Development and validation of the Cozart DDS oral fluid collection device

The testing of oral fluid for drugs of abuse has increased significantly over recent years and is now commonplace in drug rehabilitation clinics, the workplace, prisons and custody suites. The global problem of identifying drugged drivers has also led to an increase in oral fluid testing at the roadside. The main requirements for the implementation of roadside drug testing are a rapid sample collection time, collection of a known sample volume and recovery of drugs from the collection device. We report here the development of the Cozart^® DDS oral fluid collector, an oral fluid collector that combines rapid and adequate sample collection with satisfactory drug recovery. Oral fluid was collected from drug users (n = 134) and drug-free individuals (n = 137), using the Cozart^® DDS oral fluid collector. The mean time for the completion of collection (full coloration of the sample presence indicator) was 34 s for drug-free individuals and 44 s for drug users. The average volume collected was 0.34 mL (n = 271). No chemical stimulant (to induce salivation) was used to achieve the collection times observed in either the drug-free or the drug-taking sample populations. Drugs were extracted from the collector using the Cozart^® DDS buffer and drug recovery was determined by Cozart^® enzyme immunoassays. The recovery studies showed that for amphetamine, Δ⁹THC, cocaine, methadone, methamphetamine, morphine and temazepam over 90% of the drug in the sample was eluted from the collector. The Cozart^® DDS oral fluid collector provides a reliable mechanism for the collection of oral fluid at the roadside that achieves the rapid collection times required.     

Simultaneous analysis of psychotropic phenylalkylamines in oral fluid by GC-MS with automated SPE and its application to legal cases

Phenylalkylamine derivatives, such as methamphetamine (MA), amphetamine (AM), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), phentermine (PT), fenfluramine (FFA) and phenmetrazine (PM), and ketamine (KT) are widely abused recreational or anorectic drugs in Korea and are regulated under the Controlled Substance Act in Korea. Phenylalkylamines and ketamine analysis is normally performed using both urine and hair samples but there is no established method for the simultaneous analysis of all these phenylalkylamines and ketamine in oral fluids. Oral fluid is easy to collect/handle and can provide an indication of recent drug abuse. In this study, to confirm the presence of phenylalkylamine derivatives and ketamine in oral fluid after screening with an immunoassay, an analytical method using automated solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed and fully validated according to international guidelines. The applicability of the assay was demonstrated by analyzing of authentic oral fluid samples and the results of oral fluid analysis were compared with those in urine and hair to to evaluate the feasibility of oral fluid in forensic cases. The recovery of phenylalkylamines and ketamine from oral fluid collection devices was also assessed. Oral fluid specimens from 23 drug abuse suspects submitted by the police were collected using Salivette (Sarstedt, Nümbrecht, Germany), Quantisal (Immunalysis, Pomona, CA) or direct expectoration. The samples were screened using a biochip array analyzer (Evidence Investigator, Randox, Antrim, UK). For confirmation, the samples were analyzed by GC-MS in selected-ion monitoring (SIM) mode after extraction using automated SPE (RapidTrace, Zymark, MA, USA) with a mixed-mode cation exchange cartridge (CLEAN SCREEN, 130 mg/3 ml, UCT, PA, USA) and derivatization with trifluoroacetic anhydride (TFA). The results from the immunoassay were consistent with those from GC-MS. Twenty oral fluid samples gave positive results for MA, AM, PT and/or PM among the 23 cases, which gave positive results in urine and/or hair. Although large variations in the MA, AM, PT and PM concentrations were observed in three different specimens, the oral fluid specimen was useful for demonstrating phenylalkylamines and ketamine abuse as an alternative specimen for urine.     

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.     

