.0 mL/min by using a mobile phase comprising of mixture of 5mm ammonium acetate in 0.2% formic acid buffer (15:85, v/v) and acetonitrile. A linear (r2 ³ 0.99) of the calibration curve was obtained over the concentration range of 2.03–808 ng/mL.  As per FDA guidelines method validation was performed and the results met the acceptance criteria. The intra–day and inter–day precision (%CV) and accuracy results in five validation batches across five concentration levels were well within the acceptance limits. To analyze the more number of samples in short time, thus increasing the productivity a short run time of 2.25 min for each sample was applied and this was made possible. The method was successfully applied to a pharmacokinetic study in humans.

Artemether-lumefantrine (ART-LUME) off late has become the first-line treatment for uncomplicated malaria in many Sub-Saharan Africa, Asia and America. Vigorous monitoring of the therapeutic efficacy of this treatment is needed. This requires high-quality studies following standard protocols; ideally, such studies should incorporate measurement of drug levels in human plasma in biological matrices. A specific and reliable isocratic mode RP-HPLC method has been developed and validated for simultaneous determination of artemether (ART) in combination with lumefantrine (LUME) in human plasma using diode array detector (DAD) at 238 nm. The analyte was separated on NUCLEOSIC-CN Cyano coloumn (150 mm × 4.6 mm, particle size 5 μm) using a mobile phase consisting of acetonitrile and acidic buffer (adjusted to pH 2.5 with H3PO4 ­– 2 %) in the ratio of 37: 63 v/v and flow rate was 1 ml/min. The method is linear over a range of 100-1600 µg/ml (r2 ≥ 0.999) and 1-16µg/ml (r2 ≥0.998) for the assay of ART and LUME respectively. Itraconazole (ITZ) (10µg/ml) was used as internal standard. The retention times of ART and LUME was found to be 4.3 min and 14.3 respectively. Mean extraction recovery for ART and LUME were 87.3 % and 89.1 %, respectively. Inter and intraday coefficients of variation for ART and LUME were ≤ 10%. The lower limits of quantification for ART and LUME were 0.22 and 0.66 μg/ml, respectively. The results of the study showed that the proposed RP-HPLC validated method described is efficient and has the necessary accuracy and precision for the rapid quantitative simultaneous determination of ART and LUME in human plasma and is thus highly suitable for use in pharmacokinetic /bioavailability/bioequivalence studies in healthy human subjects.

This paper describes a simple, rapid and sensitive bioanalytical method based on liquid chromatography / tandem mass spectrometry (LC–MS/MS) for the determination of integrase inhibitor raltegravir in human plasma samples. Carbamazepine was used as an internal standard (IS). Analyte and the internal standard were extracted from 200 µL of human plasma via solid phase extraction. The chromatographic separation was achieved on a C18 column by using a mixture of acetonitrile and 0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The calibration curve obtained was linear (r2 ³ 0.99) over the concentration range of 20.1–4007 ng/mL. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.0 min for each sample made it possible to analyze more number of samples in short time, thus increasing the productivity. The proposed method was found to be applicable to pharmacokinetic studies in humans.

A simple and rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and validated for simultaneous quantification of two protease inhibitors ritonavir and atazanavir in human plasma. Saquinavir was used as an internal standard. The analytes were extracted from human plasma samples by solid-phase extraction technique using a Orpheus C18 extraction cartridges. The reconstituted samples were chromatographed on a C18 column by using a 85:15 (v/v) mixture of methanol and 5mM ammonium acetate as the mobile phase at a flow rate of 0.9 mL/min. The calibration curves obtained were linear (r ³ 0.99) over the concentration range of 8.0-1600.0 ng/mL for ritonavir and 50.5-5995.2 ng/mL for atazanavir. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.0 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.

  A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for simultaneous quantification of two lipid lowering agents rosuvastatin and fenofibric acid in human plasma. Lovastatin was used as an internal standard. Analytes and the internal standard were extracted from human plasma by liquid-liquid extraction technique using a 50:50, v/v mixture of ethyl acetate and diethyl ether. The reconstituted samples were chromatographed on a C18 column by using a 80:20, v/v mixture of acetonitrile and 0.1% formic acid as the mobile phase at a flow rate of 1.0 mL/min. The calibration curve obtained was linear (r2 ³ 0.99) over the concentration range of 0.10-80.00 ng/mL for rosuvastatin and 50-9003 ng/mL for fenofibric acid. The multiple reaction monitoring mode was used for quantification of ion transitions at m/z 482/258, 319/233 and 405/199 for the rosuvastatin, fenofibric acid and the internal standard, respectively. The results of the intra-day and inter-day precision and accuracy study results were well within the acceptable limits. A run time of 3.0 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was successfully applied for the determination of the fenofibric acid in real time plasma samples for pharmacokinetic studies. Key words: Rosuvastatin, Fenofibric acid, human plasma, LC-MS/MS quantification, Pharmacokinetics.