Determination of pivaloylcarnitine in human plasma and urine by high-performance liquid chromatography with fluorescence detection

Abstract

A high-performance liquid chromatographic (HPLC) method was developed for the determination of pivaloylcarnitine, one of the major metabolites of pivaloyloxymethyl (+)-(6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2- pentenamido]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2- ene-2-carboxylate hydrochloride hydrate (S-1108), an oral cephem antibiotic, in human plasma and urine. Fluorescence detection was done with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone as the labeling reagent. Pivaloylcarnitine and cyclopropylacetylcarnitine, the internal standard, were selectively fractionated from plasma or urine on a disposable cation-exchange column. Derivatization was completed in 20 min at 40 degrees C in the presence of N,N'-diisopropylethylamine as the catalyst. A column-switching device was used to remove the excess reagent for HPLC analysis. The recovery of pivaloylcarnitine was greater than 98%, and average within-day and between-day coefficients of variation were less than 5% at concentration ranges of 0.05-2 micrograms/mL for plasma and 5-500 micrograms/mL for urine. Detection limits were 0.02 micrograms/mL for plasma and 1 micrograms/mL for urine. The urinary recovery of pivaloylcarnitine was 94% after the administration of S-1108, a result that suggested that S-1108 was almost quantitatively converted to pivalic acid and then conjugated with carnitine.