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Sweat testing for heroin and metabolites in a heroin maintenance program. Pascal Kintz,1* Rudolf Brenneisen,2 Petra Bundeli,2 and Patrice Mangin1.
Clinical Chemistry 43:5 736 –739 (1997)

Drug Monitoring and Toxicology

Sweat testing for heroin and metabolites in a heroin maintenance program Pascal Kintz,1* Rudolf Brenneisen,2 Petra Bundeli,2 and Patrice Mangin1 men for testing. Thermal [2 ] or pharmacological [3 ] stimulations were proposed to help subjects secrete an unusually large amount of sweat. Occlusive bandages consisting of one to three layers of filter paper [4 ] or pieces of cotton, gauze, or towel [5 ] were proposed for capturing sweat. Significant advances have been made in developing a sweat patch technology. One of the first applications of the sweat patch was to monitor alcohol consumption [6 ]. The sweat patch design in this approach occluded the skin, leading to skin irritation, alteration of the steady-state pH of the skin, and alteration of the species of bacteria that colonize the skin [7 ]. However, new nonocclusive wound dressings have been recently developed by Sudormed™ (Santa Ana, CA) and marketed by Pharmchem™ Labs. (Menlo Park, CA) under the name Pharm-Chek™. The sweat patch acts as a specimen container for nonvolatile and liquid components of sweat, including drugs of abuse. A unique number imprinted on each patch aids with chain of custody and identification. Sweat components are collected on a special absorbent pad, located in the center of the patch. Nonvolatile substances from the environment cannot penetrate the transparent film—a semipermeable membrane over the pad that allows oxygen, water, and carbon dioxide to pass through the patch and leaves the skin beneath healthy. Worn over a period of several days, the pad becomes saturated with sweat and slowly concentrates it; drugs present in the sweat are retained. The collection pad has a surface of ;14 cm2 and collects at least 300 mL per day of insensible perspiration in a 22 °C environment. Exercise, higher temperatures, or other factors that increase sweating increase the amount of sweat collected. To date, few applications of the sweat patch have been published; these applications include tests for cocaine [8 –11 ], opiates [9, 11, 12 ], benzodiazepines, [11, 13 ], barbiturates [12 ], cannabis, buprenorphine, and methylenedioxyethylamphetamine [11 ]. In these studies, the various authors concluded that sweat testing offered a relatively noninvasive method for obtaining a cumulative estimate of drug exposure over several days. In two papers [9, 11 ], heroin exposure was docu-

Recent advances in sensitive analytical techniques have enabled the analysis of drugs in unconventional biological materials such as sweat. In a study conducted during a heroin maintenance program, 14 subjects had sweat patches applied, then received intravenously two or three doses of heroin hydrochloride ranging from 80 to 1000 mg/day. The sweat patch was applied 10 min before the first dosage and removed ;24 h later, minutes before the next dosage. Absorbent pads were stored at 220 °C in plastic tubes until analysis. The target drugs were extracted in 5 mL of acetonitrile in the presence of 100 ng each of heroin-d9, 6-acetylmorphined3, and morphine-d3. After agitation for 30 min, the acetonitrile solution was divided into two portions: 2 mL for heroin testing and the remainder for testing for the other compounds. After evaporation, the residue of the first portion was reconstituted in 35 mL of acetonitrile; the second was derivatized by silylation with 40 mL of N,O-bis(trimethylsilyl)trifluoroacetamide containing 10 mL/L trimethylchlorosilane. Drugs were analyzed by GC-MS in electron impact mode. Concentrations (nanograms per patch) ranged from 2.1 to 96.3 for heroin, 0 to 24.6 for 6-acetylmorphine, and 0 to 11.2 morphine. Except in one case, heroin was the major drug present in sweat, followed by 6-acetylmorphine and morphine. We observed no correlation between the doses of heroin administered and the concentrations of heroin measured in sweat. INDEXING TERMS: •

drugs of abuse

gas chromatography–mass spectrometry • morphine

It has been long known that many drugs are excreted in sweat. Analysis of sweat for methadone was first reported in 1973 [1 ], but until recently no one developed a practical solution to the problem of collecting an adequate speci-

1

Institut de Me´decine Le´gale, 11 rue Humann, 67000 Strasbourg, France. Institute of Pharmacy, Baltzerstr. 5, 3012 Bern, Switzerland. * Author for correspondence. Fax 33.3.88.24.00.85. Received September 10, 1996; revised December 3, 1996; accepted December 20, 1996. 2

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mented in sweat by targeting the parent drug, or 6-acetylmorphine, or both. The aim of the present study was to evaluate excretion of heroin and its metabolites in sweat after the intravenous administration of controlled heroin.

