Quantification of Benzoic, Phenylacetic, and Phenylbutyric Acids from Filter-Paper Blood Spots by Gas Chromatography–Mass Spectrometry with Stable Isotope Dilution

Abstract

An important treatment of hyperammonemia in urea cycle disorders is the stimulation of alternative pathways for nitrogen excretion (1). Salts of benzoic acid (BA), phenylacetic acid (PAA), and phenylbutyric acid (PBA) have been found to be useful vehicles for the elimination of nitrogen by alternative pathways (2)(3)(4). BA is conjugated with glycine to form hippurate, which is efficiently excreted in the urine. PBA is metabolized in the liver to phenylacetyl-CoA, which is then conjugated with glutamine to form phenylacetylglutamine and subsequently excreted in the urine. Furthermore, benzoate has been used to treat nonketotic hyperglycinemia (5), salts of PBA and PAA are used in clinical trials as anticancer agents (6)(7)(8)(9), and PBA has been found to stimulate fetal hemoglobin production and can potentially be used for treatment of hemoglobinopathies (10)(11). However, two patients have died in the US after being given the wrong doses of sodium benzoate and sodium phenylacetate (12), documenting the risk of toxicity, which could potentially be reduced by drug concentration monitoring. HPLC assays for PAA and PBA in plasma have limited sensitivity (4). A gas chromatographic–mass spectrometric method had improved sensitivity (13), but the method did not use stable isotopes as internal standards and the clinical utility of the method was not documented. Here, we describe a rapid, precise, and convenient method to measure blood concentrations of these drugs using dried filter-paper blood spots. BA, PAA, and PBA were purchased from Sigma-Aldrich Co. [ring- d 5]-BA, [ring- d 5]-PAA, and [2,2,3,3- d 4]-4-PBA were purchased from Cambridge Isotope Laboratories. Regisil RC-3 [ N , O -bis-(trimethylsilyl)trifluoroacetamide containing trimethylchlorosilane (10:1 by volume; BSTFA-TMCS)] was purchased from Regis Chemical Co. Blood collection cards (cat. no. 10538414) were purchased from Schleicher & Schuell. Blood was …