Codeine testing in sweat and saliva with the Drugwipe

With the growing interest in drug testing within different sectors of society, there has become a need for drug assays that can be performed immediately at the site of specimen collection. Recently, Securetec (Ottobrunn, Germany) has introduced the Drugwipe, a non instrument-based, on-site immunodiagnostic assay for the detection of drugs on surfaces. Different tests are available for opiates, cocaine and cannabis. To document the applications of the Drugwipe “opiate” on human biological fluids, 60 mg codeine phosphate were orally administered to 6 subjects. First, sweat testing with the Drugwipe was studied. The wiping section of the kit was used to swab the forehead of the subjects for 10 s, at 1, 4, 9 and 24 h after codeine administration. At the same time, for each period, a sweat patch (Pharmchek, USA) was applied to the outer portion of the upper arm. Codeine was then quantified in the patch by GC/MS and the measured concentrations used as reference. In all subjects except one the Drugwipe tested positive for opiates, however with few false negative results. In the second part of the study, results of the Drugwipe were compared with those obtained by GC/MS for saliva. The tongue of the subjects was carefully wiped over a period 24 h, and at the same time a specimen of saliva collected. Although codeine could be detected using the Drugwipe, numerous false negative results were observed. Codeine tested positive by GC/MS but remained negative using the Drugwipe in several cases. This can be explained by a codeine concentration which was too low to show positive with the Drugwipe, interfering substances may be present in saliva or the sampling procedure is inadequate. Received: 10 July 1997 / Received in revised form: 17 August 1997     

Immunchemische und gaschromatographisch-massenspektrometrische Befunde im Blut bei Verdacht auf Drogenkonsum – 1. Mitteilung: Opiate, Kokain, Cannabis und Amphetamine

The extension of § 24 a of the road traffic regulations is expected to lead to more frequent investigations of blood for cannabinoids, opiates, cocaine (or metabolites) and amphetamines. Nearly 200 blood (serum) samples were analysed by the inhomogenous MTP-immunoassay for cannabinoids, opiates and amphetamines and 140 for cocaine consumption. In about half the specimens a gas chromatographic-mass spectrometric analysis followed. In generally immunassay results above 10 ng/ml for opiates, cannabinoids, or amphetamines and above 25 ng/ml for benzoylecgonine resulted in additional GC-MS analyses. Moreover cases with corresponding statements in the anamnesis were analysed gas-chromatographically otherwise only random tests were performed. Cannabinoids were detected in 47.4% of the samples, opiates in 31.1%, cocaine in 25.7% and amphetamines in 13.2%. The MTP-immunoassay has proved to be a good screening method to differentiate between probably positive and negative cases. In view of the threshold values above an offence is considered to have been proven, the results of our investigations result in Cut off values of 30 ng/ml with opiates and cannabinoids, 50 ng/ml with benzoylecgonine and 10 ng/ml with amphetamines. The tested assays have proven to be practicable and neglibable susceptible to interference. However it is recommendable to make a 3-point calibration before each series of samples.     

Application of the Cozart DDS system to postmortem screening of drugs of abuse in vitreous humor

The analysis of drugs of abuse in human specimens is essential in forensic medicine. This study evaluated the use of Cozart DDS for postmortem screening of some drugs of abuse in vitreous humor (VH) prior to forensic autopsies. The Cozart DDS is an on-site drug detection system that has been validated for oral fluid. Seventy-one VH specimens were obtained from cadavers. Causes of death included injury, drug poisoning, natural disorders, and traffic accidents. The samples were tested for cannabis, cocaine, opiate, amphetamine, and methamphetamine. Thirty-three of 71 samples were positive for drugs of abuse (42% for cocaine, 28% for cannabis, 26% for opiates, and 3% for methamphetamine). The positive specimens for opiates and methamphetamine were reexamined by gas chromatography-mass spectrometry (GC-MS); benzoylecgonine (cocaine product) was detected by liquid chromatography-tandem mass spectrometry (LC-MS-MS). All positive results for cocaine or its products and opiates by the Cozart test were confirmed by MS analysis, whereas one positive result for methamphetamine was negative by MS. The Cozart DDS system is generally a useful screening test for VH specimens prior to autopsies, because of its simplicity and rapidness.     

