Lill Sun De 2008

PROCEEDINGS PAPER

Analytical Techniques for Drug Detection in Oral Fluid Pirjo Lillsunde, PhD

Abstract: Analytical techniques for detection of drugs in oral fluid (OF) are reviewed with emphasis on applications used in European Union (EU) roadside testing projects. Oral fluid is readily accessible and collectible. It has become an interesting material because no medical personnel are needed for sampling. This matrix is especially applicable for preliminary drug testing in driving under the influence controls and for monitoring illicit drug use in drug treatment. Oral fluid is also an increasingly used specimen in epidemiologic studies and in workplace drug testing. Drugs are present at lower levels in OF than in urine. The window of detection of drugs in OF reflects the corresponding window in blood, suggesting OF as a specimen of choice for roadside testing. Saliva/blood ratios vary from drug to drug, from person to person, and even intraindividually making therapeutic drug monitoring in OF challenging. Several sensitive methods for drug testing in OF have been developed during the last years. Key Words: drugs, oral fluid, analytical techniques (Ther Drug Monit 2008;30:181–187)

INTRODUCTION Recent developments in analytical techniques and methods have enabled simultaneous determination of several drug classes in small volumes of oral fluid.1–4 Oral fluid (OF) is a promising biologic matrix for drug analysis and an alternative specimen to blood and urine.5 Oral fluid sample collection is simple and noninvasive and can be done in close supervision. Opportunity for sample adulteration and substitution are minimal in comparison to urine specimens. Screening tests in OFs can be performed without medical personnel, eg, by police officers.6 The European roadside testing study (ROSITA) concluded that OF was the preferred specimen for monitoring drug use of drivers on the roadside. Oral fluid may also be useful for some applications in therapeutic drug monitoring, which so far is usually performed with plasma samples obtained by venipuncture. In the future, certain therapeutic drugs may be monitored in OF by patients themselves at home using rapid tests. A number of reviews have been published on analysis of drugs in oral fluid.7–13 The role of OF drug testing is expanding in driving under the influence and workplace drug testing areas as well as in therapeutic drug monitoring and in monitoring illicit drug Received for publication September 13, 2007; accepted December 6, 2007. From the National Public Health Institute, Drug Research Unit, Helsinki, Finland. Address for correspondence: Pirjo Lillsunde, National Public Health Institute, Drug Research Unit, Mannerheimintie 166, 00300 Helsinki, Finland (e-mail: [email protected]). Copyright Ó 2008 by Lippincott Williams & Wilkins

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use in drug treatment. Oral fluid is also an increasingly used specimen in pharmacokinetic and epidemiologic studies.7 The drug concentrations in OF depend on the latest dose, the route of administration, the salivary pH, physicochemical properties of the drug, and the degree of plasma protein binding. Oral fluid concentration reflects the detection window of these drugs in blood and free drug levels in plasma, although the reflection is not simple and can be affected by, for example, salivary pH and salivary flow.14 Nevertheless, a relationship between behavior/impairment and OF drug concentration has been suggested, making OF an interesting tool, especially for roadside control of drugs.15 Oral fluid seems especially well suited for detecting recent drug use on-site. However, saliva/plasma (S/P) ratios may vary considerably depending on the substance, and large interindividual differences in the measured S/P ratio have been reported.16–18 For amphetamines, cocaine, and heroine metabolites, the S/P ratio is greater than 1. The amphetamine OF/B ratio was reported to be especially high in cases in which amphetamine in relatively high concentrations was measured in OF and blood of drivers who were suspected of driving under the influence of drugs. The detection time window for which amphetamines was found was also longer for OF than for blood when the cutoff points 20 ng/mL in whole blood and 25 ng/mL in OF were used. The results indicated that the limits of detection for amphetamine in OF should be higher than for whole blood for the window of detection to be comparable.18 For benzodiazepines, the S/P ratio is very low. It seems that, at least in suspected driving under the influence cases, tetrahydrocannabinol (THC) in OF can be derived mainly from contamination of the OF cavity during cannabis smoking and therefore THC in OF has been measured in a wide concentration range.19,20 Heroin use can often be detected in OF better than in blood because 6-acetylmorphine/morphine ratios in OF are higher than unity.21 Enzymatic oral fluid test devices have been used to investigate the role of alcohol in a forensic praxis.22 Oral fluid alcohol content is a good indicator of plasma alcohol concentration (S/P = 1) if the sample is taken 20 minutes after ingestion to allow for absorption and distribution, preventing oral contamination, but drugs are a more complicated issue. In addition to rapid and reliable methods, use of OF in therapeutic drug monitoring requires a repeatable relationship between OF and plasma concentration. A narrow margin between the drug’s therapeutic and toxic effects is needed and low intra- and intersubject variability.7 Therefore, not all frequently monitored substances may be suitable candidates in therapeutic oral fluid monitoring. Commonly used antiepileptics phenytoin, carbamazepine, primidone, ethosuximide, levetiracetam, topiramate, and lamotrigine were considered good candidates for therapeutic oral fluid monitoring.7

