Optimization and validation of CEDIA drugs of abuse immunoassay tests in serum on Hitachi 912

Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled the definition of cutoff values with good values for sensitivity. Small rates of false-positives can be accepted in forensic cases.     

Validity testing of commercial urine cocaine metabolite assays: II. Sensitivity, specificity, accuracy, and confirmation by gas chromatography/mass spectrometry

A comprehensive validity assessment study was performed on eight commercial urine assays for detection of cocaine use. Sensitivity, specificity, and accuracy of each assay were evaluated by analyzing, in random order and under blind conditions, specimens spiked with known drug concentrations and clinical specimens obtained from human subjects after intravenous cocaine use. Commercial assay results were compared with gas chromatography/mass spectrometry (GC/MS) assay of the same specimens for benzoylecgonine. All of the assays examined were determined to have utility in screening for cocaine use, with the exception of the KDI Quik Test, which was not a reliable test for detection of cocaine use. Major differences in sensitivity, specificity, and confirmation rate by GC/MS were noted among the assays, differences which should be taken into consideration when implementing a urine screening test for cocaine use or interpreting test results involving use of these assays.     

Comparison of the MTP immunoassay with EMIT in blood screening for drugs

We compared the MTP immunoassay with EMIT for the screening of drugs of abuse (opiates, cannabinoids, cocaine metabolites and amphetamines) in whole blood samples. These blood samples were obtained from the German police, when driving under the influence of drugs of abuse was suspected. For screening with the MTP immunoassay 25 microliters of serum or blood (without any pretreatment) was pipetted into the wells of the microtiter plates and the procedure was followed as described. Prior to screening with a Cobas Mira and EMIT reagents, the samples were treated with acetone to precipitate serum proteins. The cutoff for all drugs of abuse was set at 10 ng per ml of serum or blood. In most cases there was a good agreement between the negative and positive results of the two screening assays. The agreement between the two assays in the detection of opiates and cocaine was 91% and 93%, respectively, and for cannabinoids and amphetamines approximately 80%. The MTP immunoassay was more sensitive than EMIT for the detection of cannabinoids--but at the same time the MTP immunoassay was less specific. Both screening assays have a sensitivity of 100% for the detection of opiates and cocaine, but the specificity of the EMIT--also for opiates--was substantially lower. The MTP immunoassay has in respect to amphetamines a very high sensitivity, whereas the sensitivity of EMIT for amphetamines is inacceptable due to losses during sample preparation. The specificity of MTP immunoassay for amphetamines is not optimal, because a relatively large amount of samples tested false-positive for amphetamines at the cutoff of 10 ng/ml. In summary the MTP immunoassay, although not automated, performs well in comparison with EMIT, especially if the sample preparation for EMIT testing ist considered.     

Evaluation and applicability of Alere iCup DX 14 for rapid postmortem urine drug screening at autopsy

Performing point‐of‐care urine drug screen testing at autopsy by a forensic pathologist may provide an early indication of the presence of analytes of interest during autopsy. An evaluation for the screening of 14 classes of common drugs of abuse in postmortem urine by the point‐of‐care screening device, Alere iCup DX 14, is presented. One hundred ninety postmortem urine samples were screened with the iCup occurring at autopsy by the forensic pathologist. Positive and negative results obtained from the screening kit were evaluated against confirmatory test results obtained using routine forensic toxicology analyses that employed LC‐MS/MS and GC‐MS to detect a combination of over 85 common drugs of abuse and medications. Sensitivity for each respective iCup drug class ranged from 66% (buprenorphine) to 100% (methadone, tricyclic antidepressants). Specificity for each respective iCup drug class ranged from 89% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, 3,4‐methylenedioxymethamphetamine, methadone). Positive predictive values ranged from 44% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, methylenedioxymethamphetamine, methadone), while negative predictive values ranged from 96% (methamphetamine) to 100% (barbiturates, methadone, tricyclic antidepressants). A high false‐positive rate was yielded by the benzodiazepine class. The lack of fentanyl screening in the point‐of‐care device is a significant limitation considering its prolific prevalence in forensic casework. The results obtained in the study should be acknowledged when considering the use of the Alere iCup DX 14 in the context of postmortem casework to help indicate potential drug use contemporaneously with autopsy and when requiring such preliminary results prior to the release of a final forensic toxicology report.     

