Methadone disposition in oral fluid during pharmacotherapy for opioid-dependence.

INTRODUCTION: Oral fluid testing is widely used for detecting drug exposure, but data describing methadone and metabolites in oral fluid during pharmacotherapy for opioid-dependence are relatively limited. METHODS: 414 oral fluid specimens from 16 opioid-dependent pregnant women receiving daily methadone were analyzed for methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and methadol by liquid chromatography-mass spectrometry. RESULTS: All oral fluid specimens contained methadone greater than 1 ng/mL; 88% were positive for EDDP and 12% for methadol. Over 95% of oral fluid specimens exceeded the 20 ng/mL methadone cutoff set by the European Driving Under the Influence of Drugs, Alcohol and Medicines (DRUID) study. Methadone and EDDP oral fluid concentrations were highly variable within and between participants, did not predict methadone dose, but were negatively correlated with pH. CONCLUSION: Methadone was readily identified in oral fluid at concentrations greater than 20 ng/mL following daily 30-110 mg/day methadone pharmacotherapy. As no specimens contained only EDDP or methadol, there was no advantage to including these analytes for identification of methadone exposure. As nearly all oral fluid specimens from methadone-maintained patients exceeded the DRUID guideline, the 20 ng/mL cutoff appears to be sensitive enough to detect daily methadone exposure; however, additional indicators of behavioral and/or motor impairment would be necessary to provide evidence of driving impairment.

N-demethylation of levo-alpha-acetylmethadol by human placental aromatase.

Levo-alpa-acetylmethadol (LAAM) is a methadone derivative used to treat the opiate addict. We previously reported on the kinetics for transplacental transfer of LAAM and its levels in the fetal circuit using the technique of dual perfusion of the placental lobule. The aim of this investigation was to identify the enzyme responsible for the biotransformation of LAAM and norLAAM and the metabolites formed in the term human placenta. Placental microsomes exhibited higher activities than the mitochondrial and cytosolic fractions in metabolizing LAAM to norLAAM. None of these subcellular fractions catalyzed the formation of dinorLAAM from either LAAM or norLAAM as determined by HPLC/UV. Evidence obtained from the effects of cytochrome P450 (CYP) inhibitors on the demethylation of LAAM to norLAAM by placental microsomes suggested that CYP 19/aromatase is the major enzyme involved. Out of 10 monoclonal antibodies raised against various CYP isoforms, only that for aromatase caused over 80% inhibition of norLAAM formation. The biotransformation of LAAM to norLAAM exhibited monophasic kinetics with apparent Km and Vmax values of 105 +/- 57 microM and 86.8 +/- 15.6 pmol mg(-1) protein min(-1), respectively. The kinetic profile determined for a cDNA-expressed CYP 19 metabolism of LAAM to norLAAM was similar to that determined for placental microsomes. Taken together, the above data indicate that CYP 19/aromatase is the enzyme responsible for the N-demethylation of LAAM to norLAAM in term human placentas obtained from healthy pregnant women.

Determination of l-alpha-acetylmethadol (LAAM), norLAAM, and dinorLAAM in clinical and in vitro samples using liquid chromatography with electrospray ionization and tandem mass spectrometry.

l-Alpha-acetylmethadol (LAAM) is an alternative to methadone for the maintenance treatment of opioid dependence. LAAM has a longer therapeutic half-life than methadone, primarily because it is metabolized to more active metabolites, norLAAM and dinorLAAM. We have developed a liquid chromatography-tandem mass spectrometry method capable of measuring LAAM and its metabolites, norLAAM and dinorLAAM, at lower concentrations with 1.0-mL aliquots of plasma (range of 0.25 to 100 ng/mL) or higher concentration with 0.2-mL aliquots of plasma (range 1.25 to 500 ng/mL). It has acceptable precision and accuracy across both linear ranges, as well as in the urine matrix. Results from this assay correlate well with our previously validated gas chromatograghy-mass spectrometry method. All analytes had acceptable stability after three freeze-thaw cycles, room temperature storage for 20 h, or storage of extracts either at -20 degrees C for 6 days or on the autosampler (10 degrees C) for 4 days. The pharmacokinetics of LAAM, norLAAM, and dinorLAAM were determined for the first time in three male opioid-naive individuals receiving a single oral dose of 5 mg LAAM/70 kg. Using this method, we could monitor the in vitro N-demethylation of LAAM and norLAAM at substrate concentrations in the therapeutic range of 0.5 and 1.0 microM by cDNA-expressed cytochrome P450s. This confirmed the involvement of cytochrome P450s 3A4, 2B6, 2C8, and 2C18 at therapeutic concentrations of LAAM. An accurate and precise method for determination of LAAM and its metabolites, norLAAM and dinorLAAM, that is suitable for both in vivo and in vitro metabolism studies has been developed and validated.