The frequency of alcohol,illicit and licit drug consumption in the general driving population in South-East Hungary

In the framework of the DRUID (Driving under the Influence of Drugs, Alcohol, and Medicines) EU-6 project, a roadside survey was performed in South-East Hungary to determine the incidence of alcohol and the most frequent illicit and licit drug consumption (amphetamines, THC, illicit and medical opiates, cocaine, ketamine, benzodiazepines, zopiclone and zolpidem) in the general driving population. All 3110 drivers stopped between 01 January 2008 and 31 December 2009 were checked for alcohol, and among them 2738 persons (87.7%) participated in the further examinations, on a voluntary basis. Licit and illicit drugs were determined from their oral fluid samples by GC–MS analysis.Illicit drugs were detected in 27 cases (0.99%), licit drugs in 85 cases (3.14%), and alcohol (cut off: 0.1 g/l) was found in 4 (0.13%) cases. Illicit drug consumption was the highest among men of the ages 18–34, during the spring, and on the week-end nights. With respect to licit drugs, the highest incidence was found among women over the age of 50, during the summer, and on the week-days. All alcohol positive cases were men over the age of 35. In comparison to international European averages, the alcohol and illicit drug consumption was low, but the licit drug consumption was over the European average.     

GHB and driving impairment.

Gamma hydroxybutyrate (GHB) was identified in the blood of 13 subjects arrested for impaired driving. GHB concentrations ranged from 26 to 155 mg/L (mean 87 mg/L, median 95 mg/L). In eight cases, GHB was the only drug detected, and signs of impairment were consistent with those of a CNS depressant, including erratic driving (weaving, swerving, ignoring road signs), confusion, incoherent speech, unresponsiveness, lack of balance, unsteady coordination, poor performances on field sobriety tests, and varying states of wakefulness. Given the ability of GHB to induce sleep and unconsciousness, it is evident from these cases that recreational use of the drug has the potential to impair a person's driving ability.     

Drugs in oral fluid in randomly selected drivers

There were 13,176 roadside drug tests performed in the first year of the random drug-testing program conducted in the state of Victoria. Drugs targeted in the testing were methamphetamines and Δ⁹-tetrahydrocannabinol (THC). On-site screening was conducted by the police using DrugWipe^®, while the driver was still in the vehicle and if positive, a second test on collected oral fluid, using the Rapiscan^®, was performed in a specially outfitted “drug bus” located adjacent to the testing area. Oral fluid on presumptive positive cases was sent to the laboratory for confirmation with limits of quantification of 5, 5, and 2 ng/mL for methamphetamine (MA), methylenedioxy-methamphetamine (MDMA), and THC, respectively. Recovery experiments conducted in the laboratory showed quantitative recovery of analytes from the collector. When oral fluid could not be collected, blood was taken from the driver and sent to the laboratory for confirmation. These roadside tests gave 313 positive cases following GC–MS confirmation. These comprised 269, 118, and 87 cases positive to MA, MDMA, and THC, respectively. The median oral concentrations (undiluted) of MA, MDMA, and THC was 1136, 2724, and 81 ng/mL. The overall drug positive rate was 2.4% of the screened population. This rate was highest in drivers of cars (2.8%). The average age of drivers detected with a positive drug reading was 28 years. Large vehicle (trucks over 4.5 t) drivers were older; on average at 38 years. Females accounted for 19% of all positives, although none of the positive truck drivers were female. There was one false positive to cannabis when the results of both on-site devices were considered and four to methamphetamines.     

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.     