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Oral fluid could be used in monitoring of antimicrobials isoniazid, ciprofloxacin, and gentamicin as well.7 Commercially available screening devices allow on-site testing. Immunologic methods such as the enzyme-linked immunosorbent assay are commonly used for oral fluid analysis. Gas chromatography–mass spectrometry (GC-MS) is also commonly used, but many laboratories have recently developed liquid chromatography–mass spectrometry (LCMS) or liquid chromatography–tandem mass spectrometry (LC-MS-MS) methods for analysis of drugs in oral fluid.

European Union Roadside Testing Assessment ÔROSITA 1 and 2Õ: Projects and European Union Driving Under Influence of Drugs (DRUID) Project There have been two European Union–(USA) projects (ROSITA) concerning on-site testing in road traffic control since 1998.6,21 The purpose of these projects was to evaluate on-site testing devices. In ROSITA-1, both the available urine and OF testing devices were evaluated.21,23 The survey was conducted in collaboration with the various national police officers on the road. Laboratories confirmed the drug findings in OF by using the same, most often GC-MS, methods as for blood analysis. Development of LC-MS methods in the consortium started. A clear demand for OF on-site testing arose from the police side. Therefore, the ROSITA-2 project (2003–2005) concentrated solely on the evaluation of usability and analytical reliability of on-site OF (saliva) drug testing devices.

On-Site Tests The aim of the ROSITA-2 project was to evaluate the available on-site devices for the detection of drugs in OF at the roadside or in a police station. For confirmation analysis, an additional oral fluid sample was taken with the Intercept device (OraSure Technologies, Inc., Bethlehem, PA, USA). Also, a blood sample was taken. Nine on-site devices were evaluated: Drugwipe (Securetec, Brunnthal, Germany), Lifepoint Impact (San Francisco, CA, USA), OraLab (Varian, Lake Forest, CA, USA), OraLine (Sun Biomedical Laboratories, Inc., Blackwood, NJ, USA), Oral Stat device (American Bio Medica Corporation, New York, NY, USA), Oratect II (Branan Medical Corporation, Irvine, CA, USA), RapiScan (Cozart Bioscience Ltd., Oxfordshire, UK), Saliva Screen 5 (Ultimed Products GmbH, Ahrensburg, Germany), and Uplink/Drug Test (Orasure Technologies Inc., Bethlehem, PA, USA exclusively for Dra¨ger Safety AG & Co, Lu¨beck, Germany).6 In the ROSITA-1 project, the criteria of sensitivity and specificity (greater than 90%) and accuracy (greater than 95%) was proposed. None of the devices tested in ROSITA-2 met these criteria. However, some of the devices detected a few of the substance classes well. Sensitivity, specificity, and accuracy for amphetamine screening with Drugwipe calculated based on OF confirmation results were 95.5%, 92.9%, and 95.3%, respectively (128 true-positives from 148 analyzed by OF), whereas the sensitivity for cannabis was low (52.5% ).22,24 A significant variation existed between drug classes. Theoretically, both cocaine and opiates should be more easily detected in OF because of generally higher concentrations compared