A comparison between on-site immunoassay drug-testing devices and laboratory results

The aim with this study was to evaluate the accuracy of several on-site testing devices on the market. A part of this study is included in the European Union's (EU's) roadside testing assessment project (ROSITA). An other request for this kind of study came from the Finnish prison department in the Ministry of Justice. The evaluation was performed on both urine assays and oral fluid assays. The on-site test results were compared with laboratory results (gas chromatography-mass spectrometry (GC/MS)). The samples were tested on amphetamines (AMP), cannabinoids (THC), opiates (OPI) and cocaine metabolites (COC). Some of the tests also included a metamphetamine (MET) and a benzodiazepine (BZO) test. Both positive and negative samples were tested. A total of 800 persons and eight on-site devices for urine and two for oral fluid testing were included in this study. Good results were obtained for the urine on-site devices, with accuracies of 93-99% for amphetamines, 97-99% for cannabinoids, 94-98% for opiates and 90-98% for benzodiazepines. However, differences in the ease of performance and interpretation of test result were observed. It was possible to detect amphetamines and opiates in oral fluid by the used on-site devices, but the benzodiazepines and cannabinoids did not fulfil the needs of sensitivity.     

Performance characteristics of the Cozart RapiScan Oral Fluid Drug Testing System for opiates in comparison to ELISA and GC/MS following controlled codeine administration

Oral fluid is an interesting alternative matrix for drug testing in many environments, including law enforcement, workplace drug testing, and drug treatment facilities. Performance characteristics of the FDA-cleared, qualitative, Cozart RapiScan Opiate Oral Fluid Drug Testing System (Opiate Cozart RapiScan System or Opiate CRS) were compared to the semi-quantitative Cozart Microplate EIA Opiate Oral Fluid Kit (Opiate ELISA) and to gas chromatography/mass spectrometry (GC/MS). The following oral fluid opiate cutoffs were evaluated: the GC/MS limit of quantification (LOQ) of 2.5 mg/l; 15 microg/l currently used for oral fluid testing in the United Kingdom (UK); 30 microg/l (Opiate CRS cutoff); and 40 microg/l, the proposed Substance Abuse and Mental Health Services Administration (SAMHSA) cutoff. Subjects provided informed consent to participate in this IRB-approved research and resided on the closed research ward throughout the study. Three oral codeine doses of 60 mg/70 kg were administered over a 7-day period. After a 3-week break, subjects received three doses of 120 mg/70 kg within 7 days. Oral fluid specimens (N = 1273) were analyzed for codeine (COD), norcodeine (NCOD), morphine (MOR) and normorphine (NMOR) by GC/MS with an LOQ of 2.5 microg/l for all analytes. MOR and NMOR were not detected in any sample; 26.5% of the specimens were positive for COD and 13.7% for NCOD. Opiate CRS uses a preset, qualitative cutoff of 10 microg/l; this is equivalent to 30 microg/l in undiluted oral fluid as the oral fluid collection process involves a 1:3 dilution with buffer. Sensitivity, specificity, and efficiency of Opiate CRS compared to Opiate ELISA were 98.6, 98.1, and 98.2% at a 30 microg/l cutoff and 99.0, 96.2, and 96.6% at a 40 microg/l cutoff. Compared to the much lower GC/MS LOQ of 2.5 microg/l, sensitivity, specificity and efficiency were 66.8, 99.3 and 90.7%. Increasing the GC/MS cutoff to the current UK level yielded performance characteristics of 81.5% (sensitivity), 99.3% (specificity), and 95.4% (efficiency). Using a GC/MS cutoff identical to the preset Opiate CRS cutoff yielded sensitivity, specificity, and efficiency of 88.5, 99.2, and 97.5%, respectively. At the proposed SAMSHA confirmation cutoff of 40 microg/l, sensitivity increased with little change in specificity and efficiency (91.3% sensitivity, 98.9% specificity, and 97.5% efficiency). Oral fluid is a suitable matrix for detecting drugs of abuse. Opiate CRS, with a 30 microg/l cutoff, is sufficiently sensitive, specific and efficient for oral fluid opiate analysis, performing similarly to Opiate ELISA at the same cutoff, and having performance characteristics >91% when compared to GC/MS at the proposed SAMHSA cutoff.     

