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Articles |
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Institut Universitaire de Médecine Légale, 9 Avenue de Champel, 1211 Geneva 4, Switzerland.
2
Division d’Abus de Substances, 1211 Geneva,
Switzerland.
3
Institut Universitaire de Médecine Légale,
1005 Lausanne, Switzerland.
a Author for correspondence. Fax 41-22-789-24-17; e-mail
christian.staub{at}medecine.unige.ch.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Procedures were developed for quantification of (a) morphine, 6-monoacetylmorphine (6-AM), and codeine in urine and (b) diacetylmorphine and AC in urine. Solid-phase extraction of the analytes was performed, and the extracted analytes were analyzed by selected-ion monitoring with gas chromatography–mass spectrometry. This procedure required prior derivatization with propionic anhydride.
Results: Different validation parameters were determined, such as linearity, reproducibility, extraction recoveries, and cutoffs. Seventy-one urine specimens of illicit heroin abusers and 44 urine specimens of subjects in a heroin maintenance program were analyzed. AC was detected in 85.9% of the samples of the first group but not in any of the samples from subjects taking medical heroin. In the two groups, there were 94.4% and 84.1% 6-AM positive urine specimens, respectively. Detection times were determined for AC and codeine by parallel administration of heroin containing various percentages of AC to four voluntary patients in a heroin maintenance program. The measured detection times were 8 and 23 h for AC and codeine, respectively.
Conclusions: These results indicate that, together with detection of 6-AM in urine, AC is a suitable marker of illicit heroin use.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As illustrated in Fig. 1
, both codeine and heroin are metabolized into morphine, which
is then excreted in the urine. Therefore, detection of morphine in
urine can result from intake of heroin, morphine, codeine, or poppy
seeds.
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6-Monoacetylmorphine (6-AM)1 in urine has been suggested as a specific marker of heroin abuse, and several methods for its detection have been reported (5)(6)(7)(8). The detection time measured for 6-AM is short (<8 h), whereas the detection times for other metabolites, such as morphine, are longer.
In addition to 6-AM, acetylcodeine (AC) has been suggested recently as
another marker of illicit heroin use (9). AC is a
manufacturing impurity (1–15%) of heroin (10) and
is metabolized into codeine and, subsequently, into morphine (Fig. 1
).
In 1994, the Swiss Federal Office of Public Health started a new heroin maintenance program in which addicted patients receive heroin under governmental supervision. Here, AC could be useful in monitoring addicts enrolled in such programs. Because the maintenance heroin administered is pure (<0.1% AC), the presence of AC in the urine of these patients indicates that they may be supplementing their supervised heroin doses with illicit heroin.
The role of AC in urine as a specific indicator of illicit heroin use depends on two main factors: (a) the availability of a reliable and sensitive analytical method for AC detection and, subsequently, availability of other opiate detection methods; and (b) an adequate detection time for AC and codeine in urine. Two separate procedures were validated and applied to urine specimens from patients in a heroin maintenance program and from illicit heroin users.
This report also describes a study on AC excretion in urine. Four volunteer patients in a heroin maintenance program agreed to take heroin with various percentages of AC, which allowed us to determine detection times for AC and codeine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dose and specimen collection
Urine specimens were obtained from illegal heroin users and
subjects enrolled in the Prove Program, a medical heroin treatment
program set up by the Swiss Federal Office of Public Health. Volunteers
of the Prove Program received 120–750 mg of heroin intravenously per
day. Urine specimens were collected at least 3 months after the
beginning of the treatment. For the group of illegal heroin consumers,
the indicated heroin daily dose was based on the consumers’
declarations.
Immunoassay screening was performed on all urine specimens using the
following tests, cutoff values, and reference substances: amphetamines
(d-methamphetamine, 1000 µg/L); barbiturates
(secobarbital, 200 µg/L); benzodiazepines (nordazepam, 100 µg/L);
cannabis (11-nor-
-9-THCCOOH, 50 µg/L); cocaine (benzoylecgonine,
300 µg/L); methadone (300 µg/L); methaqualone (300 µg/L);
tricyclic antidepressants (nortriptyline, 300 µg/L); and
D-lysergic acid diethylamide (LSD, 0.5
µg/L).