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with D9-THC and benzodiazepines. Sensitivity for amphetamines, opiates, and cocaine (S/P ratios 1) were generally better than for THC and benzodiazepines. In the ROSITA projects, the importance of size, practicability, rapidity, and portability of the testing device was emphasized from the operational point of view. In addition to thin lines in the test display and sometimes difficult interpretation of the test result, practical problems had been noted, eg, with using cold water for testing and/or test devices in winter. Furthermore, the education of police officers was important. Most of the police officers who performed the tests saw the OF on-site test devices as valuable tools for helping in the identification and confirming of initial suspicion of drug use. The police officers with more experience of using on-site devices were generally more satisfied with the device. In a few countries, legislation already allows the use of OF for screening and/or confirmation in suspected driving under influence of drugs cases. Although at the end of the ROSITA-2 project, no device was considered reliable enough to be recommended for roadside screening of drivers, on-site tests are used routinely in some countries by police officers. In Australia, random roadside tests for drugs are allowed and drivers are tested by Drugwipe (Securetec) and RapiScan (Cozart Bioscience Ltd.). If both devices give a positive drug result, an OF sample is sent to the laboratory for confirmation analysis. The police in Finland started using Drugwipe in 2003 as an on-site test in cases in which police officers already suspect drug use. Amphetamine is the most commonly found illicit drug among Finnish drivers. The police are satisfied with the ability of the device to detect amphetamine users. Since then, the number of amphetamine findings has more than doubled among driving under the influence suspected drivers.25

Oral Fluid Sample Collection, Storage, and Pretreatment for Confirmatory Analysis Oral fluid collection is a critical step in the validity of the OF drug testing process. Oral fluid can be collected by adsorbent swabs, by spitting, draining, or suction. The flow can be stimulated mechanically (chewing gum, parafilm, Teflon, rubber band) or chemically (citric acid). Although stimulation allows collection in a shorter time, changing the pH of OF can affect the concentrations of drugs and metabolites and affect the S/P ratio of drugs. Citric acid stimulation of OF, for example, produced a fivefold decrease of cocaine, benzoylecgonine (BE), and ecgonine methyl ester (EME) in OF. It can also cause interference in some immunoassays. Absorption of highly lipophilic substances may occur when using parafilm or other stimulation devices, leading to decreases in the amount of measured drugs.7 Oral fluid specimen collectors are valuable tools for collecting OF. Collecting a valid and representative OF is a challenge. If drugs are administered orally or smoked, OF may be contaminated and elevated concentrations of drugs may be found. These elevated concentrations affect adversely S/P and S/B ratios, which make reliable interpretation of OF results difficult.26 The preservation buffer solution of devices may cause long-term problems in GC-MS analysis and poor q 2008 Lippincott Williams & Wilkins

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precision and accuracy in the form of ion suppression (see subsequently). Oral fluid samples should be stored at +4°C and be analyzed as soon as possible. If longer storage is needed, samples should be stored at –20°C. Freezing lowers the viscosity and after thawing, samples can be easily centrifuged. For pretreatment, both liquid–liquid and solid-phase extraction is used. The variability of the recoveries of different substances from the different collection devices was not well known at the time of the ROSITA-2 project. All the ROSITA partners agreed to use the Intercept collector, although the drug stability in the buffer and absorption of drugs in the cotton roll of the specimen collector was not known. The average amount collected with the device was low, only 224 mL (minimum, 0; maximum, 795 mL) in roadside conditions. In laboratory conditions, however, much higher volumes were collected (500 to 700 mL).24 There was a wide variability in the volumes of OF collected. Collection of OF was much more difficult, for example, from amphetamine abusers, whose oral fluid secretion is lowered and who experienced dry mouth.5 Oral fluid was diluted with buffer in the Intercept device, which complicated the quantitative determination, especially because the amount of buffer solution was not constant. Furthermore, some nonvolatile components in the Intercept buffer coextracted with the analytes, increasing the background interference in GC-MS analysis and decreasing the column life.1 A study27 was done find out the best choice of OF collection device for the European Union project, DRUID. The criteria were that the device should be suitable for roadside collection (fast, easy to use), provide enough sample for toxicologic analysis, be able to measure sample volume, and enable good recovery and stability of drugs and medicinal drugs. The tested analytes were amphetamine, MDMA, THC, cocaine, morphine, codeine, diazepam, alprazolam (all 1000

