The detection of acetylcodeine and 6-acetylmorphine in opiate positive urines

Acetylcodeine (AC), an impurity of illicit heroin synthesis, was investigated as a urinary biomarker for detection of illicit heroin use. One hundred criminal justice urine specimens that had been confirmed positive by GC/MS for morphine at concentrations >5000 ng/ml were analyzed for AC, 6-acetylmorphine (6AM), codeine, norcodeine and morphine. The GC/MS analysis was performed by solid phase extraction and derivatization with propionic anhydride. Total codeine and morphine concentrations were determined by acid hydrolysis and liquid/liquid extraction. AC was detected in 37 samples at concentrations ranging from 2 to 290 ng/ml (median, 11 ng/ml). 6AM was also present in these samples at concentrations ranging from 49 to 12 600 ng/ml (median, 740 ng/ml). Of the 63 specimens negative for AC, 36 were positive for 6AM at concentrations ranging from 12 to 4600 ng/ml (median, 124 ng/ml). When detected, the AC concentrations were an average of 2.2% (0.25 to 10.2%) of the 6AM concentrations. There was a positive relationship between AC concentrations and 6AM concentrations (r=0.878). Due to its very low concentration in urine, AC was found to be a much less reliable biomarker for illicit heroin use than 6AM in workplace or criminal justice urine screening programs. However, AC detection could play an important role in determining if addicts in heroin maintenance programs are supplementing their supervised diacetylmorphine doses with illicit heroin.     

Detection of drugs of abuse in simultaneously collected oral fluid, urine and blood from Norwegian drug drivers

Blood and urine samples are collected when the Norwegian police apprehend a person suspected of driving under the influence of drugs other than alcohol. Impairment is judged from the findings in blood. In our routine samples, urine is analysed if morphine is detected in blood to differentiate between ingestion of heroin, morphine or codeine and also in cases where the amount of blood is too low to perform both screening and quantification analysis. In several cases, the collection of urine might be time consuming and challenging. The aim of this study was to investigate if drugs detected in blood were found in oral fluid and if interpretation of opiate findings in oral fluid is as conclusive as in urine. Blood, urine and oral fluid samples were collected from 100 drivers suspected of drugged driving. Oral fluid and blood were screened using LC-MS/MS methods and urine by immunological methods. Positive findings in blood and urine were confirmed with chromatographic methods. The analytical method for oral fluid included 25 of the most commonly abused drugs in Norway and some metabolites. The analysis showed a good correlation between the findings in urine and oral fluid for amphetamines, cocaine/benzoylecgonine, methadone, opiates, zopiclone and benzodiazepines including the 7-amino-benzodiazepines. Cocaine and the heroin marker 6-monoacetylmorphine (6-MAM) were more frequently detected in oral fluid than in urine. Drug concentrations above the cut-off values were found in both samples of oral fluid and urine in 15 of 22 cases positive for morphine, in 18 of 20 cases positive for codeine and in 19 of 26 cases positive for 6-MAM. The use of cannabis was confirmed by detecting THC in oral fluid and THC-COOH in urine. In 34 of 46 cases the use of cannabis was confirmed both in oral fluid and urine. The use of cannabis was confirmed by a positive finding in only urine in 11 cases and in only oral fluid in one case. All the drug groups detected in blood were also found in oral fluid. Since all relevant drugs detected in blood were possible to find in oral fluid and the interpretation of the opiate findings in oral fluid was more conclusive than in urine, oral fluid might replace urine in driving under the influence cases. The fast and easy sampling is time saving and less intrusive for the drivers.     