Phencyclidine and cannabinoids in vitreous humor

Vitreous humor (VH) is routinely collected at autopsy for the testing of electrolytes and ethanol. In recent years drugs of abuse have been detected in this specimen. In this study the authors assayed 30 VH samples for phencyclidine (PCP) and 50 specimens for cannabinoids. Specimens were screened by immunoassay and then assayed for PCP by GC-FID and cannabinoids by GC/MS. Eighteen (60%) specimens screened positive for PCP using a cutoff of 25ng/mL. Quantitative analysis showed PCP concentrations in VH that screened positive ranged 30-290ng/mL. Corresponding blood concentrations were 50-600ng/mL. VH PCP concentrations in the 12 cases which screened negative were 40-470ng/mL. False negative results were probably due to matrix effects. All VH specimens screened for cannabinoids were negative. Ten negative screening specimens assayed by GC/MS yielded 1 11-nor-9-carboxy-delta-9-tetrahydrocannabinol positive result at 2ng/mL. These data indicated that VH maybe a useful specimen for the detection of PCP but not for cannabinoids.     

Applicability of an on-site test for its use in post-mortem blood

The number of deaths related to drugs of abuse makes necessary the use of an on-site test for those cases in which a rapid detection of the consumed drug is required. Cozart® DDS test provides a simple, fast and reliable tool for the qualitative on-site analysis in post-mortem blood. Owing that this test is prepared for oral fluid samples, a validation becomes essential in order to use it for a different matrix than the established one. According to that, results obtained by Cozart® DDS test used in post-mortem blood samples have been compared with a qualitative gas chromatography–mass spectrometry (GC/MS/MS). Positive results for cocaine family compounds (COC-F) were 43.75%, for opiates family compounds (OPI-F) 25.78%, and for cannabis family compounds (THC-F) 2.34%. Negative results were 28.13%. No amphetamines (AMP) or methamphetamines (MA) were found. Sensitivity and specificity was available for cocaine and opiates but not for cannabis because only five cases were detected. Sensitivity, specificity, predictive positive value and predictive negative value for cocaine were 98%, 91%, 88% and 99%, respectively. Sensivilty, specificity, predictive positive value (PPV) and predictive negative value (NPV) for opiates were 93%, 92%, 76% and 98%, respectively. Likelihood positive ratios for cocaine and opiates have been 10.92 and 11.69, respectively, while likelihood negative ratios have been 0.02 and 0.08, respectively. Results show the suitability of Cozart® DDS test for the qualitative detection of cocaine and opiates in post-mortem blood.     

Interpretation of forensic cases based on empirical data

Over the course of the last years the importance of hair analysis increased obviously. For example work place testing, driving licence cases, so-called health checks, and especially in criminal acts the analysis of illicit substances is common. With the modern multiplex analytical methods (GC/MS, GC/MS/MS, LC/MS) the routine analytical probe of hair is in general unproblematic. But one of the major problems in hair analysis is the interpretation of the results. To solve this difficult and challenging task statistical data as a source of information could be a helpful tool. Examination of more than 1,000 hair segments within the past 2 years using gas chromatography/tandem-mass spectrometry (GC/MS/MS) as the preferred method for conducting the daily routine detection of the most prevalently abused drugs and metabolites, such as cannabinoids, cocaine, some synthetic drugs from the amphetamine group, and opiates serve as the basis for the statistics. The quantity of analytical results and additional essential facts have been documented in the publication to present our sources of information and supplementary data to prepare forensic reports. We hereby state that we do not have a significant financial interest or other relationship with any product manufacturer or provider of services discussed in this article.     

