Rapid Determination of Acetaminophen levels in Human plasma by High performance liquid chromatography

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.