Fast confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine by LC/MS/MS using negative atmospheric-pressure chemical ionisation (APCI)

A fast method using automated solid-phase extraction (SPE) and short-column liquid-chromatography coupled to tandem mass-spectrometry (LC/MS/MS) with negative atmospheric-pressure chemical ionisation (APCI) has been developed for the confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine samples. This highly specific method which combines chromatographic separation and MS/MS-analysis can be used for the confirmation of positive immunoassay results with a NIDA cut-off of 15ng/ml. The conjugates of THC-COOH were hydrolysed prior to SPE, and a standard SPE was performed using C18-SPE columns. No derivatisation of the extracts was needed as in GC/MS analysis, and the LC run-time was 6.5min by gradient elution with a retention time of 2.4min. Linearity of calibration was obtained in the range between 0 and 500ng/ml (correlation coefficient R(2)=0.998). Using linear regression (0-50ng/ml) the limit of detection (LOD) was 2.0ng/ml and the limit of quantitation (LOQ) was 5.1ng/ml; day-to-day reproducibility and precision were tested at 15 and 250ng/ml and were 13.4ng/ml+/-3.3% and 255.8ng/ml+/-4.5%, respectively.     

Opiate concentrations following the ingestion of poppy seed products – evidence for `the poppy seed defence'

The universally accepted 300 ng/ml cut-off limit for opiate assays stated to be mandatory for all drug screening laboratories by the Substance Abuse and Mental Health Services Administration, has been questioned recently due to positive results being obtained following the ingestion of poppy seed containing food products. To establish the plausibility of the `the poppy seed defence' the concentrations of codeine, norcodeine, morphine, normorphine and thebaine (a potential marker for seed ingestion) in several varieties of poppy seeds from different countries were quantified by GC–MS. The country of origin of the seed specimen analysed and the preparation of the seeds prior to their culinary use was found to influence the alkaloid concentration determined. The maximum morphine and codeine concentrations determined in the seeds were found to be 33.2 and 13.7 μg/g seed respectively. In addition, thebaine concentrations were found to vary with each seed sample analysed. Following the consumption of bread rolls (mean 0.76 g seed covering per roll) by four subjects, all urine specimens analysed produced negative results (using the Dade Bebring EMIT II opiate screening assay) with the exception of one subject (body weight 63.0 kg) who consumed two poppy seed rolls. In this subject opiate positive screening results were obtained for up to 6 h post ingestion with maximum urinary morphine and codeine concentrations of 832.0 ng/ml (@ 2–4 h post ingestion) and 47.9 ng/ml (@ 0–2 h post ingestion) respectively being achieved. Following the ingestion of poppy seed cake containing an average of 4.69 g of seed per slice by four individuals, opiate positive screening results were obtained for up to 24 h. In one subject (dose equivalent to 0.07 g poppy seed/kg body weight) maximum urinary morphine and codeine concentrations of 302.1 ng/ml (@ 0–2 h) and 83.8 ng/ml (@ 2–4 h) respectively were recorded. The elimination of thebaine was found to vary widely between individuals, therefore suggesting that its absence from a specimen is not necessarily indicative of opiate abuse. These findings demonstrate that the poppy seed defence could be used as an argument in medico-legal and employment medical cases. Great care should therefore be taken when interpreting the data produced when screening for opiates.     

Gas chromatographic/mass spectrometric analysis of morphine and codeine in human urine of poppy seed eaters

In this study, poppy seeds were examined for a natural constituent that might serve as a maker for the seeds' ingestion as opposed to opiate abuse. Thebaine was selected as possible marker, since it was found to be a component of all poppy seeds examined and was not a natural component of different heroin samples. During the course of this investigation, a new extraction and cleanup procedure was developed for the gas chromatographic/nitrogen phosphorus detection (GC/NPD) and gas chromatographic/mass spectrometric (GC/MS) analysis of morphine and codeine in urine. A linear response, over a concentration range of 25 to 600 ng/mL, was obtained for codeine and morphine (r = 0.9982 and 0.9947, respectively). The minimum detectable level (LOD) and limit of quantitation (LOQ) for morphine were 10 and 30 ng/mL, respectively; whereas LOD and LOQ for codeine were 2 and 8 ng/mL, respectively. The coefficients of variance (CV, n = 6) for morphine and codeine analyses at the 100-ng/mL level were 13.3 and 4.6%, respectively. This procedure was used for the analysis of urine samples from five poppy seed eaters who each ingested 200 g of poppy seed cake. Results indicated that significant amounts of morphine and codeine are excreted in urine and that in all subjects, at least at one point in time, the apparent morphine concentration as determined by radioimmunoassay (RIA) analysis exceeded the cutoff value (300 ng/mL) established for screening. Thebaine was not detected in urine specimens collected following poppy seeds ingestion and thus could not be used as a marker.     