Evaluation of four oral fluid devices (DDS®, Drugtest 5000®, Drugwipe 5+® and RapidSTAT®) for on-site monitoring drugged driving in comparison with UHPLC-MS/MS analysis

New Italian legislation on driving under the influence of drugs considers oral fluid (OF) as a possible alternative drug testing matrix. On this basis, the present research was carried out to evaluate the applicability of four commercial on-site OF drug screening devices, namely DDS(?), Drugtest 5000(?), Drugwipe 5+(?) and RapidSTAT(?), in a real operative context. Preliminarily trained police officers tested randomly stopped drivers with two different kits side-by-side during roadside patrols. A central laboratory confirmed on-site kits' results by UHPLC-MS/MS analysis of the saliva specimen remaining after the screening analysis. 1025 drivers were submitted to the OF tests: 11.6% were positive for cocaine and metabolites, 11.1% for THC, 6% for amphetamines and amphetamine-type designer drugs and 2.3% for ketamine. The sensitivities of the kits were 81% (RapidSTAT(?)), 82% (DDS(?)), 90% (Drugwipe 5+(?)) and 97% (Drugtest 5000(?)) for cocaine and 38% (DDS(?)), 47% (Drugwipe 5+(?)), 72% (RapidSTAT(?)) and 92% (Drugtest 5000(?)) for THC. Drugtest 5000 was the only kit showing an acceptable sensitivity for on-site application. Only Drugtest 5000(?) and RapidSTAT(?) could be evaluated for amphetamines and methamphetamines: Drugtest 5000(?) showed a sensitivity of 100% in the case of amphetamines and 86% for methamphetamines, while RapidSTAT(?) 90% and 76% respectively. Nowadays, ketamine is not included in the target analytes of any on-site devices, but it was systematically included in the UHPLC-MS/MS confirmatory analysis. To ensure adequate reliability, MS confirmation of on-site OF screening tests is anyway always necessary, due to the presence of a significant number of false positive results even when using the commercial kit with the best performance.     

生物检材中苯丙胺类兴奋剂和氯胺酮的LC-MS/MS分析

目的建立生物检材中苯丙胺类兴奋剂和氯胺酮的液相色谱-串联质谱(LC-MS/MS)分析方法。方法生物检材包括血液、尿液和毛发,采用稀释法和液液提取的前处理方法,应用两个不同的液相柱,优化LC-MS/MS分析方法,并考察了血液和尿液基质的离子抑制作用。结果同时分析苯丙胺和MDA,液相1在3m in内完成,液相2可用于确认分析或复杂基质分离。尿液稀释法检材用量少,前处理简便快速。毛发中苯丙胺类兴奋剂和氯胺酮的最低检测限(LOD)为0.005~0.05ng/mg。对送检案例检材产妇头发和胎毛进行苯丙胺类兴奋剂和氯胺酮的分析。结论本方法可用于生物检材中苯丙胺类兴奋剂和氯胺酮的同时分析,血、尿等生物检材的离子抑制作用是影响本方法灵敏度的主要原因。     

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.     

Evaluation of the IDS One-Step™ ELISA kits for the detection of illicit drugs in hair