Toxicologic aspects of heroin substitution treatment.

Heroin abuse is an international problem with which all countries must continually cope. Many countries have implemented heroin substitution therapy as an effective means of decreasing illicit heroin use, crime, HIV risk, and death, and in improving employment and social adjustment. Although methadone is the most commonly used medication for heroin substitution, other agonists in current use include levomethadyl acetate (LAAM), buprenorphine, and pharmaceutical-grade heroin. This report reviews toxicologic issues that arise in these programs. A broad array of testing methodologies are available that allow selection of on-site testing or laboratory-based methodology. Urine specimens may be monitored for nonprescribed drugs on a qualitative or semiquantitative basis. Methods for differentiating opiate sources by urinalysis have been proposed to distinguish poppy seed consumption from heroin abuse and for distinguishing pharmaceutical-grade heroin from illicit heroin. Therapeutic drug monitoring for methadone in plasma continues to be evaluated for use in establishing adequate dosing and detecting diversion, and new methods have been devised for measurement of the optical isomers of methadone in plasma. Biologic specimens, in addition to plasma and urine, have been evaluated for use in drug monitoring, including sweat, hair, and oral fluid, with promising results. Overall, the many recent developments in testing methodology provide more effective means to assess patients in heroin substitution programs and should contribute to improvements in public health.

Incorporation of drugs for the treatment of substance abuse into pigmented and nonpigmented hair.

Hair analysis for drugs may be useful for the long-term monitoring of recidivism and treatment compliance. L-alpha-Acetylmethadol, buprenorphine, and methadone are drugs that are used for the treatment of substance abuse. The purpose of this study was to study the relationship between dose, plasma concentration, hair concentration, and hair pigmentation for these compounds and their major metabolites in an animal model. Male Long-Evans rats received either L-alpha-acetylmethadol (1 and 3 mg/kg; n = 6), buprenorphine (1 and 3 mg/kg; n = 5), or methadone (4 and 8 mg/kg; n = 5) by intraperitoneal injection daily for 5 days. Fourteen days after beginning drug administration, newly grown hair was collected and analyzed for either L-alpha-acetylmethadol and two metabolites (L-alpha-acetyl-N-normethadol and L-alpha-acetyl-N,N-dinormethadol), methadone and two metabolites (D,L-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium and D,L-2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline), or buprenorphine and one metabolite (norbuprenorphine). The plasma time course (AUC) for each compound was also determined after a single administration of each drug at the specified doses. There was an approximate dose-dependent increase in measured hair concentration of each parent drug in pigmented hair. The concentrations of L-alpha-acetylmethadol, methadone, and buprenorphine in nonpigmented hair were significantly less than that measured in pigmented hair at either the high or low dose. The metabolites L-alpha-acetyl-N-normethadol and D,L-2-ethyl-1,5dimethyl-3,3-diphenylpyrrolinium were detected at lower concentrations than their respective parent compounds (L-alpha-acetylmethadol or methadone) in pigmented hair. However, the L-alpha-acetyl-N,N-dinormethadol metabolite concentrations in pigmented hair were significantly greater than those of the parent drug after either the low or the high L-alpha-acetylmethadol dose. These data demonstrate that L-alpha-acetylmethadol, methadone, buprenorphine, and metabolites are distributed into hair in a dose-related manner with a preference for pigmented hair.

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.

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.

Cumulation of active metabolites of levo-alpha-acetylmethadol in the rat fetus and neonate.