Robotic method for the analysis of morphine and codeine in urine.

A totally automated procedure has been developed for the detection and quantitation of morphine and codeine in urine case samples. The samples were initially screened for these drugs by a Syva EMIT Toxicology System (ETS). A Zymate laboratory robotic system confirms positive samples from Syva ETS by performing the hydrolysis, extraction, and derivatization of morphine and codeine. The derivatized morphine and codeine were detected using gas chromatography/mass spectrometry (GC/MS). Enzymatic hydrolysis conditions were experimentally optimized during method development. The automation of these procedures has proven to be reliable and efficient.     

Fatal versus non-fatal heroin "overdose": blood morphine concentrations with fatal outcome in comparison to those of intoxicated drivers

The study was performed to distinguish fatal from non-fatal blood concentrations of morphine. For this purpose, blood levels of free morphine and total morphine (free morphine plus morphine conjugates) in 207 cases of heroin-related deaths were compared to those in 27 drivers surviving opiate intoxication. The majority of both survivors and non-survivors were found to show a concomitant use of depressants including alcohol or stimulants. Blood morphine levels in both groups varied widely, with a large area of overlap between survivors (free morphine: 0-128 ng/ml, total morphine: 10-2,110 ng/ml) and non-survivors (free morphine: 0-2,800 ng/ml, total morphine: 33-5,000 ng/ml). Five (18.5%) survivors and 87 (42.0%) non-survivors exhibit intoxication only by morphine. In these cases, too, both groups overlapped (survivors-free morphine: 28-93 ng/ml, total morphine: 230-1,451 ng/ml; non-survivors-free morphine: 0-2,800 ng/ml, total morphine: 119-4,660 ng/ml). Although the blood levels of free or total morphine do not allow a reliable prediction of survival versus non-survival, the ratio of free/total morphine may be a criterion to distinguish lethal versus survived intoxication. The mean of the ratio of free to total morphine for all lethal cases (N=207) was 0.293, for those that survived (N=27) 0.135, in cases of intoxication only by morphine 0.250 (N=87) and 0.080 (N=5), respectively. Applying a cut-off of 0.12 for free/total morphine and performing ROC analyses, fatal outcome can be predicted in 80% of the cases correctly, whereas 16% of the survivors were classified as dead. Nevertheless, in this study, all cases with a blood concentration of 200 ng/ml and more of free morphine displayed a fatal outcome.     

Hair and urine analysis: relative distribution of drugs and their metabolites

This work studies the distribution of cocaine and heroin metabolites in hair and urine of living polidrug abusers. Cocaine, benzoylecgonine (BEG), ecgonine methyl ester (EME), morphine, codeine and 6-monoacetylmorphine (6-MAM) were simultaneously extracted and analyzed by GC/MS in SIM mode. The results obtained show a different distribution of heroin and cocaine metabolites in urine and hair. In urine, we generally find BEG and EME for cocaine abuse, and morphine for heroin abuse. In hair, we detect cocaine and MAM as major metabolites for cocaine and heroin abuse, respectively.     

Validation of LUCIO-Direct-ELISA kits for the detection of drugs of abuse in urine: application to the new German driving licence re-granting guidelines

LUCIO-Direct-enzyme linked immunosorbent assay (ELISA) tests were validated for the screening of drugs of abuse cannabis, opiates, amphetamines and cocaine in urine for the new German medical and psychological assessment (MPA) guidelines with subsequent gas chromatographic-mass spectrometric (GC-MS) confirmation. The screening cut-offs corresponding to 10 ng/mL 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), 50 ng/mL amphetamine, 25 ng/mL morphine and codeine and 30 ng/mL benzoylecgonine were chosen at the point where the number of false negatives was lower than 1%. Due to their accuracy, ease of use and rapid analysis, these ELISA tests are very promising for cases where a large proportion of the tests are expected to be negative such as for abstinence monitoring as part of the driving licence re-granting process.     