This work presents the validation of a new immunological assay, the One-Step™ enzyme-linked immunosorbent assay (ELISA) tests from International Diagnostic Systems Corp. for the screening of drugs of abuse (cannabis, amphetamines, opiates, and cocaine) in human hair, with subsequent GC–MS confirmation. After decontamination and segmentation into small pieces, 50 mg of hair sample were incubated in 1 ml of methanol during 16 h at 40 °C. A 100 μL aliquot was collected and evaporated to dryness in presence of 100 μL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 μL of the “sample and standard diluent” solution included in the kit. This solution was submitted to analysis according to the recommended instructions of the manufacturer. During the validation phase, GC–MS confirmations were conducted according to our fully validated and published methods for opiates, cocaine, cannabis, and amphetamines determinations in hair. In a last development step, these procedures were slightly modified to directly confirm ELISA results by GC–MS using the methanolic extract. Ninety-three specimens were simultaneously screened by the ELISA tests (103 for tetrahydrocannabinol (THC)) and confirmed by GC–MS. Twenty were found positive for cannabis (THC: 0.10–6.50 ng/mg), 21 for cocaine (0.50–55.20 ng/mg), 24 for opiates (6-acetylmorphine (6-AM): 0.20–11.60 ng/mg, MOR: 0.20–8.90 ng/mg, codeine (COD): 0.20–5.90 ng/mg), and 13 for amphetamines (AP: 0.20 and 0.27 ng/mg, methamphetamine (MAP): 0.30 and 1.10 ng/mg, methylenedioxymethamphetamine (MDMA): 0.22–17.80 ng/mg). No false negative results were observed according to the Society of Hair Testing's (SoHT) cutoffs (0.5 ng/mg for cocaine, 0.2 ng/mg for opiates and amphetamines, and 0.1 ng/mg for THC). The One-Step™ ELISA kits appear suitable due to their sensitivity and specificity for drug of abuse screening in hair. This technology should find interest in workplace drug testing or driving license regranting, especially when many samples have to be tested with a high rate of negative samples, as ELISA is an easy and high-throughput method.     

Evaluation of two immunoassay procedures for drug testing in hair samples

A preliminary initial enzyme-linked immunosorbent assay (LUCIO-Direct ELISA kit) and a preliminary DRI enzyme immunoassay were evaluated for drug detection in head hair with respect to lowered cutoff values recommended in Germany for the control of abstinence in cases of re-granting of drivers' licences. Following drug classes were included: cannabinoids, opiates, cocaine like substances, amphetamine, methamphetamine (and methylenedioxyamphetamines), methadone, and benzodiazepines. 759 analyses were performed using LUCIO-Direct ELISA kits and 936 analyses using DRI enzyme immunoassay tests. Sample size for each drug group and immunoassay test reached from 74 to 178. The LUCIO-Direct ELISA kit revealed a sensitivity of 91% for amphetamine up to 98% for methadone (methamphetamine 92%, cocaine 94%, opiates 94%, benzodiazepines 96%) and values of specificity of 72% for methadone up to 89% for amphetamine and benzodiazepines. The test was not useful for a preliminary screening for tetrahydrocannabinol (sensitivity of 65%) in consideration of a suggested cutoff of 0.02 ng/mg. The DRI enzyme immunoassay test was only useful for morphine and cocaine testing at low recommended new cutoff values (0.1 ng/mg) revealing sensitivities of 94% and 99%, respectively.     

Ethyl glucuronide in hair - A highly effective test for the monitoring of alcohol consumption

In Germany drink driving offenders lose their license and must prove abstinence for one year in order to regain it. In this paper we assess the newly introduced ethyl glucuronide (EtG) tests in urine and hair in this alcohol abstinence monitoring. 20% (80 out of 386) of the 3cm long hair samples were tested positive for EtG in hair, compared to only 2% (92 out of 4248 samples) in urine in the same time period. Additionally 50% of the samples positive for EtG in hair had EtG values greater than 30pg/mg hair, indicating chronic alcohol consumption in the last three months. This study shows that four EtG tests in 3cm hair lengths reveal a significantly higher percentage of drink driving offenders who fail to be sober in the rehabilitation period, than do six random EtG tests in urine. Presumably, the hair test is more adequate to monitor long term alcohol abstinence than the urine test as defined by the new driving license re-granting medical and psychological assessment (MPA) in Germany.     

Practical aspects of roadside tests for administrative traffic offences in Germany

In the context of the European project ROSITA, the Institut of Legal Medicine Homburg/Saar has co-operated with the Saarland traffic police in order to assess different roadside drug tests for their functionality and reliability in traffic controls, and for their analytical force of evidence. In 254 cases within the time period from June 1999 to December 1999, police officers performed a (voluntary) roadside drug testing in saliva/sweat, or urine, to confirm or refute their initial suspicion that a driver had used drugs. Whereas in 45 cases the tests gave negative results (which were confirmed by lab urinalysis), in 209 cases the police officers ordered blood samples after a positive outcome of the tests.In 203 of the 209 positive cases, the results could be confirmed by GC/MS analysis. Regarding the prevalence of used drugs, a single consumption was found in 156 cases (113 cannabis, 38 amphetamines/methamphetamines, three opiates, two cocaine), and a consumption of two drugs was found in 44 cases (34 cannabis+amphetamines/methamphetamines, five cannabis+opiates, three cannabis + cocaine, two cocaine+amphetamines/methamphetamines). In three cases, multi-consumption was found.In six cases, the performed tests gave an incorrect prediction to the police officer at the roadside.The roadside tests gave 97.6% correct assistance to the police officers in the right direction (79.9% correct positive predictions and 17.7% correct negative predictions). As a consequence, the performed tests can be seen as a positive and needful tool for the police to get an immediate response to their initial suspicion and to take the right steps concerning a following legal action.     