Konsum von Amphetamin, Methylendioxyamphetaminen und Beigebrauch anderer Drogen (Köln 1991–1996)

The amount of analyses positive for amphetamine or amphetamine derivatives carried out in Köln showed an increase in the years 1991–1996. Among the users there were more male than female individuals and the age was about 25 years. In addition to an increase in the total amount of these drugs, the additional consumption of other drugs also increases (> 90%) of predominantly cannabinoids and cannabinoids combined with alcohol or cocaine were detected in the samples. Since 1994 the total amount of samples positive for methylenedioxyamphetamines has shown a clear increase. A comparison of the drugs additionally used by consumers of amphetamine or methylenedioxyamphetamines showed – in addition of the abuse of opioides or medicaments such as benzodiazepines – no significant differences.     

Immunchemisches Screening von Serumproben: Vergleich der Cannabinoid- und Opiat-Assays Mahsan MTP und Abbott FPIA mit Kontrolle durch Gaschromatographie-Massenspektrometrie

By the update of §24?a of the German road traffic act driving under the influence of drugs of abuse can now be fined even in ¶absence of an actual driving impairment. ¶The only requisite is the detection of certain drugs/metabolites in a blood sample. Therefore for pretesting there is a need for fast and reliable immunological screening methods in blood/serum. The new Mahsan microtiterplate enzyme immunoassay (MTP-EIA) is claimed to be sensitive and independent of the matrix. For an evaluation we carried out a comparison of the cannabinoid and opiate assays from Mahsan (MTP-EIA) and the routinely used Abbott fluorescence polarization immunoassay (FPIA) with a batch of routine serum samples. For the MTP-EIAs we used the cut-off recommendations of the supplier (cannabinoids 2 ng/mL, opiates 10 ng/mL). For the Abbott FPIAs the serum samples had to be pretreated (deproteinization with acetone) and appropriate cut-offs were defined by our investigations (cannabinoids 20 ng/mL, opiates 30 ng/mL). Out of 136 samples screened for cannabinoids 42% were positive with GC-MS and 18% from 123 samples were positive for opiates. The Abbott cannabinoid FPIA gave false negative results in 23% of GC-MS positive cases and 9% false negatives for opiates while Mahsan MTP-EIA gave no false negatives. Each of the two cannabinoid assays gave false positive results in 13% of the cases and for the MTP opiate assay 2% of the positives could not be confirmed with GC-MS. As a result of our study the cut-off values for the MTP-EIAs have proved to be suitable for a distinction between potential positives and true negatives.     

Perspiration versus saliva – basic aspects concerning their use in roadside drug testing

Various aspects concerning the practical application and forensic interpretation of data obtained by saliva drug testing and drug monitoring from the skin surface are discussed. Basic information on the composition of saliva and skin secretions and their particular transport mechanisms, as far as known, are given. For drugs of abuse secretion into saliva is suggested to be by passive diffusion and to depend on lipid solubility, pKa, plasma protein binding and on the pH of saliva. Drug molecules from blood are considered to reach the skin surface by various routes such as by sweat and sebum as well as by inter- and/or transcellular diffusion. The role of the stratum corneum as a temporary drug reservoir exceeding positive drug findings in urine is outlined. Current data on opioids, cocaine metabolites, cannabinoids and amphetamines detected in saliva and on the skin surface are reviewed. Aspects of collection, processing and analysis of the samples for implementation in roadside testing are addressed. The requirement of test sensitivity covering the broad concentration ranges and the importance of test specificity bearing in mind that the parent drug is the main analyte present in those specimens is stressed. Theoretical and practical findings on frequently abused drugs are discussed with regard to the possibilities and limitations of drug monitoring from saliva and perspiration to support a suspicion of actual or recent drug administration. Received: 13 August 1997 / Received in revised form: 25 August 1998     

Automated solid-phase extraction and two-step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC/MS

A combination of automated solid-phase extraction (SPE) and subsequent two-step derivatisation has been developed for the simultaneous analysis of basic drugs of abuse and cocaine metabolites in serum samples. Substances included in this procedure are morphine, codeine, methadone, cocaine, benzoylecgonine, methylecgonine, amphetamine, methamphetamine, MDMA, MDEA and MDA. SPE with mixed-mode cartridges (RP-C8 and cation-exchange) was fully automated with a Zymark RapidTrace SPE robot. GC/MS analysis was performed after derivatisation with a new two-step reaction by trifluoroacetic anhydride and 2,2,3,3,3-pentafluoropropanol. High recoveries (> 85%) with high reproducibility (CV 1.1-3.8%) were found for all drugs. High correlation coefficients (r > 0.998) were obtained due to the addition of deuterated standards prior to extraction. Experience obtained over 2 years of applying this method to drug analysis in serum is discussed.     