Poppy seed consumption and toxicological analysis of blood and urine samples

Poppy seeds contain morphine in different amounts. Reported concentrations are up to 294 mg morphine/kg poppy seeds. Since penalties based on Street Traffic Law (parapgraph 24a StVG) in Germany (administrative offence) require definitive proof of morphine in blood samples, and the "Grenzwertkommission" in consultation with the Ministry of Transportation recommended a threshold of free morphine of 10 ng/mL, the question arose whether the consumption of poppy seeds can lead to a blood concentrations equal or higher than 10 ng/mL of free morphine. Therefore, five volunteers ate poppy seed products (50 mg morphine/kg poppy seeds). In urine, all on-site tests were enzyme immunologically positive for opiates and were positive to morphine by GC/MS. All the blood samples were negative to morphine by EIA and to free morphine by GC/MS. However, after hydrolysis, morphine was detected by GC/MS in all cases. Accordingly, in Germany, penalties based on parapgraph 24a StVG are not likely to cause road users any concerns should they have consumed poppy seeds. Driver Licensing Authorities, however, should be advised of this problem to avoid unjustified legal measures.     

尿样中海洛因代谢物的测定及海洛因滥用的确认

用ＳＰＥ－ＧＣ－ＮＰＤ法建立了尿样中吗啡、６－单乙酰吗啡及可待因的定性分析方法,适用于海洛因滥用者的尿样分析。尿样中吗啡及可待因的最小检测限均为５０ｎｇ／ｍｌ。方法的相对标准偏差分别为：吗啡１１．３％（ｎ＝５）,可待因１４．２％（ｎ＝５）。方法简便、灵敏、快速,１５ｍｉｎ可完成一例尿样的分析。研究了服用含可待因成分的复方甘草合剂后,尿样中的吗啡及可待因的峰面积比为０．４５７±０．１９７（Ｐ＝９９％）;统计了４０例明确滥用海洛因尿液的分析结果,吗啡与可待因的峰面积比为３．４６±０．８９４,Ｐ＝９９％。可作为判断海洛因滥用的依据。同时与免疫板法比较,附５５例免疫板法阳性尿样的分析结果     

Concentrations of unconjugated morphine, codeine and 6-acetylmorphine in urine specimens from suspected drugged drivers

Concentrations of unconjugated morphine, codeine and 6-acetylmorphine (6-AM), the specific metabolite of heroin, were determined in urine specimens from 339 individuals apprehended for driving under the influence of drugs (DUID) in Sweden. After an initial screening analysis by immunoassay for 5-classes of abused drugs (opiates, cannabinoids, amphetamine analogs, cocaine metabolite and benzodiazepines), all positive specimens were verified by more specific methods. Opiates and other illicit drugs were analyzed by isotope-dilution gas chromatography-mass spectrometry (GC-MS). The limits of quantitation for morphine, codeine and 6-AM in urine were 20 ng/mL. Calibration plots included an upper concentration limit of 1000 ng/mL for each opiate. We identified the heroin metabolite 6-AM in 212 urine specimens (62%) at concentrations ranging from 20 ng/mL to > 1000 ng/mL. The concentration of 6-AM exceeded 1000 ng/mL in 79 cases (37%) and 31 cases (15%) were between 20 and 99 ng/mL. When 6-AM was present in urine the concentration of morphine was above 1000 ng/mL in 196 cases (92%). The concentrations of codeine in these same urine specimens were more evenly distributed with 35% being above 1000 ng/mL and 21% below 100 ng/mL. These results give a clear picture of the concentrations of unconjugated morphine, codeine and 6-acetylmorphine that can be expected in opiate-positive urine specimens from individuals apprehended for DUID after taking heroin.     