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.     

Evaluation of the One-Step ELISA kit for the detection of buprenorphine in urine, blood, and hair specimens

A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in the range 5.3-15.6 ng/mL), did not cause a false negative response by the immunoassay.     

Roadside oral fluid testing: comparison of the results of drugwipe 5 and drugwipe benzodiazepines on-site tests with laboratory confirmation results of oral fluid and whole blood

Drugged drivers pose a serious threat to other people in traffic as well as to themselves. Reliable oral fluid screening devices for on-site screening of drugged drivers would be both a useful and convenient means for traffic control. In this study we evaluated the appropriateness of Drugwipe 5 and Drugwipe Benzodiazepines oral fluid on-site tests for roadside drug screening. Drivers suspected of driving under the influence of drugs were screened with the Drugwipe tests. Oral fluid and whole blood samples were collected from the drivers and tested for amphetamine-type stimulant drugs, cannabis, opiates, cocaine and benzodiazepines by immunological methods, GC and GC-MS. The performance evaluations of the tests were made by comparing the results of the Drugwipe tests with laboratory GC-MS confirmation results of oral fluid or whole blood. In addition to the performance evaluations of the Drugwipe tests based on laboratory results, a questionnaire on the practical aspects of the tests was written for the police officers who performed the tests. The aim of the questionnaire was to obtain user comments on the practicality of the tests as well as the advantages and weak points of the tests. The results of the performance evaluations were: for oral fluid (sensitivity; specificity; accuracy) amphetamines (95.5%; 92.9%; 95.3%), cannabis (52.2%; 91.2%; 85.1%), cocaine (50.0%; 99.3%; 98.6%), opiates (100%; 95.8%; 95.9%), benzodiazepines (74.4%; 84.2%; 79.2%) and for whole blood accordingly, amphetamines (97.7%; 86.7%; 95.9%), cannabis (68.3%; 87.9%; 84.9%), cocaine (50.0%; 98.5%; 97.7%), opiates (87.5%; 96.9%; 96.6%) and benzodiazepines (66.7%; 87.0%; 74.4%). Although the Drugwipe 5 successfully detected amphetamine-type stimulant drugs and the police officers were quite pleased with the current features of the Drugwipe tests, improvements must still be made regarding the detection of cannabis and benzodiazepines.     

Evaluation of immunoassay methods for the screening of cocaine metabolites in urine

Immunoassay kits for urine cocaine (and metabolite) screening, obtained from two commercial sources, were examined for correlation of their results, expressed in terms of equivalent benzoylecgonine concentration, with the gas chromatography/mass spectrometry (GC/MS) concentration of benzoylecgonine. The correlation coefficients obtained, based on 62 (out of a total sample population of 3295) highly relevant samples, were 0.467 and 0.766 for Abuscreen (ARIA) and TDx (TDX), respectively. The preliminary screen cutoff values, which correspond to 150 ng/mL benzoylecgonine (as determined by GC/MS), were calculated based on the resulting regression equations and found to be 380 and 190 ng/mL for ARIA and TDX, respectively. With these cutoff values, ARIA generates 5 false negatives and 16 unconfirmed presumptive positives, while TDX results in 3 false negatives and 6 unconfirmed presumptive positives.     