Zum Suchtmittelnachweis in Haaren

Hair samples collected from drug addicts (n=25) were analysed for opiates and cocaine by GC/MS. A decrease in drug concentration was observed even after a single bleaching or permanent wave application. The stability of the drug molecules in hair seemed to be different and cocaine was less affected than morphine or 6-acetylmorphine. When drug concentrations were low, the drug content often declined below the detection limit after cosmetic treatment. In hair samples which showed a drug content above 2 ng/ mg hair the qualitative detection of drugs was regularly successful. However, a distinct rule could not be established mainly due to influences of the individual hair matrix.     

Determination of cocaine in human hair by gas chromatography/mass spectrometry

Summary A qualitative method for the determination of cocaine alone without its metabolites in human hair by gas chromatography/mass spectrometry (GC/MS) was developed. The assay used helium as carrier gas, a 30-m bonded phase fused silica OV-1 capillary column, and solid injection at 290°C evaporator temperature.The cocaine concentrations in hair were determined also by radio-immunoassay (RIA). The values obtained are the sum of cocaine and its metabolites.Both GC/MS and RIA meet the requirements for the determination of drug abuse by two different methods in forensic science.     

Comparison of the Cozart DDS 801 on-site drug test device and gas chromatography/mass spectrometry (GC/MS) confirmation results of cannabis and cocaine in oral fluid specimens

Driving under the influence of drugs (DUID) is a problem around the world. The objective of this study is to assess the reliability of the oral fluid screening device the Cozart DDS 801 by comparing the on-site results with confirmatory gas chromatography/mass spectrometry (GC/MS) oral fluid analysis. The study was carried out in Catalonia (Spain) with a sample of 2180 oral fluid specimens taken from subjects suspected of driving under the influence of drugs of abuse, and collected by police officers during 2009–2010. Statistical parameters of the tests were determined for cannabis and cocaine. The sensitivity, specificity, predictive positive value, predictive negative value, likelihood positive ratio and likelihood negative ratio for cocaine were 92%, 90%, 95%, 85%, 9.44, and 0.09 respectively. Sensitivity, specificity, predictive positive value, predictive negative value, likelihood positive ratio and likelihood negative ratio for cannabis were 87%, 86%, 94%, 73%, 6.15 and 0.6 respectively. Accuracy was 91% for cocaine and 86% for cannabis. The Cozart DDS 801 drug test system is a simple to use screening tool for cocaine and cannabis in oral fluid, at initial screening cut-off established by the manufacturer, confirmed with a GC/MS analysis. The system has demonstrated its acceptable performance.     

