Morphine and 6-monoacetylmorphine in hair of heroin users: use of invalid extraction procedures generates erroneous conclusions.

A paper in the October 2003 issue of the Journal, Opiate Recidivism in a Drug-Treatment Program: Comparison of Hair and Urine Data, by B.K. Charles et al. (1) concludes the following: 1. hair results of heroin use disagreed with the urine data in most cases; 2. hair was a poor matrix for routine use in recidivism monitoring; and 3. urine appeared to be a more sensitive indicator of changes in the pattern of drug use during the course of clinical treatment. The conclusions of the authors are unwarranted in light of serious flaws in their procedures. First, as stated in the paper, recoveries of morphine from fortified or spiked drug-free hair samples were very low, between 32% and 38%. Further, in the more challenging case of hair containing opiates because of use, the authors did not attempt to validate the efficiency or completeness of recovery of morphine from the matrix of the hair with their method (overnight shaking in acetonitrile at room temperature). Additionally, because they did not wash the hair samples before analysis, they could not distinguish between Table I. Use of Acetonitrile in Extraction of Morphine drug on the surface of the hair and that deposited by drug use. and 6-MAM from Hair of Heroin Users To test their procedures, we attempted to extract heroin users' hair samples using their method. Four known opiateCodeine Morphine 6-MAM positive samples (12 mg each) were washed by the wash Sample Analysis (ng/10 mg hair) procedures used routinely in our laboratory (2,3). For this experiment, all the washes were saved and analyzed by liquid 1 Drug in all washes 0.00 0.00 1.87 chromatography-tandem mass spectrometry (LC-MS-MS). The Drug in acetonitrile 0 0 0 hair was dried after the washes, to replicate the physical state of Drug in digested hair 0.8 6.28 11.65 unwashed samples used by Charles et a]. (1). In accordance with the procedures presented in the paper, after being cut into small 2 Drug in all washes 2.57 5.45 20.28 pieces, the hair in each tube was covered with 1 mL of Drug in acetonitrile 0 0 0.14 Drug in digested hair 1.66 5.42 6.95 acetonitrile and the tubes mixed by rotation overnight at room temperature (~ 22~ After 15 h acetonitrile extraction, the 3 Drug in all washes 0.08 0.53 2.56 acetonitrile was removed from the hair and analyzed by Drug in acetonitrile 0 0 0 LC-MS-MS for the opiate class of drugs, including codeine, Drug in digested hair 3.54 16.11 30.69 morphine, and 6-acetylmorphine (6-MAM). The hair was then enzymatically digested at pH 6.65 for 6 h (4), after which the 4 Drug in all washes 0.38 2.50 28.36 digested hair was centrifuged and the supernatants extracted for Drug in acetonitrile 0 0 0.07 LC-MS-MS analysis. Drug in digested hair 3.8 4.76 5.13 The results in Table I show the amounts of codeine, morphine, and 6-MAM removed from the hair by the washes, by the acetonitrile, and by extraction of digested hair after washing and acetonitrile extraction. The phosphate buffer washes removed some surface contamination from all four samples, less in the case of morphine (0, 5.45, 0.53, and 2.50 ng/10 mg hair) than for 6-MAM (1.87, 20.28, 2.56, and 28.36 ng/10 mg hair). With thorough washing having removed the surface contamination, the acetonitrile 15-h "extraction" contained essentially no drug. After digestion of the hair sample, which ensures release of the drug from the hair matrix, the true amount of the drugs in the hair can be measured: 6.28, 5.42, 16.11, and 4.75 ng morphine/10 mg hair and 11.65, 6.95, 30.69, and 5.13 ng 6-MAM/10 mg hair in samples 1-4, respectively.