Blind trials of an onsite saliva drug test for marijuana and opiates

The objective of these clinical trials was to calculate the performance, limit of detection, specificity and sensitivity of a novel, semi-quantitative immunoassay for drugs of abuse in saliva and to determine operator bias when measured blind by four different operators. The test is based on lateral flow gold particle technology coupled with digital photography to provide a semi-quantitative end point. The performance of the test was compared with that of enzyme immunoassays and GC/MS methods. Volunteers consumed marijuana or codeine and their saliva was collected 0.25 to 24 h later with the Cozart RapiScan collection device. The sensitivity and specificity of the opiate test were both 100%+/-10.4% for codeine for 9 h after dosing. The cutoff of the marijuana test at 10 ng/mL THCA was too high to detect marijuana use for more than a few hours after smoking. There was no operator bias because the results were presented in written form either as "positive" or "negative" for each of the five drug classes on the screen of the hand-held reader.     

Evaluation of the IDS One-Step ELISA kits for the detection of illicit drugs in hair

This work presents the validation of a new immunological assay, the One-Step enzyme-linked immunosorbent assay (ELISA) tests from International Diagnostic Systems Corp. for the screening of drugs of abuse (cannabis, amphetamines, opiates, and cocaine) in human hair, with subsequent GC-MS confirmation. After decontamination and segmentation into small pieces, 50 mg of hair sample were incubated in 1 ml of methanol during 16 h at 40 degrees C. A 100 microL aliquot was collected and evaporated to dryness in presence of 100 microL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 microL of the "sample and standard diluent" solution included in the kit. This solution was submitted to analysis according to the recommended instructions of the manufacturer. During the validation phase, GC-MS confirmations were conducted according to our fully validated and published methods for opiates, cocaine, cannabis, and amphetamines determinations in hair. In a last development step, these procedures were slightly modified to directly confirm ELISA results by GC-MS using the methanolic extract. Ninety-three specimens were simultaneously screened by the ELISA tests (103 for tetrahydrocannabinol (THC)) and confirmed by GC-MS. Twenty were found positive for cannabis (THC: 0.10-6.50 ng/mg), 21 for cocaine (0.50-55.20 ng/mg), 24 for opiates (6-acetylmorphine (6-AM): 0.20-11.60 ng/mg, MOR: 0.20-8.90 ng/mg, codeine (COD): 0.20-5.90 ng/mg), and 13 for amphetamines (AP: 0.20 and 0.27 ng/mg, methamphetamine (MAP): 0.30 and 1.10 ng/mg, methylenedioxymethamphetamine (MDMA): 0.22-17.80 ng/mg). No false negative results were observed according to the Society of Hair Testing's (SoHT) cutoffs (0.5 ng/mg for cocaine, 0.2 ng/mg for opiates and amphetamines, and 0.1 ng/mg for THC). The One-Step ELISA kits appear suitable due to their sensitivity and specificity for drug of abuse screening in hair. This technology should find interest in workplace drug testing or driving license regranting, especially when many samples have to be tested with a high rate of negative samples, as ELISA is an easy and high-throughput method.     

A high-throughput urinalysis of abused drugs based on a SPE-LC-MS/MS method coupled with an in-house developed post-analysis data treatment system

A rapid urinalysis system based on SPE-LC-MS/MS with an in-house post-analysis data management system has been developed for the simultaneous identification and semi-quantitation of opiates (morphine, codeine), methadone, amphetamines (amphetamine, methylamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA)), 11-benzodiazepines or their metabolites and ketamine. The urine samples are subjected to automated solid phase extraction prior to analysis by LC-MS (Finnigan Surveyor LC connected to a Finnigan LCQ Advantage) fitted with an Alltech Rocket Platinum EPS C-18 column. With a single point calibration at the cut-off concentration for each analyte, simultaneous identification and semi-quantitation for the above mentioned drugs can be achieved in a 10 min run per urine sample. A computer macro-program package was developed to automatically retrieve appropriate data from the analytical data files, compare results with preset values (such as cut-off concentrations, MS matching scores) of each drug being analyzed and generate user-defined Excel reports to indicate all positive and negative results in batch-wise manner for ease of checking. The final analytical results are automatically copied into an Access database for report generation purposes. Through the use of automation in sample preparation, simultaneous identification and semi-quantitation by LC-MS/MS and a tailored made post-analysis data management system, this new urinalysis system significantly improves the quality of results, reduces the post-data treatment time, error due to data transfer and is suitable for high-throughput laboratory in batch-wise operation.