6-acetylmorphine detection in postmortem cerebrospinal fluid.

Opiate testing is one of the most frequently requested assays in the death investigation of suspected drug overdoses. Screening assays typically target morphine, whereas more specific assays are able to positively identify and quantitate the particular opiate present in the specimen. Because many opiates are metabolized to morphine, it is often desirable to differentiate heroin-related deaths from other opiate fatalities. The only definitive method of achieving this is to monitor heroin or 6-acetylmorphine (6-AM) or both. Because of its short half-life and instability in aqueous media, heroin is not typically monitored. Therefore, the detection of its active metabolite, 6-AM, is of primary importance in order for the pathologist to determine if recent heroin exposure caused or was a contributing factor in an individual's death. The usefulness of 6-AM in blood as a specific marker for recent heroin use depends upon several factors. These include the availability of a sensitive and specific analytical method for the detection of 6-AM in biological specimens and an adequate detection time for 6-AM in blood. Recently, several assays have been reported for the detection of 6-AM in biological matrices (1,2). The second factor is more problematic. Studies have reported various elimination half-lives for 6-AM in blood depending upon the route of drug administration. Jenkins et al. (3) reported the mean elimination half-lives of 6-AM as 5 and 9 rain, respectively, after smoked and intravenous administration of heroin. After intranasal administration, the half-life of 6-AM was reported as 11 rain (4). A controlled clinical study (3) investigating the pharmacokinetics of smoked and intravenously administered heroin found that 6-AM appeared in blood 1 rain after drug administration and peaked 1-2 rain after smoking and 2-5 rain after intravenous administration. In addition, concentrations of 6-AM reached the limit of detection (1 ng/mL) of the assay within 2 h of administration by both routes. Another study (4) investigating the pharmacokinetics of intranasal heroin found peak 6-AM concentrations occurred 5-10 rain after heroin administration. Blood 6-AM concentrations reached the assay limit of detection between 30 and 90 rain. 6-AM has been detected in urine for 2-8 h after a single 6-rag intramuscular dose of heroin hydrochloride (5). However, detection of 6-AM in urine only indicates exposure and cannot be correlated with pharmacological effects. These data indicate that a sensitive assay is necessary to reliably detect 6-AM in blood and that the window of detection after exposure is short. We recently conducted a study to evaluate the usefulness of cerebrospinal fluid (CSF) in detecting 6-AM. Data were collected from cases submitted to the Cuyahoga County Coroner's Office (Cleveland, OH) for autopsy. We collected data on cases (N = 65) in which blood and CSF were assayed for opiates. Blood was collected from the heart and placed in screw-cap glass tubes containing sodium fluoride (200 rag) and potassium oxalate (400 rag). CSF was collected by cisternal puncture and stored in greytop VACUTAINER | tubes containing 20 mg potassium oxalate and 25 mg sodium fluoride. Specimens were refrigerated at 4~ until analysis. Injection site tissue was analyzed from cases in which it was submitted. These specimens were soaked in methanol for 24 h before extraction. 6-AM and other opiates were identified and quantitated in these matrices by liquid-liquid extraction using deuterated internal standards followed by gas chromatographic-mass spectrometric selected ion monitoring of the trimethylsily] derivatives. The limit of quantitation for morphine, codeine, hydrocodone, and 6-AM was i ng/mL. The assay was linear, 1-I000 ng/mL, for all analytes except 6-AM; it was 1-200 ng/mL for 6-AM. Results are illustrated in Table I. Cases are listed ckronologically. Morphine was detected in the blood of every case at concen-