Rapid Determination of Cefazolin Levels in Human Plasma by High Performance Liquid Chromatography

A simple, precise, and rapid high performance liquid chromatography (HPLC) method for the determination of cefazolin level in human plasma using ceftriaxone as an internal standard (IS) was developed and validated. 0.25ml plasma samples containing cefazolin were mixed with 17.5 µg of the IS. After adding 0.3 ml of cold methanol kept in fridge at 4 °C, the mixture was vortexed for two min and then centrifuged for 15 min at 16000 rpm at room temperature. The clear supernatant was transferred into an auto-sampler vial and 100 µl was injected into the HPLC system with a run time of 18.0 min. The compounds of interest were efficiently separated on 4.6 x 150 mm Atlantis dC18-5µm steel column, using a Guard Pak pre-column module with Nova-Pak C185-µm insert, and detected using Waters 2998 photodiode array detector set at 270 nm. The mobile phase consisted of a mixture of 0.02 M of cetyltriethyl ammonium bromide and 0.01 M dipotassium hydrogen phosphate (pH = 6.5, adjusted with phosphoric acid) and acetonitrile, 60:40 (v:v) for 6 min, 50:50 for 6 min, and 60:40 for 6 min. It was delivered at a flow rate of 1.5 ml/min. No interference in blank plasma or by commonly used drugs was observed; and the detection limit of cefazolin was 0.1 µg/ml. The relationship between cefazolin concentration in plasma and peak area ratio of cefazolin/IS was linear (r2 ≥ 0.9979) in the range of 0.2–200 µg/ml. Intra- and inter-day coefficient of variation (CV) was ≤ 7.4%  and  ≤ 5.7%, with the corresponding bias of ≤ 4.9% and ≤ 5.1%. Mean extraction recovery of cefazolin and IS were 97%, respectively. Using the method, cefazolin was found to be stable in processed samples for 48 hrs at -20°C ( ≥ 96%), and in unprocessed samples for 8 weeks at -20°C (≥ 98%), Further, the method was successfully used to measure cefazolin level in plasma samples.