Intoxikation mit Liquid Ecstasy

Zusammenfassung Ein 15-jähriges Mädchen trank in einer Diskothek eine unbekannte Flüssigkeit. Nach 3-stündigem Klinikaufenthalt erwachte sie ohne Nachwirkungen. Es standen 2 Blutproben (Abnahme ca. 6 h und 8,5 h nach Ingestion) und 2 Urinproben (Abnahme ca. 6 h und 9,5 h nach Ingestion) für die toxikologischen Untersuchungen zur Verfügung. Das Drogenscreening auf Kannabinoide, Opiate, Kokain und Amphetaminderivate und das Arzneimittelscreening mit HPLC/DAD verliefen im Blut und im Urin negativ. Mit GC/MS wurde %-Hydroxybuttersäure nachgewiesen. Folgende Konzentrationen wurden ermittelt: Etwa 6 h nach Ingestion im Serum: 4,1 µg/ml. Etwa 8,5 h nach Ingestion im Serum: Konzentration unter der Nachweisgrenze von 1 µg/ml. Etwa 6 h nach Ingestion im Urin: 365 µg/ml. Etwa 9,5 h nach Ingestion im Urin: 176 µg/ml. Die relativ niedrige Konzentration in der 1. Blutprobe und das Nichtauffinden von %-Hydroxybuttersäure (GHB) in der 2. Blutprobe sind auf die kurze Halbwertszeit (durchschnittlich 27 min) zurückzuführen. Die Konzentration in der 1. Blutprobe lässt aber den Rückschluss zu, dass diese zum Ingestionszeitpunkt oberhalb des therapeutischen Bereiches lag (50-120 µg/ml). An GHB als Noxe sollte immer gedacht werden, wenn trotz einer für Opiate typischen Symptomatik das Drogenscreening und das mit HPLC/DAD durchgeführte Arzneimittelscreening negativ verlaufen. Abstract A 15-year-old girl drank an unknown liquid in a discotheque and woke up after a stay of 3 h in a hospital without any after-effects. Screening for illicit drugs such as cannabinoids, opiates, cocaine, derivates of amphetamines and screening of drugs with HPLC/DAD in blood and urine was negative but %-hydroxybutyric acid (GHB) was detected by GC/MS at different concentrations depending on the time after ingestion. Approximately 6 h after ingestion the level measured in serum was 4.1 µg/ml, after approximately 8.5 h the level was below the detection limit of 1 µg/ml. Approximately 6 h after ingestion a level of 365 µg/ml was detected in urine and after approximately 9.5 h the level was 176 µg/ml. The relatively low concentration in the first blood sample as well as the non-detection of GHB in the second, can be explained by the short half-life period (on average 27 min). The concentration in the first blood sample is indicative of a level above the therapeutic range of 50-120 µg/ml shortly after ingestion. In such cases where the patient presents symptoms similar to those caused by opiates but screening for drugs is negative, the ingestion of GHB should be considered.     

Bestimmungsgrenzen für den Nachweis von Cannabinoiden in Serum

The limits of quantitation for cannabinoids in serum using automated solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC/MS) have been determined. Extraction was performed automatically using C18-cartridges, extracts were methylated and analysed with selected-ion monitoring (SIM). The limits of quantitation (LOQ) in serum were determined according to Euronorm EN 45001 and German Industrial Norm DIN 32645 and were 0.1 and 0.7 ng/ml for tetrahydrocannabinol, 0.5 and 0.7 ng/ml for 11-hydroxy-tetrahydrocannabinol and < 0.25 and 1.6 ng/ml for tetrahydrocannabinolcarboxylic acid, respectively. The use of these LOQ’s in forensic cases and the necessity of drug identification prior to quantitation are discussed in detail. Due to the newest amendment of the German traffic law (§24a “Straßenverkehrsgesetz”) the quest for nationwide standardisation of the forensic analysis of illicit drugs in serum or blood arises. By automation of several manual sample preparation steps we could get closer to this aim. Taking into account a margin of safety, a qualitative cut-off at 1 ng/ml THC and a limit of quantitation of 2 ng/ml THC in serum should be achievable in all forensic toxicology laboratories.     

Zum Suchtmittelnachweis in Haaren. VII. Untersuchungen bei Methadonpatienten unter konstanter Wirkstoffeinnahme

Hair samples and perspiration collected from long term drug addicts (n = 18) under constant methadone treatment were investigated. Hair segments representing the individual hair growth of 4 weeks were analyzed for methadone, opiates and cocaine by GC/MS. The results obtained from the transdermal collection devices confirmed the presence of drug substances in variable amounts on the skin surface, methadone being the main analyte. The perspiration test period lasted for 4 days. For six persons illicit drugs similar to those in the corresponding hair samples were found. Overall, a poor relationship (r = 0,602) was found between the methadone dose under steady state conditions and the methadone level in hair. The results are discussed in the light of pharmacokinetic aspects, drug uptake by perspiration and forensic interpretation.     