ng/mL in OF), and ethanol (0.2% in OF). Analytes were quantified from OF or OF buffer solution by GC-MS (all except for ethanol) and gas-chromatography flame ionization detector (GC-FID) (ethanol). Collected OF volume was tested with test persons (n = 6). Collection time of different collectors was recorded simultaneously by the test persons and also user comments were requested. The summary of the results are presented in Table 1. The ordinary plastic tube did well in the experiments, but it was considered somewhat messy to collect OF by spitting. Other collection devices with no buffer were not good enough in terms of recovery or stability. The Greiner was considered to be too complicated to use at the roadside. The Statsure collector gave the best results for recovery (greater than 80% for all substances) and good results for stability. It was quick to use (less than 2-minute collection time) and received good user feedback. The buffer solutions seem to contain macromolecules that coextract with the analytes in the extraction procedure and cause long-term problems in the GCMS systems.1 Therefore, the buffer solutions from the devices, including Statsure, contaminated the GC liner significantly. The only exception was the Greiner that provided a clean background in the chromatograms. None of the collection devices was thus perfect, but the Statsure was chosen as the OF collection device of the DRUID project.27 Buffer macromolecules seemed to coextract with target drugs and caused interfering background in chromatograms both after solidphase extraction as well as after liquid–liquid extraction.1,27

Screening Tests Used by Laboratories The analytical approaches for OF testing of laboratories in ROSITA project were immunologic, GC-MS, and LC-MS (-MS) methods. Some laboratories used immunologic screening like the enzyme-linked immunosorbent assay before confirmation, but most laboratories did screening and confirmation simultaneously by chromatographic methods. Table 2

TABLE 1. Summary of Evaluation of Collection Devices27 Parameter

Best

In Between

Worst

Collection time (test persons) persons) Best: 0–2 minutes In between: 2–4 minutes Worst: .4 minutes Recovery Best: .80% In between: THC ,80% Worst: THC and any other(s) ,80% Stability (28 days storage) Best: ,15% units decrease In between: 15–29% units decrease Worst: .30% units decrease Contamination of the analysis equipment (gas chromatography–mass spectrometry)

Quantisal Statsure OralCol Salivette

Oratube Intercept

Greiner Cozart Salicule Tube

Statsure

Quantisal Intercept Greiner Salicule Tube Intercept Statsure Tube Salicule Oracol Greiner

Cozart Oracol Oratube Salivette Quantisal Greiner

Cozart

Devices without buffer

Statsure Cozart Intercept Quantisal

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TABLE 2. Confirmation Techniques for Drugs in Oral Fluid in ROSITA-26 Belgium Finland Germany Norway Spain USA/Florida USA/Utah (Washington)

LC-MS-MS GC-MS GC-MS LC-MS-MS LC-MS LC-MS and GC-MS GC-MS and LC-MS-MS

LC-MS-MS, liquid chromatography–mass spectrometry–mass spectrometry; GC-MS, gas chromatography–mass spectrometry; LC-MS, liquid chromatography–mass spectrometry.

lists the laboratory methods used in ROSITA-2.24 Advantages of microtiter plate enzyme immunoassay are that low-sample volumes are enough for analysis (usually 25 mL), no sample pretreatments are needed, the methods are sensitive, and the antibody crossreacts for the parent drug.28,29

Confirmation Analysis To evaluate the on-site testing devices in the ROSITA projects, reliable confirmation methods were needed. New equipment, especially LC-MS-(MS) instruments, was purchased and massive development work started in the participating laboratories. The common list of target substances and cutoff values (Table 3) were set in a meeting in Strasbourg 2003. For several substances, the target concentrations were far below the cutoff concentrations that the laboratories had used for analyzing drugs in blood. In addition to the lower cutoffs than before, the laboratories had to aim to

TABLE 3. Cutoffs (ng/mL) for ROSITA-2 participating Laboratories6 Substance

Blood

Oral Fluid

Amphetamine Methamphetamine MDMA MDA MDEA Cocaine Benzoylecgonine Morphine Codeine THC THCCOOH 11-OH-THC Diazepam Nordiazepam Temazepam 7-aminoflunitrazepam Bromazepam Lorazepam Clonazepam Zopiclone/zolpidem

20 20 20 20 20 20 20 10 10 1 5 1 50 50 50 1 10 10 5 10

25 25 25 25 25 8 8 20 20 2

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5 5 5 2 5 5 5

analyze approximately 30 substances in approximately 0.5 mL of OF. The common cutoff values in OF and whole blood were set in the European Union, DRUID project. Also, the Substance Abuse Mental Health Services Administration (SAMSHA) has proposed cutoff values for oral fluid (Table 4).

