Abstract

DETERMINATION OF CEPHALEXIN LEVEL AND STABILITY IN HUMAN PLASMA BY FULLY VALIDATED RAPAID HPLC ANALYSIS

Rajaa Farhan Hussein and Muhammad M. Hammami*

ABSTRACT

We optimized and validated a simple, precise, and rapid reversed phase high performance liquid chromatographic (HPLC) assay for cephalexin measurement in human plasma and applied it in a clinical and laboratory settings. Plasma samples containing cephalexin were spiked with cefuroxime (internal standard, IS), and precipitated with trichloroacetic acid in methanol. After centrifugation, the clear supernatant was injected into the HPLC system. The components of interest were efficiently separated on 4.6 x 150 mm, Atlantis dC18, 5 µm (particle-size) steel column, using a Guard Pak pre-column module with Nova-Pak C18, 4 µm insert. The mobile phase consisted of a mixture of equal volumes of 0.01 M of cetyltrimethylammonium bromide and 0.01 M dipotassium hydrogen phosphate (pH = 6.5, adjusted with phosphoric acid), acetonitrile, and triethylamine (60:40:0.001, v:v:v). The analytes were detected using Waters 2998 photodiode array detector set at 260 nm. The response was linear (R2  0.9951) in the range of 0.5–120 µg/ml, the intra- and inter-day coefficient of variations (CV) were ≤ 3.1% and ≤ 3.4%, respectively. Extraction recoveries for cephalexin and the IS were ≥ 94% and 88%, respectively, whereas bias of cephalexin measurement was 1 %-5% using quality control samples and 3%-12% as determined by back calculation from peak area ratios of calibration curves. In human plasma, cephalexin was stable (≥ 93%) for at least 24 hours at room temperature in plasma; for at least 24 hours at room temperature (≥ 96%) or 48 hours at – 20ºC (≥ 98%) after processing; after 3 cycles of freeze at –20ºC and thaw at room temperature (≥ 92%); and for at least 8 weeks at –20ºC (≥ 99%). Stock solutions of cephalexin and the IS (1 mg/ml in methanol) were stable for at least 8 weeks at –20ºC (101% and 89%). The method was successfully used to measure cephalexin level in samples obtained from a healthy volunteer.

Keywords: Cephalexin, Cefuroxime, HPLC, Validation, Stability.