Muhammad M. Hammami

A simple, precise, and rapid high performance liquid chromatography (HPLC) method for the determination of acetaminophen level in human plasma using caffeine as an internal standard (IS) was developed and validated. 0.5 ml plasma samples containing acetaminophen were mixed with 50 µg of the IS. After adding 30 µl of 50% perchloric acid, the mixture was vortexed for one minute and then centrifuged for 5 minutes at 13200 rpm. The clear supernatant was transferred into an auto-sampler vial and 100 µl was injected into the HPLC system with a run time of 7.0 min. The compounds of interest were efficiently separated on Symmetry C18 (4.6 x 150 mm, 5-µm) column, and were detected with a photodiode array detector set at 245 nm. The mobile phase consisted of water, methanol, and acetonitrile (80:10:10, v:v:v) and was delivered at a flow rate of 0.9 ml/min. No interference in blank plasma or by commonly used drugs was observed; and the detection limit of acetaminophen was 0.05 µg/ml. The relationship between acetaminophen concentration in plasma and peak area ratio of acetaminophen /IS was linear (r2 ≥ 0.9991) in the range of 0.1– 40 µg/ml. Intra- and inter-day coefficient of variations (CV) and biases were ≤11.6%  and  ≤10.8%, and ≤≤14.0 and ≤12.8, respectively. Extraction recovery of acetaminophen and the IS from the plasma samples was ≥99% and 86%, respectively. Using the method, acetaminophen was found to be stable under conditions generally encountered in the clinical laboratory (≥99% and 91% in processed and unprocessed samples, respectively). Further, the method was successfully used to measure acetaminophen level in plasma samples from a healthy volunteer.

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of meloxicam in human plasma was developed and validated. Using piroxicam as an internal standard (IS), separation was achieved on Symmetry shield RP-18 column. 0.5 ml plasma samples were prepared by protein precipitation using trifluoroacetic acid and acetonitrile. The mobile phase consisted of 0.025 M dibasic potassium phosphate (pH=6.0, adjusted with phosphoric acid), methanol, and acetonitrile (73:5:22, v:v:v) and was delivered at a flow rate of 1.5 ml/min. Eluents were measured using photodiode array detector set at 355 nm. Under these conditions, no interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of meloxicam in plasma and peak height ratio of meloxicam to the IS was linear over the range of 0.05-2.0 μg/ml. Intra-day and inter-day coefficient of variations (CV) and biases were ≤ 6.0% and ≤ 8.6%, and ±5.9% and ±5.3%, respectively. Extraction recovery of meloxicam and the IS from plasma was ≥80% and 98%, respectively. The method was applied to assess the stability of meloxicam under various clinical laboratory conditions. In processed samples, meloxicam was stable for at least 24 hours at room temperature (≥ 82%) and 48 hours at -20C (≥ 95%). In unprocessed sample it was stable for at least 24 hours at RT (≥ 82%), 16 weeks at -20ºC (≥ 87%), and after three freeze-thaw cycles (≥ 90%). The method is suitable for clinical and bioavailability investigation involving meloxicam concentration in the therapeutic range.

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of hydrochlorothiazide (HCT) in human plasma was developed and validated. Using hydroflumethiazide as an internal standard (IS), separation was achieved on Atlantis dC 18 column. The mobile phase, 10 mM monobasic potassium phosphate and acetonitrile (80:20, v: v), was delivered at a flow rate of 1.2 ml/min. The eluent was monitored by photodiode array detector, with the wavelength set at 272 nm. Plasma samples containing HCT and IS were extracted with methyl tert butyl ether and reconstituted in mobile phase. No interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of HCT in plasma and peak area ratio of HCT to the IS was linear over the range of 5-300 ng/ml. The intra-day and inter inter-day coefficient of variation and bias were