Analysis of cannabis in oral fluid specimens by GC-MS with automatic SPE

Methamphetamine (MA) is the most commonly abused drug in Korea, followed by cannabis. Traditionally, MA analysis is carried out on both urine and hair samples and cannabis analysis in urine samples only. Despite the fact that oral fluid has become increasingly popular as an alternative specimen in the field of driving under the influence of drugs (DUID) and work place drug testing, its application has not been expanded to drug analysis in Korea. Oral fluid is easy to collect and handle and can provide an indication of recent drug abuse.In this study, we present an analytical method using GC–MS to determine tetrahydrocannabinol (THC) and its main metabolite 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid. The validated method was applied to oral fluid samples collected from drug abuse suspects and the results were compared with those in urine. The stability of THC and THC-COOH in oral fluid stored in different containers was also investigated.Oral fluid specimens from 12 drug abuse suspects, submitted by the police, were collected by direct expectoration. The samples were screened with microplate ELISA. For confirmation they were extracted using automated SPE with mixed-mode cation exchange cartridge, derivatized and analyzed by GC-MS using selective ion monitoring (SIM).The concentrations of THC and THC-COOH in oral fluid showed a large variation and the results from oral fluid and urine samples from cannabis abusers did not show any correlation. Thus, detailed information about time interval between drug use and sample collection is needed to interpret the oral fluid results properly. In addition, further investigation about the detection time window of THC and THC-COOH in oral fluid is required to substitute oral fluid for urine in drug testing.     

Legal issues in oral fluid testing

The use of oral fluid for drugs of abuse testing has received increased attention with the availability of accurate methods for the collection and analysis of drugs in oral fluid specimens. Already used in the transportation and insurance industries, there is increasing interest in oral fluid drug testing in the workplace, schools, roadside driving under the influence of drugs, and criminal justice. Given that sanctions may accrue from positive test results, legal challenges are to be expected. However, with its established scientific base, demonstrated accuracy and reliability of collection and test methods, and current positive regulatory developments, it seems clear that the use of oral fluid as a specimen for drugs of abuse testing will be able to withstand judicial scrutiny.     

Legal issues in oral fluid testing

The use of oral fluid for drugs of abuse testing has received increased attention with the availability of accurate methods for the collection and analysis of drugs in oral fluid specimens. Already used in the transportation and insurance industries, there is increasing interest in oral fluid drug testing in the workplace, schools, roadside driving under the influence of drugs, and criminal justice. Given that sanctions may accrue from positive test results, legal challenges are to be expected. However, with its established scientific base, demonstrated accuracy and reliability of collection and test methods, and current positive regulatory developments, it seems clear that the use of oral fluid as a specimen for drugs of abuse testing will be able to withstand judicial scrutiny.     

Detection of cocaine and its metabolites in breast milk

A method was developed for measuring cocaine and its metabolites, benzoylecgonine, ecgonine methyl ester, norcocaine, ecgonine ethyl ester, cocaethylene, and m-hydroxybenzoylecgonine, in breast milk by gas chromatography/mass spectrometry. Limits of detection for this method ranged from 2.5 to 10 ng/mL, and limits of quantitation ranged from 5 to 50 ng/mL. For each of the compounds measured by this method, linear response was demonstrated to 750 ng/mL. Breast milk was collected from 11 mothers who admitted to drug use during pregnancy and ten drug-free volunteers serving as control subjects. Cocaine was detected in six of the specimens obtained from drug-exposed subjects, and in none of the drug-free control subjects. In breast milk specimens where cocaine and one or more of its metabolites were detected, the concentration of parent compound was greater than any of the metabolites. The highest cocaine concentration found was over 12 microg/mL. Breast-fed infants of cocaine abusing mothers may be exposed to significant amounts of drug orally.     

Comparison of point-of-collection screening of drugs of abuse in oral fluid with a laboratory-based urine screen.

Oral fluid is becoming increasingly useful for the detection of drugs, since it is a non-invasive specimen to collect and, because collection is directly observed, it is difficult to adulterate. A point-of-collection (POCT) oral fluid drug analysis kit has been developed for use in many drug testing situations. This paper summarizes the results of field evaluations of the ORALscreen System for screening of drugs in oral fluid. The ORALscreen System consists of an oral fluid collection device and a test device containing a lateral flow membrane immunoassay system. Paired samples (oral fluid and urine) were collected from drug users and the results from the ORALscreen POCT system were compared to urine screening results conducted in a licensed laboratory. The results demonstrate that the ORALscreen System has excellent percent agreement with the laboratory-based urine screening test results for the detection of cocaine and opiates through 2.5-3 days following drug use, respectively. Tetrahydrocannabinol (THC) was detected by ORALscreen on the day of use and 1 day after use. Good correlation between urine and oral fluid screening results was observed for the methamphetamine positive samples; however, the number of days following drug use was not determined.     