Three heroin consumers were excluded from the study because only morphine was found in their urine and the concentration was <100 µg/L. This lower concentration could be explained by too long of a period between heroin consumption and urine sampling.
Subject characteristics and parallel consumption (results of immunoassay screening) for the two groups are summarized in Tables 1 and 2. A third group included four voluntary patients in a heroin maintenance program who agreed to take heroin containing various percentages of AC. To simulate parallel consumption of illicit heroin, they received heroin containing various percentages of AC during 36 days in the following manner: (a) between days 2 and 9, heroin contained 9% AC; (b) during days 12 and 13, heroin contained 5% AC; and (c) between days 19 and 26, heroin contained 3% AC. Pure heroin was administered on days 1, 10, 11, 14–18, and 27–36.
All patients received their daily heroin in three doses, and only the
morning dose contained AC. Table 3
summarizes dose characteristics for the four patients. During
days 2, 12, and 19, urine specimens were collected at 0, 1, 3, 6,
12, and 24 h after the morning dose. Urine collections
performed at 0, 6, 12, and 24 h were just prior to administration
of the next dose of heroin. All urine specimens were stored at
-20 °C until analysis. We found that AC was very stable in these
storage conditions because there was no loss after 6 months of storage.
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The study was conducted according to the guidelines for the protection of human subjects, and each volunteer provided informed consent.
sample preparation
Nalorphine (300 µL of a 25 mg/L solution) was added to 1 mL of
urine for procedure A (morphine, 6-AM, and codeine), and nalorphine
(200 µL of a 2.5 mg/L solution) was added to 2 mL of urine for
procedure B (heroin and AC).
Urine specimens were then extracted by the same technique with Bond Elut Certify columns. This consisted of conditioning the column with 2 mL of methanol, followed by 2 mL of deionized water. Samples were added to the columns, and the columns were then rinsed with 2 mL of deionized water, 2 mL of 0.1 mol/L acetate buffer (pH 4), and 2 mL of methanol. Columns were dried under reduced pressure (10 mmHg) for 5 min, then eluted with 2 mL of methylene chloride–isopropanol (4:1 by volume) containing 20 mL/L ammonium hydroxide. The eluate was dried under nitrogen.
Pyridine and propionic anhydride (100 µL each) were then added, and samples were heated at 60 °C for 30 min. After derivatization, the reagent was dried under nitrogen, and the samples were reconstituted with 50 µL of ethyl acetate.
gas chromatography–mass spectrometry (gc-ms)
For GC, a DB-5MS capillary column (15 m x 0.25 mm; 0.25-µm
film thickness; J & W Scientifics) was used with helium as
the carrier gas. The following temperatures were applied: 170 °C
maintained for 1 min; ramped to 240 °C at 20 °C/min, to 256 °C
at 2 °C/min, and to 270 °C at 10 °C/min; and then held at
270 °C for 0.6 min. The injector temperature was 270 °C, and
injection was made in splitless mode. The interface temperature was
280 °C.
The sample (2 µL) was injected into the GC-MS system, which was operating in selected-ion monitoring mode. The electron multiplier voltage was set at the EI-tune voltage for procedure A and at +200 V above EI-tune voltage for procedure B.
The following ions were monitored: for procedure A, morphine (m/z 397 and 341), 6-AM (m/z 383 and 327), codeine (m/z, 355 and 282), and nalorphine (m/z 423 and 367); and for procedure B, heroin (m/z 369 and 327), AC (m/z 341 and 282), and nalorphine (m/z 423 and 367).
The internal ratio for each compound was monitored. Quantification was based on the peak-area ratios (the first ion listed for each compound was used) of the analytes to the internal standard (nalorphine).
validation protocol
Extraction recovery was determined by adding the analytes to
drug-free urine at low and high concentrations (n = 6). After
extraction, nalorphine was added as an external standard, and peak-area
ratios were then compared with unextracted calibrators of equal
concentrations in methanol or in acetonitrile for AC.
