Quantitative analysis of l-alpha-acetylmethadol, l-alpha-acetyl-N-normethadol, and l-alpha-acetyl-N,N-dinormethadol in human hair by positive ion chemical ionization mass spectrometry.

Abstract

A sensitive and specific method was developed for the quantitative analysis of l-alpha-acetylmethadol (LAAM), l-alpha-acetyl-N-normethadol (norLAAM), and l-alpha-acetyl-N,N-dinormethadol (dinorLAAM) in hair. In the development of this method, it was determined that sample pretreatment methods performed by the laboratory greatly affect the measured concentrations of drug and metabolite in hair. Deuterated internal standards were added to 20-mg hair samples and the samples digested overnight in a buffered solution of Protease Type VIII enzyme. Digests were extracted by modification of a liquid-liquid extraction procedure developed previously in our laboratory for the analysis of plasma and tissues. Derivatized extracts were analyzed on a Finnigan MAT 4500 mass spectrometer in positive ion chemical ionization mode using methane and ammonia reagent gases, helium carrier gas, and a DB-5MS (30 m, 0.25-micron film thickness) capillary column. The assay was linear to 50 ng/mg hair (r = 0.99) for all three compounds with a limit of quantitation experimentally determined to be 0.5 ng/mg for LAAM and 0.3 ng/mg for norLAAM and dinorLAAM. Intra-assay precision ranged from 1.0 to 10.5% for the three analytes at concentrations of 0.5, 5.0, and 25.0 ng/mg of hair. Interassay precision ranged from 4.7 to 12.9%. The performance of the method was also evaluated for its utility in detecting and quantitating LAAM, norLAAM, and dinorLAAM in hair from rats (n = 6) that had been administered 3 mg/kg LAAM intraperitoneally once daily for five days. LAAM, norLAAM and dinorLAAM were detectable in pigmented hair at concentrations of 1.27 ng/mg (+/-0.04), 1.28 ng/mg (+/-0.014), and 2.89 ng/mg (+/-0.014), respectively. Five laboratory wash solvents were then evaluated for their effect on the measured concentration of LAAM and metabolites in the rat hair. Phosphate buffer and 1% SDS washes substantially reduced the measured LAAM, norLAAM, and dinorLAAM concentrations by at least 30%, which suggests that drug incorporated into hair is removed (extracted) during the laboratory wash procedures. Wash procedures using methanol, methylene chloride, or water reduced the measured concentrations by no more than 20%. Because measured concentrations of LAAM, norLAAM, and dinorLAAM in hair appear to depend on the specific wash procedures used by a laboratory, quantitative data must be interpreted cautiously based on the sample pretreatment conditions.