Levo-alpha-acetylmethadol (LAAM, 0.2 or 2.0 mg/kg/day) was orally administered to female Sprague-Dawley rats for one month prior to and throughout pregnancy. The rats were killed on the 18th day of gestation along with a group of 18-day pregnant females given a single oral 2.0 mg/kg dose of LAAM 24 hours earlier. Although cumulation of LAAM or its active metabolites was not seen in plasma or brain of pregnant rats given drug chronically, significant cumulation was observed in whole fetus and in fetal brain. In addition, a 2-3 fold elevation in the concentrations, and an even greater elevation of total content, was noted in the newborn pup. These data suggest that opiate intoxication soon after birth may be a factor responsible for the increased morbidity and mortality of rat pups prenatally exposed to LAAM.

Determination of acetylmethadol and metabolites by use of high-performance liquid chromatography.

A method is described for the simultaneous determination of 1, alpha-acetylmethadol (LAAM) and five active metabolites--noracetylmethadol, dinoracetylmethadol, methadol, normethadol, and dinormethadol--in biofluids by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are recovered from biofluids by a multistep liquid--liquid extraction. The mobile phase is methanol--acetonitrile (70:30, v/v) containing 0.015% ammonium hydroxide as the modifier. Retention times can be varied by adjusting the composition of the mobile phase to maximize peak height for quantitation using l-propranolol as the internal standard or peak separation for the collection of fractions. Using a UV detector the lower limit of sensitivity is 10 ng/ml of biofluid. Using fraction collection of radiolabeled drug and metabolites followed by liquid scintillation counting the lower limit of sensitivity is 1.0 ng/ml. Commonly used or abused narcotics including morphine, heroin, meperidine, methadone and propoxyphene do not interfere with the analysis. The method has been applied to plasma and urine samples from humans, sheep and rats. Extracts of urine from patients receiving maintenance treatment with LAAM contain LAAM and each of the five active metabolites.

Stereospecific radioimmunoassay of alpha-l-acetylmethadol (LAAM).

A sensitive and specific radioimmunoassay has been developed which allows quantitation of picogram amounts of alpha-l-acetylmethadol (LAAM) in biological fluids without prior extraction. Antiserum was obtained from rabbits immunized with antigen prepared by conjugation of alpha-l-methadol hemisuccinate with thyroglobulin. The antiserum was stereospecific, distinguishing LAAM from its three stereoisomers. In addition, there was little cross-reaction in the assay by metabolites of LAAM and no interference with the assay by other narcotic analgesics. The sensitivity of the assay was 50 pg, requiring serum sample sizes as small as 10 microliters. The radioimmunoassay was used to determine serum concentrations of LAAM in five female dogs receiving i.v. injections of 2 mg/kg of LAAM. The decay of LAAM in serum appeared to be multiexponential, with a mean terminal half-life of 35 hr. The dogs were behaviorally depressed for at least 9 hr and hypothermic for at least 24 hr after injection. The persistence of these pharmacological effects was probably due to the slow elimination of LAAM and the presence of major metabolites which are pharmacologically active.

Isotope dilution technique for the estimation of l-alpha-[2-3H]-acetylmethadol and its metabolites in biological samples.

An isotope dilution technique for the determination of l-alpha-(2-3H)-acetylmethadol and its metabolites from biological samples is described. The parent drug and metabolites were extracted from biological samples with chlorobutane after the addition of unlabeled internal standards. The extracts were purified by two-dimensional thin-layer chromatography for quantification by gas-liquid chromatography and scintillation counting. Dinoracetylmethadol (NNAM) was found to be converted to 6-acetamido-4,4-diphenyl-3-heptanol (AMIDE) during the extraction procedure. The conversion of NNAM to AMIDE was confirmed by gas chromatography-mass spectrometry.

The quantitative analysis of 1-alpha-acetylmethadol and its principal metabolites in biological specimens by gas chromatography-chemical ionization-multiple ion monitoring mass spectrometry.

1-alpha-acetylmethadol (LAAM) is a new drug under development for the treatment of heroin dependence. A new analytical method applicable to the accurate biodispositional study of the drug and its metabolities is described and critically discussed in this report. The procedure involves sample preparation and direct organic solvent extraction using eta-butyl chloride, amide derivatization by molecular rearrangement, and gas chromatography-chemical ionization mass spectrometry-selected ion monitoring, with methane as the carrier and ammonia as reagent gases. Deuterated (d3 stable isotopes of LAAM and its metabolites are used as internal standards. The method is free from qualitative interferences and has quantitative sensitivity to 5 ng/ml for 2.0 ml samples with 10-15% accuracy and precision in the range 5-100 ng/ml; and 2-5% at concentrations up to 750 ng/ml. Specimens of plasma, whole blood, urine, bile, brain, liver, and other visceral samples have been successfully analyzed, as well as in vitro preparations such as hepatic microsomes. By appropriate data processing, the method lends itself to routine analysis and high volume work; even manually the method is capable of at least 50 samples per week. A simplified procedure for the analysis of LAAM and its metabolites in urine only is also presented and discuet up and use the methods.