Usefulness of multiple opiate and amphetamine analysis of hair segments under methadone therapy using LC-APCI-MS-MS

A detailed procedure for simultaneous analysis of morphine, codeine, 6-monoacetylmorphine, amphetamine, methadone, and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human hair segments by liquid chromatography (LC)-atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry (MS-MS) was established. Hair samples were pulverized and extracted with methanol. The blank hair obtained from healthy subjects showed no interfering impurity peaks. Good linearity was obtained for all compounds in the range of 0.2–20 ng/mg. Accuracy and precision data were also satisfactory. Using the established method, the opiates, amphetamine, methadone, and EDDP in hair segments were measured for 20 patients undergoing methadone therapy. Complete abstinence was achieved by only 6 of the 20 patients. Other patients failed to abstain from opiate(s) and/or amphetamine. Our data show that the present hair analysis of multiple drugs of abuse by LC-APCI-MS-MS is useful for monitoring the success or failure of methadone therapy.     

Concentrations of Δ9-tetrahydrocannabinol,cocaine and 6-monoacetylmorphine in hair of drug abusers

Hair samples taken from 850 individuals with presumed drug abuse were tested simultaneously forΔ⁹tetrahydrocannabinol (THC), cocaine, heroin, the primary heroin metabolite 6-monoacetylmorphine (6-MAM) and morphine. The drugs were extracted with methanol under sonication. Compared to other extraction procedures this solvent extraction technique provides high extraction yields and less experimental effort. The analyses were carried out using gas chromatography - mass spectrometry (GCMS) in selected ion monitoring (SIM) mode. This procedure allows the simultaneous detection of amphetamine, methylenedioxyamphetamine (MDA), methylenedioxymetbamphetamine (MDMA) and methylenedioxyethylamphetamine (MDE). THC was found in 104 (12.2%), cocaine in 230 (27%) and 6-MAM in 141 (16.6%) samples. In addition to 6-MAM, morphine was detected in 87 (10.2%) and heroin in 38 samples (4.5%). The concentrations found were in a range 0.009-16.7 ng/mg for THC, 0.037-129.68 ng/mg for cocaine, 0.028-79.82 ng/mg for 6-MAM, 0.045-53.14 ng/mg for heroin and 0.011-7.800 ng/mg for morphine. The statistical distribution of the drug concentrations compared with the self-reported consumption behaviour of the users may possibly lead to a better understanding of the relationship between drug dosage and corresponding concentrations in hair.     

SPME–GC–MS analysis of α-pyrrolidinovaleorophenone in blood in a fatal poisoning case

We provided toxicological analytical support for a fatal case of abuse of α-pyrrolidinovaleorophenone (α-PVP). Solid-phase microextraction (SPME) and capillary gas chromatography coupled to mass spectrometry (GC–MS) was employed to quantify the drug in whole blood. The whole blood concentration of the drug in the heart was 486 ng/ml. This is the first report of α-PVP intoxication as ascertained by mass spectrometric identification of α-PVP in whole blood.     

Determination of 1-phenyl-2-propanone (P2P) by HS-GC/MS in a material sold as “wet amphetamine”

In this paper, we describe the approach to the characterization of an unusual material seized by the judicial authority, near Brescia City in Northern Italy. Usual analyses such as thin-layer chromatography, gas chromatography (GC)–flame ionization detection, and GC/mass spectrometry (MS) did not show the presence of drugs of abuse, in particular amphetamine-like compounds. The major solid component was identified as cornstarch; then taking into account the strong aromatic scent of the seized material; a preliminary experiment for volatile organic compounds was carried out by headspace (HS)-GC/MS. This analysis tentatively evidenced the presence of 1-phenyl-2-propanone (P2P), an amphetamine precursor. Therefore, we developed and optimized a new analytical method for determination of P2P in seized materials by HS-GC/MS. We also synthesized P2P, with the permission of the Ministry of Health, to have it as reference standard, because of its being illegal and the difficulty in obtaining it. This case had some analogies with the cases referred to as “wet amphetamine” by the judicial authority, in which amphetamines are sold mixed with P2P. The possible use of the material could be the production of tablets made of cornstarch with an aromatic scent similar to that of amphetamines to deceive consumers and to sell them as a drug of abuse.