Determination of benzyl alcohol and its metabolite in plasma by reversed-phase high-performance liquid chromatography

A study undertaken following recent reports of deaths in neonatal children associated with the use of benzyl alcohol resulted in the development of a stability-indicating high-performance liquid chromatographic assay of benzyl alcohol in plasma using benzocaine as internal standard. Thawed plasma samples were diluted and subjected to solid-phase extraction using Extrelut and eluted with ethyl acetate. The evaporated eluate was reconstituted with mobile phase and chromatographed on a C18 column with water-acetonitrile-glacial acetic acid as mobile phase and detection at 254 nm. Baseline separation was achieved within 12 min for benzyl alcohol, benzaldehyde, benzoic acid, hippuric acid and benzocaine. Peak-height ratios were linear over 80-640 ng of benzyl alcohol injected (r = 0.998) and over 10-80 ng of benzoic acid injected (r = 0.999). Benzaldehyde and hippuric acid were not quantitated because these compounds were not detectable in actual dog plasma. Validation studies by spiking dog plasma with benzyl alcohol and benzoic acid gave overall percent recoveries (+/- relative standard deviation, n = 4) of 98.3 +/- 3.0 and 101.4 +/- 7.6%, respectively. The method was applied to the assay of actual plasma samples. Since benzyl alcohol is very susceptible to oxidation to benzaldehyde and benzoic acid, its purity in bulk liquid samples can be determined by this method.