氯胺酮滥用的研究进展

氯胺酮为非竞争性NMDA受体阻断剂,作为一种分离性麻醉药应用于临床,其作用机制复杂,由于其具有一定的致幻作用而被滥用于各种娱乐场所,是目前药物滥用中的一个新问题。本文在阐述氯胺酮的药理学及毒理学特性的基础上,复习了氯胺酮与NMDA受体、多巴胺受体等神经生物学相关受体的相互作用,综述氯胺酮滥用的现状、滥用的可能生理机制及其滥用后的检测方法等方面的研究进展。     

Comparison of GC-MS and EIA results for the analysis of methadone in oral fluid

The purpose of these studies was to evaluate the performance characteristics of the Cozart Microplate Enzyme Immunoassay (EIA) for the determination of methadone in oral fluid from patients in a drug misuse treatment program. Oral fluid specimens were collected using the Cozart RapiScan Collection system from 198 donors who were receiving treatment for their addiction and were monitored for drug misuse. Oral fluid specimens were also collected from forty volunteer donors who were not drug users. The specimens were analyzed in the laboratory by EIA and then analysed for methadone and its main metabolite EDDP by gas chromatography-mass spectrometry (GC-MS). A total of 103 samples were confirmed positive for methadone. The Cozart Microplate EIA for d-Methadone in oral fluid using a cutoff of 30 ng/mL in diluted oral fluid had a sensitivity of 91.3% +/- 2.8% and a specificity of 100% +/- 1.0% vs. GC-MS.     

The behavioral and cognitive effects of two benzodiazepines associated with drug-facilitated sexual assault

Recently, sexual assaults have included the use of benzodiazepines to impair the victim. Our aim was to examine the physiological, cognitive, and behavioral effects of flunitrazepam (FN) and clonazepam (CLO). In the first study, ten healthy volunteers received a single oral dose of 2 mg of FN. Mini Mental State Examination (MMSE), behavioral reports and staff observations were then collected. In the second study, ten healthy volunteers received a single oral dose of 3 mg of CLO. Vital signs, performance on the MMSE and Digit Symbol Substitution Test, and behavioral changes were examined. FN significantly decreased systolic and diastolic blood pressure 4 h post drug ingestion with diastolic remaining low at 6 h. CLO was associated with changes in temperature and decreased systolic pressure. FN affected memory and attention 4 h following ingestion. CLO affected memory and attention throughout the study (6 h), and psychomotor performance was decreased 2 h post ingestion. In both studies, subjects were disinhibited and did not perceive their own impairment.     

Validation of the Cozart microplate EIA for analysis of opiates in oral fluid

The purpose of this study was to determine the performance characteristics of the Cozart microplate EIA for detecting opiates in oral fluid from patients in a drug misuse treatment program. Oral fluid samples were collected using the Cozart RapiScan Collection System from 216 donors who were receiving treatment for their addiction and were monitored for drug abuse. A further 40 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory by using the Cozart microplate EIA for opiates and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. The intra-assay precision for the Cozart microplate oral fluid EIA for opiates over 40 assays was 0.43% to 9.13% CV (within assay) and 2.9% to 9.1% CV (within day). A total of 109 samples were positive for various opiates. The Cozart microplate EIA for opiates in oral fluid, using a cut-off of 30 ng/ml morphine equivalents in neat oral fluid, had a sensitivity of 99.1 +/- 2.1% and a specificity of 94.4 +/- 2.2% versus GC-MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