Gas Chromatography–Mass Spectrometry and Gas Chromatography–Mass Spectrometry–Mass Spectrometry The drugs have to be extracted from oral fluid and, in most cases, then to be derivatized to make them volatile before introducing them into the GC-MS instrument. Molecules are ionized, commonly using electron ionization (EI) at 70 eV. Mass-to-charge ratio and abundances of fragments are created and a unique fingerprint of ion fragments is detected for compound identification.30,31 There are numerous GC-MS methods published for analyzing drugs in oral fluids.28 More stable and less extensive fragments are produced by chemical ionization.32,33 EI produces more ions and better selectivity but poorer sensitivity than negative ion chemical ionization. For confirmation analysis, GC-MS is usually operated in selected ion monitoring mode and deuterated internal standards are recommended. Three ion and two ion ratios are normally followed, but in chemical ionization mode, there may not always be three ions available for selection. The benefits of GC-MS techniques are robustness and inexpensiveness. Different instruments produce comparable fragmentation and spectra. Reliable libraries are thus available, which is an advantage in comparison to LC-MS. Drug concentrations, eg, for THC and benzodiazepine, are much lower in OF than those in blood. More sensitive methods may be needed. Detection limits can be lowered by using techniques called Dean Switch (Agilent Technologies, Santa Clara, CA). By connecting this switching valve to the GC-MS, many interfering substances can be eliminated and the signalto-noise ratio can be increased.31 Lower detection limits can be achieved also by using MS-MS.34,35 Increased sample throughput and decreased costs of the method can be achieved also by using fast gas chromatography. Chromatographic run times of approximately 20 minutes by conventional GC are shown to be reduced approximately 5 minutes by fast GC ystems.36 Kankaanpa¨a¨ et al developed and fully validated a rapid GC-EI assay for the simultaneous determination of 15 amphetamine-type stimulants and related drugs using only 100 mL of OF.4 Extraction and derivatization were performed in a single step using buffer and toluene with internal standard (methylmexiletine) and heptafluorobutyric anhydride (HFBA) as an extraction-derivatization reagent. The mixture was centrifuged and injected into a GC-MS. The run time in GC-MS for 15 stimulants was 15 minutes. Gunnar et al1 presented a multicomponent procedure for determination of 30 various types of drugs in oral fluid using 250 mL of sample. Solid phase extraction was used and three different derivatization procedures in three fractions were used. Overview of OF sample pretreatment is presented in Figure 1. A technician can work up approximately 30 samples per day by this method. q 2008 Lippincott Williams & Wilkins

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TABLE 4. Confirmatory Tests’ Cutoff Values for Drugs in Undiluted Oral Fluid Proposed by European Union (EU) DRUID Project and Substance Abuse Mental Health Services Administration (SAMSHA)47 Substance

Whole Blood EU/DRUID Cutoff (ng/mL)

Oral Fluid EU/DRUID Cutoff (ng/mL)

Oral Fluid Cutoffs (ng/mL) Proposed by SAMSHA

Ethanol Morphine Amphetamine MDMA MDA Cocaine THC THCCOOH Diazepam Alprazolam Clonazepam Benzoylecgonine Codeine 6-acetylmorphine Methamphetamine Methadone Oxazepam Nordiazepam Zopiclone MDEA Lorazepam Flunitrazepam Zolpidem

0.1 g/L 10 20 20 20 10 1 5 20 10 10 50 10 10 20 10 50 20 10 20 10 2 20

0.1 g/L 20 25 25 25 10 1

40 50 50 50 8 2

Liquid Chromatography–Mass Spectrometry Some of the drugs are present in low concentrations in OF and are thus difficult to analyze by GC-MS methods. The advantage of LC-MS-MS is better sensitivity than that of GCMS. Therefore, a smaller volume of sample is needed for analysis, which is important for analyzing drugs in OF. Also, substances of low extraction recovery can be detected. In addition, thermally labile and polar compounds can be detected by LC. LC-MS methods are beneficial, especially because of simple sample pretreatment without complicated sample cleanup and derivatization steps. Oiestadt et al used simple and rapid liquid–liquid extraction (ethylacetate:hexane 4:1) for screening 32 substances in OF.3 Although the extraction recovery of benzoylecgonine was as low as 0.2%, it was successfully screened and detected with LC–tandem mass spectrometry. Analysis time in LC-MS-MS was approximately 20 minutes for 32 substances. After a nonlaborious protein precipitation procedure, significant ion suppression was detected.37 Interfering coeluting residual matrix components can influence the ionization of the target compound even to the extent that the compound will not be detected. It was concluded that to overcome matrix suppression, more exhaustive sample preparation and increased chromatographic separation was needed. Ion suppression was also noticed after using OF collection devices.2 The buffer of collection devices contained macromolecules, which partially coextracted with the drug molecules and caused ion suppression. Ion suppression or enhancement can influence q 2008 Lippincott Williams & Wilkins