Materials and Methods specimen collection Sweat was collected from 14 Caucasian subjects [13 men, 1 woman, ages 27– 45 years and weighing 57 kg (the woman) and 65–94 kg (the men)] by using the PharmChek sweat patch. These subjects were drug addicts taking part in the Swiss Heroin Maintenance Program in Bern; they had been in the heroin program at least 6 months (most for 9 months). The subjects self-administered, intravenously under controlled conditions, heroin hydrochloride in 2 or 3 doses per day: from 0730 to 0900, from 1200 noon to 1315, and from 1730 to 2015. Heroin dosage totaled 80 –1000 mg/day, with the highest single dose being 150 mg. Subjects had to wait at least 30 min to get another dose, to avoid depression in breathing. The sweat patch was applied to the outer portion of the upper back 10 min before the first administration. The selected skin site for patch placement was gently cleaned with a 70% isopropanol swab before application. The patch was removed 24 later, just before a new heroin administration, by pulling an edge of the adhesive backing, taking care not to touch the absorbent pad. After removal of the patch, the pads were stored separately in sealed plastic tubes at 220 °C until analysis within 3 weeks.

chemicals Acetonitrile was of HPLC grade (Merck, Darmstadt, Germany). All drugs and deuterated internal standards (heroin-d9, 6-acetylmorphine-d3, and morphine-d3) were purchased from Radian (Austin, TX). N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) plus 10 mL/L trimethylchlorosilane (TMCS) was purchased from Interchim (Montluc¸on, France).

with an HP 5989 B engine mass selective detector (all from Hewlett-Packard, Les Ulis, France). Injector temperature was 260 °C, and splitless injection was used with a splitvalve off-time of 0.75 min. The flow of helium through the column was 1 mL/min. Column temperature was programed to rise from an initial temperature of 60 °C (held 1 min), to 290 °C at 30 °C/min, and held at 290 °C for the final 6 min. The ions monitored and typical retention times of the various analytes and the deuterated internal standards are presented in Table 1.

Results and Discussion Calibration curves were constructed for each drug by adding known concentrations of drugs (2, 10, 20, 50, 100, and 200 ng) and of internal standards (100 ng) to drugfree absorption pads. The assay results were linear with the drug concentrations in the range tested (Table 2). The extraction recoveries were 71.3%, 76.4%, and 70.9% for morphine, 6-acetylmorphine, and heroin, respectively. These recoveries were lower [11 ] than those observed after methanol extraction (;90% for all of the drugs). However, when methanol was used, ;10 –20% of the heroin was converted to 6-acetylmorphine. Typically, when using acetonitrile, hydrolysis of heroin was 5%, whereas 6-acetylmorphine was stable under these conditions, giving no evidence of hydrolysis to morphine. Moreover, to ensure stability of heroin, which in contrast of 6-acetylmorphine and morphine does not require derivatization, we divided the extract into two portions for GC/MS assay. This split procedure was reported to be suitable for heroin testing in blood, urine, and saliva [14 ]. Under the chromatographic conditions used, there was no interference with the target compounds or the deuterated standards by any extractable endogenous materials present in sweat. Within-run and between-run CVs, studied after addition of 20 and 50 ng of pure substances to drug-free absorbent pads, were 13% for all drugs tested. The limits of detection were ;1.0, 1.0, and 0.5 ng/patch for mor-

analysis of sweat patches The target drugs were extracted from the absorbent pad in 5 mL of acetonitrile in the presence of 100 ng of the following deuterated internal standards: heroin-d9, 6-acetylmorphine-d3, and morphine-d3. The samples were shaken for 30 min on an orbital shaker at 200 rpm. Then the acetonitrile solution was divided into two portions: 2 mL for heroin testing and the remainder for the other compounds (6-acetylmorphine and morphine). The acetonitrile was evaporated to dryness in both vials. Heroin was not derivatizated, and the residue from the first portion was reconstituted with 25 mL of acetonitrile. The other drugs (in the second portion) were derivatizated by silylation for 20 min at 60 °C with 40 mL of BSTFA containing 10 mL/L TMCS. A 1.5-mL portion of each extract was injected through a HP5-MS capillary column [5% phenyl–95% methyl siloxane, 30 m 3 0.25 mm (i.d.)] into a Model 5890 gas chromatograph coupled

Table 1. Retention times and selected ions monitored. Ions Compound

Retention time, min

Undeuterated

Deuterated

Morphine-2-TMS 6-Acetylmorphine-TMS Heroin

10.01 10.33 10.73

401, 414, 429 340, 399 310, 327, 369

432 402 378

The ions in bold type were used for quantification.

