Syed Alvi

A simple, precise, and rapid reversed-phase high performance liquid chromatography (HPLC) method for the determination of ibuprofen (IBU) in human plasma using gemfibrozil as an internal standard (IS) was developed and validated. Plasma samples containing ibuprofen were spiked with the IS, precipitated with methanol, and centrifuged. The compounds of interest were efficiently separated on Symmetry Shield RP18 column maintained at a temperature of 40C, and detected with multi-wave length fluorescence detector (excitation/emission wavelength 230/ 275 nm). The mobile phase consisted of 0.01M dibasic ammonium phosphate (pH adjusted to 5.5 with phosphoric acid) and acetonitrile (45:55, v:v), and was delivered at a flow rate of 1.5 ml/min. The relationship between ibuprofen concentration in plasma and peak area ratio of ibuprofen / IS was linear (R2 0.9997) in the range of 0.25 – 60 μg/ml, the intra-and inter-day coefficient of variations (CV) were ≤ 3.8% and ≤ 2.9%, respectively. Extraction recoveries of ibuprofen and the IS were > 90%, whereas the accuracy (relative recovery) of ibuprofen measurement was 98% to 107% using quality control samples and 92% to 105% as determined by back calculation from peak area ratios of the calibration curves. The method was used to assess the stability of ibuprofen in human plasma under various conditions in the clinical laboratory. Further, it was successfully employed to measure ibuprofen levels in samples obtained normal volunteer.

A simple, precise, and rapid reversed-phase high performance liquid chromatography (HPLC) method for the determination of methotrexate (MTX) level in human plasma using sulfamethoxazole as an internal standard (IS) was developed and validated. Plasma samples containing MTX were spiked with the IS, vortex-mixed with methanol, and centrifuged. After evaporation, supernatant residue was dissolved in 0.01 M HCl and injected into the HPLC system. The compounds of interest were efficiently separated on Symmetry C-8 column at room temperature, and detected with a photodiode array detector wavelength at 303 nm. The mobile phase consisted of 0.03 M dibasic potassium phosphate (pH adjusted to 6.5 with phosphoric acid), methanol, and acetonitrile (82:14:4, v:v:v), and was delivered at a flow rate of 1.0 ml/min. No interference in blank plasma or of commonly used drugs was observed, and the detection limit of MTX was 0.02 µg/ml. The relationship between MTX concentration in plasma and peak area ratio of MTX /IS was linear (R 2 ≥ 0.9997) in the range of 0.05 – 20 µg/ml. Intra-and inter-day coefficient of variations (CV) were ≤ 8.2%. Extraction recoveries of MTX and the IS were ≥ 93%, whereas Intra-day and inter-day bias (relative recovery) of MTX measurement was-8.0% to 0.8% using quality control samples and-4.0% to 1.2% as determined by back calculation from peak area ratios of the calibration curves. The method was used to assess the stability of MTX in human plasma under conditions generally encountered in clinical laboratory. Further, it was successfully applied to measure MTX level in samples obtained from a patient on MTX therapy.

A simple, precise, and rapid high performance liquid chromatography (HPLC) method for the determination of hydroxyzine level in human plasma using omeprazole as an internal standard (IS) was developed and validated. Plasma samples containing hydroxyzine were spiked with the IS; 3 ml of tert. Butyl methyl ether were added; and mixture was vortexed for 1 min and then centrifuged for 15 min at 4200 rpm. The organic layer was transferred to a clean tube and dried under and a gentle stream of nitrogen, and the residue was reconstituted in 200 µl mobile phase. The compounds of interest were efficiently separated on 4.6 x 150 mm, Atlantis dc18, 5-µm, steel column, maintaining temperature at 30°C, and were detected with a photodiode array detector set at 230 nm. The mobile phase consisted of 0.05 M Ammonium Acetate buffer (pH ꞊ 4.0, adjusted with phosphoric acid), acetonitrile, and methanol (50:35:15, v:v:v) and was delivered at a flow rate of 1.0 ml/min. No interference in blank plasma or by commonly used drugs was observed, and the detection limit of hydroxyzine was 0.015 µg/ml. The relationship between hydroxyzine concentration in plasma and peak area ratio of hydroxyzine /IS was linear (R 2 ≥0.986) in the range of 0.04 – 0.6 µg/ml. Intra-and inter-day coefficient of variations (CV) and biases were ≤10.9% and ≤11.3%, and ≤10.0 and ≤5.0 respectively. Mean extraction recovery of hydroxyzine and the IS from the plasma samples was ≥86%, Using the method, hydroxyzine was found to be stable under conditions generally encountered in the clinical laboratory (≥89% and ≥92% in processed and unprocessed samples, respectively).

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of ranitidine in human plasma was developed and validated. Using 4-aminoantipyrene as an internal standard (IS), separation was achieved on Symmetry Shield RP-18 column. The mobile phase, 0.02 M potassium phosphate (dibasic), acetonitrile, and methanol (80:10:10, v/v) was delivered at a flow rate 1.0 ml/min. The eluent was monitored spectrophotometric at 317 nm. Plasma samples were prepared using centrifree filters. No interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of ranitidine in plasma and peak area ratio of ranitidine to the IS was linear over the range of 0.03–2.0 µg/ml. The intra-day and inter-day coefficients of variation were < 8.0% and < 11.0%, and corresponding biases were < 13.4% and < 8.3%, respectively. Mean extraction recovery of ranitidine and IS from plasma samples was 99.4% and 87%, respectively. The method was used to determine ranitidine level in plasma samples obtained from healthy subject and to assess stability of ranitidine in plasma under various conditions encountered in the clinical laboratory.

A reliable ultra-performance liquid chromatography (UPLC) assay was developed for simultaneous quantification of vitamin D-2 (VD-2) and vitamin D-3 (VD-3) levels in VD-fortified milk, using laurophenone as an internal standard (IS). Milk samples were prepared by overnight incubation with a mixture of ethanol and 50% potassium hydroxide, extracted with hexane, and cleaned-up with ethanol and 5% potassium hydroxide. After evaporation, the residue was dissolved in methanol, centrifuged, and 5.0 µl of the clear solution was injected into Acquity UPLC BEH C 18 column. The mobile phase, composed of methanol, acetonitrile, and water (80:10:10, v:v:v), was used at a flow rate of 0.35 ml/min and a run time of 15 minutes Eluents were monitored by photodiode array detector, with the wavelength set at 265 nm. The relationship between the concentration of VD-2 and VD-3 and their corresponding peak area ratios to the IS was linear over the range of 2.5 – 60 ng/ml. Mean extraction recovery for VD-2, VD-3, and IS was 93%, 94% and 70%, respectively. Stability of VD-2 and VD-3 before and after extraction was studied under various laboratory conditions. Further, the method was successfully used to measure vitamin D levels in milk samples from the local market.