5 1 1 10 20 5 25 20 5 1 10 25 1 1 10

40 4 50

assay sensitivity, reproducibility, and linearity in quantitative LC-MS(-MS).38 Therefore, the matrix effect should be studied. For identification of unknown substances, in-house libraries are used and those libraries are applicable only on a single apparatus or apparatus type. The most commonly used ionization method in LC-MS is electrospray ionization (ESI) for drug analysis in OF. The alternate ionization (interface) is atmospheric pressure chemical ionization. Ion suppression and enhancement problems have been reported especially with ESI, but problems can occur also with atmospheric pressure chemical ionization.39 When OF was pretreated with four sample preparation procedures/direct injection, dilution, protein precipitation, solid-phase extraction, and analyzed by LC-ESI-MS-MS and LC–atmospheric pressure chemical ionization–MS-MS, both ionization types showed matrix effect, but ESI was more vulnerable. Preconcentration of OF was needed and acetonitrile protein precipitation was found to provide sufficient preconcentration and protein removal.40 Modern instrumental methods in forensic toxicology have been reviewed recently by several authors.31,37–39,41,42 Samyn et al reviewed extensively the techniques and methods used for determination of drugs of abuse in oral fluid.30 Several LC-MS(-MS) methods have been developed for analysis of drugs in oral fluids.43–45

Validation Integral components for method validation are the signal-to-noise ratio, limit of detection, lower limit of quantification, upper limit of quantification, accuracy, precision, and

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drugs at very low concentrations in a small volume of OF. There remain challenges in OF collection to avoid absorption. Long-term contamination of columns and ion source in the GC-MS instrument because of interfering substances in preservation buffer solutions is a problem as well as ion suppression and/or enhancement in LC-MS-(MS) instruments. On-site tests are a helpful tool for police officers but their sensitivity, especially for THC and benzodiazepines, still needs improvement.

REFERENCES

FIGURE 1. An example of sample preparation for gas chromatography–mass spectrometry analysis.1

interference.46 In LC-MS methods, ion suppression and enhancement should be studied.

CONCLUSIONS Oral fluid is a preferred specimen for preliminary drug testing on the roadside. The purpose of testing may be driving under the influence of drugs controls performed by police officers or epidemiologic studies in which prevalence of different drugs in drivers in the traffic flow is studied. Sensitive and specific analytical methods have enabled measurements of