氯胺酮、甲基苯丙胺和吗啡金标单抗试剂盒的研制

目的建立同步检测氯胺酮、甲基苯丙胺和吗啡的方法。方法将胶体金标记的抗氯胺酮、抗甲基苯丙胺和抗吗啡单克隆抗体浸涂在玻璃纤维膜上，将氯胺酮、甲基苯丙胺和吗啡的完全抗原以及羊抗鼠多克隆抗体喷涂在硝酸纤维素膜上，分别标定为检测区（T）和质控区（C）。样本中游离的氯胺酮、甲基苯丙胺和吗啡分别与包被的完全抗原免疫竞争结合胶体金标记抗氯胺酮、抗甲基苯丙胺和抗吗啡单克隆抗体。以质控区和检测区是否出现紫红色条带判读结果。结果对66种药品和毒品的特异性测试表明，该试剂盒仅识别氯胺酮及其代谢物、甲基苯丙胺及其衍生物和吗啡类；对人体尿样中的氯胺酮、甲基苯丙胺和吗啡检测阈值分别为1000ng／ml、1000ng／ml和300ng／ml；与GC／MS对照试验结果一致；试剂盒稳定性较好，在常温下可较长时间保存。结论本文研制的试剂盒可用于样本中氯胺酮、甲基苯丙胺和吗啡成分定性的同步检测。     

度冷丁滥用者尿中原体及其代谢产物的分析研究

本研究利用ＧＣ／ＭＳ（ＥＩ．ＰＣＩ）技术，在度冷丁滥用者尿中确认了６种代谢产物。并建立了ＧＣ／ＦＩＤ分析度冷丁及其代谢产物的方法。方法线性范围为０．１～２０μｇ／ｍｌ，变异系数小于１０％，最小检出量为５０ｎｇ／ｍｌ，适用于度冷丁成瘾者尿中含量的测定。     

Detection of the synthetic drug 4-fluoroamphetamine (4-FA) in serum and urine

4-Fluoroamphetamine (4-FA) was detected in the blood and urine of two individuals suspected for driving under the influence (DUI). The test for amphetamines in urine subjected to immunoassay screening using the CEDIA DAU assay proved positive. Further investigations revealed a 4-FA cross-reactivity of about 6% in the CEDIA amphetamine assay. 4-FA was qualitatively detected in a general unknown screening for drugs using GC/MS in full scan mode. No other drugs or fluorinated phenethylamines were detected. A validated GC/MS method was established in SIM mode for serum analysis of 4-FA with a limit of detection (LOD) of 1 ng/mL and a lower limit of quantification (LLOQ) of 5 ng/mL. Intra-assay precision was approx. 4% and inter-assay precision approx. 8%. Applying this method, the 4-FA serum concentrations of the two subjects were determined to be 350 ng/mL and 475 ng/mL, respectively. Given the pharmacological data of amphetamine, 4-FA psychoactive effects are to be expected at these serum levels. Both subjects exhibited sympathomimetic effects and psychostimulant-like impairment accordingly.     

生物检材中吗啡类生物碱的LC-MS／MS分析

目的针对滥用药物分析鉴定实践中亟待解决的问题,开展LC-MS/MS分析生物检材中吗啡类生物碱的应用研究。方法满足不同的鉴定需要,分别建立血液、尿液、唾液和头发等生物检材的样品前处理方法,确定同时分析海洛因、单乙酰吗啡、吗啡、可待因、乙酰可待因、二氢可待因酮和氢吗啡酮等吗啡类生物碱的LC-MS/MS方法。将方法应用于实际案例。结果所建立的方法对吗啡类生物碱分离良好。尿液稀释法、尿液提取法和头发中吗啡的最低检测限(LOD)分别为10ng/mL、0.01ng/mL和0.01ng/mg。结论所建立的方法简便、快速、特异性强、灵敏度高。目标物中加入二氢可待因酮和氢吗啡酮扩大了方法的实用范围。     