新型毒品氯胺酮分析方法研究进展

本文介绍了氯胺酮的提取及检测现状,比较了液液提取、固相萃取、固相微萃取等提取方的优劣和GC/NPD、GC/FID、GC/MS以及薄层色谱等不同分析方法的应用情况。     

氯胺酮成瘾机制的研究进展

氯胺酮为苯环己哌啶（PCP）衍生物,是非竞争性N-甲基-D-天门冬氨酸（NMDA）受体桔抗剂。除作为临床上常用的静脉麻醉药外,氯胺酮还具有一定的致幻作用和成瘾性,非法滥用氯胺酮的人数呈现快速增长趋势。本文根据氯胺酮的药理及毒理学特性与目前公认的药物成瘾机制．从与药物成瘾机制有关的强化效应和奖赏系统、解剖结构、相关受体及个体差异等方面,对氯胺酮成瘾的可能机制作一综述。     

Elimination of 7-aminoflunitrazepam and flunitrazepam in urine after a single dose of Rohypnol

The hypnotic benzodiazepine flunitrazepam (Rohypnol) has been identified as the drug of choice for the purposes of "drugging" unsuspecting victims and raping them while they are under the influence of this substance. The objective of this paper was to study elimination of flunitrazepam and 7-aminoflunitrazepam in urine collected from ten healthy volunteers who received a single 2 mg oral dose of Rohypnol, to determine how long after drug administration 7-aminoflunitrazepam can be detected. A highly sensitive NCI-GC-MS method for the simultaneous quantitation of flunitrazepam (LOQ 100 pg/mL) and 7-aminoflunitrazepam (LOQ 10 pg/mL) in urine was developed. All samples were screened for benzodiazepines using optimized micro-plate enzyme immunoassay. The highest concentrations of 7-aminoflunitrazepam (70-518 ng/mL) and flunitrazepam (0.7-2.8 ng/mL) in urine were observed 6 h after drug administration in nine subjects and after 24 h in one subject. In six subjects 7-aminoflunitrazepam was detected up to 14 days after flunitrazepam administration, in one subject up to 21 days and in three subjects up to 28 days. In urine samples collected from six volunteers, flunitrazepam was detected three days after Rohypnol intake, in three subjects 24 h, and in one subject 5 days later. Benzodiazepine micro-plate enzyme immunoassay kit allowed the detection of flunitrazepam and metabolities 5 to 21 days after drug administration.     

Driving under the influence of drugs -- evaluation of analytical data of drugs in oral fluid, serum and urine, and correlation with impairment symptoms

A study was performed to acquire urine, serum and oral fluid samples in cases of suspected driving under the influence of drugs of abuse. Oral fluid was collected using a novel sampling/testing device (Dr?ger DrugTest System). The aim of the study was to evaluate oral fluid and urine as a predictor of blood samples positive for drugs and impairment symptoms. Analysis for cannabinoids, amphetamine and its derivatives, opiates and cocaine was performed in urine using the Mahsan Kombi/DOA4-test, in serum using immunoassay and gas chromatography-mass spectrometry (GC-MS) confirmation and in oral fluid by GC-MS. Police and medical officer observations of impairment symptoms were rated and evaluated using a threshold value for the classification of driving inability. Accuracy in correlating drug detection in oral fluid and serum were >90% for all substances and also >90% in urine and serum except for THC (71.0%). Of the cases with oral fluid positive for any drug 97.1% of corresponding serum samples were also positive for at least one drug; of drug-positive urine samples this were only 82.4%. In 119 of 146 cases, impairment symptoms above threshold were observed (81.5%). Of the cases with drugs detected in serum, 19.1% appeared not impaired which were the same with drug-positive oral fluid while more persons with drug-positive urine samples appeared uninfluenced (32.7%). The data demonstrate that oral fluid is superior to urine in correlating with serum analytical data and impairment symptoms of drivers under the influence of drugs of abuse.