Reproducibility (within-run precision) was determined by analysis of a low and high concentration of each analyte on the same day (n = 6). Seven-point calibration curves for each analyte were analyzed to determine method linearity. The limit of quantification (LOQ) was determined as the lowest concentration yielding a result within ± 20% of the target concentration and with a CV <10%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Satisfactory validation data were achieved for linearity, recovery, and
reproducibility. Linearity, accuracy, and precision for the two
procedures were measured at the LOQ values, which are listed in Table 4
. Assigned LOQ values were therefore used as cutoffs in the rest
of the study with the additional condition that the internal ion ratio
should correspond to ± 20% of the value (given in Table 4
) for
reference standards. Extraction recovery and precision are given in
Table 5
for two concentrations. CVs were generally 
7.4%.
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Seventy-one urine specimens from illicit heroin users and 44 urine specimens from medical heroin users were analyzed by the two procedures described above. Quantitative results, as well as the respective median, mean, and extreme values for all opiates, are listed in Tables 6 and 7. In these two groups, respectively, 94.4% and 84.1% 6-AM-positive urine specimens were found, again demonstrating that 6-AM is an good biomarker of heroin use. AC was detected in 85.9% of the samples from illicit heroin users but not in any of the samples from patients undergoing heroin maintenance. These results indicate that AC is a good biomarker of illicit heroin use, as demonstrated previously by O’Neal and Poklis (10). These authors found a positive relationship between AC and 6-AM concentrations in urine (r = 0.878). In our case, a positive relationship between AC and 6-AM was also found, but with a slightly lower correlation (r = 0.702; slope, 5.38; intercept, 1453.7).
The quantitative excretion patterns for free morphine, 6-AM, codeine, AC, and heroin were determined by GC-MS after administration of pure heroin containing 0–9% AC to four male subjects. Data are presented in Tables 8 and 9. Peak concentrations of free morphine, 6-AM, and codeine occurred within 1–2 h after intake. Peak concentrations of AC were not different from those of other opiates, and only free morphine and 6-AM were detected in the morning urine specimens.
The quantitative excretion patterns for all previously mentioned opiates were determined after administration of pure medical heroin. Neither AC nor codeine was detected, which demonstrates that AC is excreted in urine only when AC is present in heroin and that codeine is the main metabolite of AC.
Detection times are important markers in forensic drug testing because
they indicate how long after drug administration a subject excretes
a drug or a metabolite at a concentration above a specific assay
cutoff (i.e., they show how long a subject tests positive for a drug)
(7). The detection times by GC-MS for AC and codeine in four
male subjects are shown in Tables 8
and 9
. The two assigned cutoffs
were 1 and 10 µg/L, respectively. Mean detection times (± SD) were
8 ± 4 h for AC and 23 ± 4 h for codeine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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86% in our study. This difference in rates could be
explained by the longer period between heroin consumption and urine
sampling or by the lower AC content in the heroin administered in their
study. Since 1994, when the Swiss Federal Office of Public Health started the heroin maintenance program, this is the first time that monitoring of urinary AC has been applied during such a maintenance program. No AC was found in the urine of these patients, whereas concentrations of other opiates were higher than in the urine of illicit heroin users. In addition, detection times were determined for AC and codeine by parallel administration of heroin containing various percentages of AC to patients in the heroin maintenance program who had volunteered for the experiment. The measured detection time of 8 ± 4 h was close to that of 6-AM (7). This study also clearly demonstrates that the presence of codeine in urine could be caused by either codeine or illicit heroin consumption. However, with a measured detection time of 23 ± 4 h, codeine remains a much less specific marker of illicit heroin use than AC.
In conclusion, this study shows that, together with the detection of 6-AM in urine, AC is a suitable marker of illicit heroin use.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  G. Ceder and A. W. Jones Concentration Ratios of Morphine to Codeine in Blood of Impaired Drivers as Evidence of Heroin Use and not Medication with Codeine Clin. Chem., November 1, 2001; 47(11): 1980 - 1984. [Abstract] [Full Text] [PDF]
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