Table 2. Analytical parameters after addition of drug analytes to drug-free absorbent pads. Compound

Linearity, r

Recovery, %

Limit of detection, ng/patch

Morphine 6-Acetylmorphine Heroin

0.996 0.994 0.995

71.3 76.4 70.9

1.0 1.0 0.5

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phine, 6-acetylmorphine, and heroin, respectively, at a signal-to-noise ratio .3. Once stored at 220 °C, drug-free pads (n 5 4) supplemented with 100 ng of each drug were stable for at least 1 month. Typically, hydrolysis of heroin and 6-acetylmorphine was ,4% over that period. Subjects wore the patch with minimal discomfort for 24 h, and each subject could continue his or her normal hygiene practices; nobody accidentally abraded the patch. No special precautions were observed during patch wear except to avoid rubbing with a towel after bathing. Tampering with the patch would be quite evident because once a patch has been applied to the skin, no more adhesive remains, so that one cannot remove and then reapply the patch. Results of the sweat patch analysis are presented in Table 3, along with the administered heroin doses. The ability of the patch to collect and contain heroin and metabolites from sweat was demonstrated. All the patches contained heroin, and except for one patch, the major analyte excreted in sweat was heroin. Concentrations ranged from 2.1 to 96.3 ng/patch for heroin, from not detected to 24.6 for 6-acetylmorphine and from not detected to 11.2 for morphine. Heroin was present at concentrations ;2– 4 times higher than 6-acetylmorphine concentrations and ;5–20 times higher than morphine. As with other drugs, the parent compound appears to be the major analyte in sweat after injection. Therefore, care is necessary to prevent the conversion of heroin or 6-acetylmorphine to morphine. No morphine 3-glucuronide or morphine 6-glucuronide was detected in the sweat patch, as analyzed by liquid chromatography with fluorometric detection (limits of detection for both compounds were ;1.0 ng/patch). Heroin was also identified in sweat collected from street addicts, as mentioned by Cone et al. [9 ] and Kintz et al. [11 ]. In those studies, Table 3. Administered heroin doses and results of sweat patch analyses. Concn., ng/patch Subject

Daily dose, mg

Heroin

6-AM

Morphine

1 2 3 4 5 6 7 8 9 10 11 12 13 14

80 650 500 400 600 310 440 200 170 500 650 1000 750 300

17.7 29.7 37.5 8.5 52.7 96.3 11.4 55.2 11.4 7.2 28.3 38.4 6.4 2.1

5.8 14.6 17.0 ND 16.5 24.6 8.6 16.4 ND 4.1 13.1 13.4 11.6 ND

ND 1.9 2.7 2.0 1.5 11.2 4.9 5.8 ND 5.9 ND ND 5.2 ND

6-AM, 6-acetylmorphine; ND, not detected (below the limit of detection).

however, heroin was seldom the major analyte identified, probably because of hydrolysis in patches worn for several days. The unique finding of heroin in sweat is of particular interest for documenting drug exposure of the subject. Administration of heroin to these subjects was done under close medical supervision, but no correlation could be established between daily doses and heroin concentrations (r 5 2.59 3 1025) or between daily doses and total opiates concentrations (r 5 3.48 3 1023). Substantial intersubject variability was associated with the excretion of heroin and its metabolites in sweat. Therefore, sweat testing seems to be a qualitative rather than a quantitative test to estimate the amount of drug administered. Similar observations were described for testing buprenorphine [11 ]; for cocaine [9 ] or diazepam [13 ], however, the concentrations in sweat increased in apparent relation to the administered dose. Patches can be worn continuously and used to monitor drug use during that period. However, sweat patches are not suitable for rapid screening purposes; there, urine, which is immediately available, appears to be the specimen of choice. Nonetheless, sweat testing offers the advantage of being a noninvasive method for obtaining a cumulative estimate of drug exposure, whereas urine presents an incremental measure with high invasiveness. The patch, easily applied and removed, is also easily stored [15 ]. In conclusion, sweat analysis may be an useful adjunct to conventional drug testing. Sweat specimens can be obtained more easily and with less embarrassment to subjects than urine specimens. This new technology may find useful applications in the treatment and monitoring of drug abusers.

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