Confirmation of Syva enzyme multiple immunoassay technique (EMIT) d.a.u. and Roche Abuscreen radioimmunoassay (RIA) (125I) urine cannabinoid immunoassays by gas chromatographic/mass spectrometric (GC/MS) and bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) methods

Thirty human urines screened positive by the Syva enzyme multiple immunoassay technique (EMIT) d.a.u. urine cannabinoid assay were also positive for the major marijuana urinary metabolite 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) when assayed by gas chromatographic/mass spectrometric (GC/MS) and a noninstrumental qualitative bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) technique. The noninstrumental BPA-TLC procedure was the simpler of the two techniques to perform and interpret. Assay of these same samples by the Roche Abuscreen radioimmunoassay (RIA) for cannabinoids (125I) revealed that reliance on the 100-ng/mL equivalent positive calibrator yielded a high incidence of false negative results (10 out of 30). The performance of these same 4 assays on 30 true negatives also was evaluated. All samples were negative for cannabinoids by EMIT and RIA, and for THC-COOH by BPA-TLC. GC/MS assay, however, detected spurious low levels of approximately 5-ng/mL THC-COOH in two instances. Because of this, a reliability level of 10 ng/mL was set for the routine quantitative confirmation of THC-COOH by the GC/MS method.     

Correlations on radioimmunoassay, fluorescence polarization immunoassay, and enzyme immunoassay of cannabis metabolites with gas chromatography/mass spectrometry analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in urine specimens.

Results obtained from three commercial immunoassay kits, Abuscreen, TDx, and EMIT, commonly used for the initial test of urine cannabinoids (and metabolites) were correlated with the 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (9-THC-COOH) concentration as determined by GC/MS. Correlation coefficients obtained based on 26 (out of 1359 total sample population) highly relevant samples, are 0.601 and 0.438 for Abuscreen and TDx. Correlation coefficients obtained from a parallel study on a different set of 47 (out of 5070 total sample population) highly relevant specimens are 0.658 and 0.575 for Abuscreen and Emit. The immunoassay concentration levels, that correspond to the commonly used 15 ng/ml GC/MS cutoff value for 9-THC-COOH, as calculated from the regression equations are 82 ng/ml and 75 ng/ml for TDx and EMIT and 120 ng/ml and 72 ng/ml for Abuscreen manufactured at two different time periods. The difference of these calculated corresponding concentrations provides quantitative evidence of the reagent specificity differences.     

固相萃取／LC-MS／MS测定尿液中吗啡类药物

目的 建立尿液中吗啡类药物的固相萃取／LC—MS／MS方法。方法采用OASIS MCX3cc（60mg）固相萃取柱进行提取,应用LC—MS／MS方法进行检测,运用保留时间和MRM方式对尿液中吗啡类药物及其代谢物进行定性定量分析。结果磷酸盐缓冲液pH4．0时,海洛因、6-MAM、可待因、吗啡、M3G的固相萃取回收率分别达64．33％-70．21％,96．95％～117．57％,83．60％～123．63％,68．82％～91．03％,94．64％～107．33％;最低检测限（LOD）分别为5、10、5、5、2pg,线性范围0．005～10μg／mL;相关系数分别为0．9998、0．9958、0．9992、0．9994、0．9997。结论本文所建方法,适用于尿液中吗啡类药物的分析。     

尿中氯胺酮及其代谢物检测的研究

目的建立氯胺酮滥用者尿中氯胺酮及其代谢物检测方法。方法尿液用有机溶剂液-液萃取,气相色谱/氮磷检测器、电子捕获检测器、氢火焰检测器和气-质联用仪测定。结果确认了尿液中氯胺酮的主要代谢物,尿液中氯胺酮及去甲氯胺酮的最小检测限均为2ng/mL,脱氢去甲氯胺酮的最小检测限为5ng/mL。结论所建方法快速、灵敏、准确,能够满足氯胺酮滥用者尿液检测的需要。