Carbon-13 nuclear magnetic resonance study of the alpha- and beta- isomers of methadol and acetylmethadol hydrochlorides.

The 13C-NMR spectra reported in these studies give direct evidence for the presence of two contributing conformers for alpha-methadol hydrochloride, alpha-acetylmethadol hydrochloride and beta-acetylmethadol hydrochloride. Indirect evidence is also available for the presence of more than one conformer for beta-methadol hydrochloride. However, in order to be more descriptive about the structures of the conformers, it is necessary to obtain 13C-NMR spectra that have a higher degree of resolution than is available from our present NMR system. Such systems are available, and plans are currently in progress to obtain 13C-NMR spectra or these compounds at high enough magnetic fields and low enough temperatures to give us the necessary data.

Comparative study on the derivatization of l-alpha-acetyl-methadol metabolites for electron capture gas-liquid chromatography.

Six reagents-trichloroacetyl chloride, trichloroacetic anhydride, pentafluorobenzoyl chloride, heptafluorobutyryl chloride, heptafluorobutyric anhydride, and trifluoroacetic anhydride- were evaluated as potential derivatizing reagents for quantitating the metabolites of l-alpha-acetylmethadol (LAAM) -noracetylmethadol, dinoracetylmethadol, methadol, and normethadol-by electron capture gas-liquid chromatography. All of the reagents studied reacted quantitatively with all of the metabolites except methadol; however, trichloroacetyl chloride was found to be the most satisfactory general reagent for analyzing these metabolites in biological fluids. A gas-liquid chromatographic method is presented which combines both flame ionization and electron capture detection for quantitating plasma and urine levels of methadone, l-alpha-acetylmethadol and its metabolites.

Extraction method and thin-layer chromatographic system for the determination of alpha-l-acetylmethadol and metabolites in biological fluids.

An extraction method and thin-layer chromatographic (TLC) system for the determination of alpha-l-acetylmethadol and its known metabolites (methadol, noracetylmethadol, dinoracetylmethadol, normethadol, 6-acetamide-4,4-diphenyl-3-heptanol, and N-methyl-6-acetamido-4,4-diphenyl-3-heptanol) are described. The parent drug and metabolites are extracted from biological fluids with ethyl acetate and separated by TLC using silica gel plates and a developing system of ethyl acetate-methanol-water-ammonia (85:10:1:1). This system may be used to quantitatively determine levels of radiolabeled drug and metabolites by scraping the TLC plates into 3-mm zonal fractions and measuring the amount of radioactivity by scintillation counting. A representative radiochromatogram obtained from an extract of monkey urine is shown.

Rapid TLC determination of methadyl acetate and some in vitro metabolites.

Methadyl acetate was metabolized by microsomal preparations of rat liver to yield nor-methadyl acetate and 6-(dimethylamino)-4,4-diphenyl-3-heptanol. The identification and separation of these three compounds was established by TLC, using iodoplatinate spray as a visualizing agent.

Simultaneous determination of acetylmethadol and its active riotransformation products in human biofluids.

A method employing solvent extraction and gas-liquid chromatography has been developed for the quantitative determination of acetylmethadol simultaneously with its two major biotransformation products, noracetylmethadol and dinoracetylmethadol. Noracetylmethadol and dinoracetylmethadol are analyzed following their conversion to the corresponding amides. The amide structure is confirmed by the use of chemical ionization mass spectroscopy and infrared spectroscopy. The method can be used to determine the concentration of acetylmethadol and these compounds in plasma samples from acetylmethadol maintenance subjects. Methadol and normethadol do not attain neasurable plasma levels. Urine contains predominantly noracetylmethadol and dinoracetylmethadol. Evidence was also obtained for the urinary excretion of acetylmethadol, methadol and normethadol. A mean quantity equal to 28% of the administered dose was excreted in the urine of a 48-h dosing interval as acetylmethadol and metabolites.