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1. Gunnar T, Ariniemi K, Lillsunde P. Validated toxicological determination of 30 drugs of abuse as optimized derivatives in oral fluid by long column fast gas chromatography/electron impact mass spectrometry. J Mass Spectrom. 2005;40:739–753. 2. Wood M, Laloup M, del Mar Ramirez Fernandez M, et al. Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography–tandem mass spectrometry. Forensic Sci Int. 2005;150:227–38. 3. Oiestadt EL, Johansen U, Christophersen AS. Clin Chem. 2007;53:2: 300–309. 4. Kankaanpa¨a¨ A, Gunnar T, Ariniemi K, et al. Single-step procedure for gas chromatography–mass spectrometry screening and quantitative determination of amphetamine-type stimulants and related drugs in blood, serum, oral fluid and urine samples. J Chromatogr B. 2004;810:57–68. 5. Samyn N, Verstraete A, van Haaren C, et al. Analysis of drugs of abuse in saliva. Forensic Sci Int. 1999;11:11–19. 6. Verstraete AG, Raes E. ROSITA-2 Project. Final Report. Ghent, Belgium: University of Ghent; 2006. 7. Choo RE, Huestis MA. Oral fluid as a diagnostic tool. Clin Chem Lab Med. 2004;42:1273–1287. 8. Samyn N, Verstrate A, van Haeren C, et al. Analysis of drugs of abuse in saliva. Forensic Sci Rev. 1999;11:1–19. 9. Kidwell DA, Holland JC, Athanaselis S. Testing for drugs of abuse in saliva and sweat. J Chromatogr B. 1998;713:11–135. 10. Schram W, Smith RH, Graig PA, et al. Drugs of abuse in saliva: a review. J Anal Toxicol. 1992;16:1–9. 11. Kintz P, Samyn N. Use of alternative specimens: drugs of abuse in saliva and doping agents in hair. Ther Drug Monit. 2002;24:239–246. 12. Verstraete AG. Oral fluid testing for driving under the influence of drugs: history, recent progress and remaining challenges. Forensic Sci Int. 2005; 50:143–150. 13. Ho¨ld KM, de Boer D, Zuidema J, et al. Saliva as an analytical tool in toxicology. Int J Drug Testing. 1996;1:15–36. 14. Navarro M, Pichini S, Farre M, et al. Usefulness of saliva for measurement of 3,4-methylenedioxymethamphetamine and its metabolites: correction with plasma drug concentrations and effect of salivary pH. Clin Chem. 2001;47:1788–1795. 15. Kidwell DA. Discussion: caveats in testing for drugs of abuse. NIDA Res Monogr. 1992;117:98–120. 16. Samyn N, van Haeren C. On-site testing of saliva and sweat with Drugwipe and determination of concentrations of drugs of abuse in saliva, plasma and urine of suspected users. Int J Legal Med. 2000;113: 150–154. 17. Drummer OH. Review: pharmacokinetics of illicit drugs in oral fluid. Forensic Sci Int. 2005;150:143–150. 18. Engblom C, Gunnar T, Rantanen A, et al. Driving under influence of drugs—amphetamine concentrations in oral fluid and whole blood sample. J Anal Toxicol. 2007;31:276–280. 19. Gro¨nholm M, Lillsunde P. A comparison between on-site immunoassay drug-testing devices and laboratory results. Forensic Sci Int. 2001;121: 37–46. 20. Pehrsson A, Gunnar T, Engblom C, et al. 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. Forensic Sci Int. 2008;175:140–148. 21. Verstraete A. ROSITA 1. ROSITA Roadside Testing Assessment EU Project Report. Ghent, Belgium: University of Ghent; 2001.

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22. Penttila¨ A, Karhunen PJ, Pikkarainen J. Alcohol screening with the Alcoscan test trip in forensic praxis. Forensic Sci Int. 1990;44:43–48. 23. Leino U, Saarimies J, Gro¨nholm M, et al. Comparison of eight commercial on-site screening devices for drugs of abuse testing. Scand J Clin Lab Invest. 2001;61:325–332. 24. Gunnar T, Rantanen A, Engblom C, et al. ROSITA2 in Finland. In: Verstraete AG, Raes E, eds. ROSITA-2 Project. Final Report. Ghent, Belgium: University of Ghent; 2006:55–95. 25. Lillsunde P, Pehrsson A, Engblom C, et al. The combined Ôzero tolerance lawÕ and Ôimpairment lawÕ for drugs and driving in Finland. T 2007. In: Logan BK, Isenschmid DS, Walsh JM, et al, eds. Abstracts of the Joint International Meeting of ICADTS/TIAFT/IIS. Confrence Abstracts; 2007:P111. 26. Crouch DJ. Oral fluid collection: The neglected variable in oral fluid testing. Forensic Sci Int. 2005;150:165–173. 27. Pehrsson A, Engblom C, Langel K, et al. T 2007. In: Logan BK, Isenschmid DS, Walsh JM, et al, eds. Abstracts of the Joint International Meeting of ICADTS/TIAFT/IIS. Confrence Abstracts; 2007:126. 28. Samyn N, Laloup M, De Boeck G. Bioanalytical procedures for determination of drugs of abuse in oral fluid. Anal Bioanal Chem. 2007;388: 1437–1453. 29. Laloup M, Tilman G, Maes V, et al. Validation of an ELISA-based screening assay for the detection of amphetamine, MDMA and MDA in blood and oral fluid. Forensic Sci Int. 2005;153:29–37. 30. Lillsunde P, Korte T. Thin layer chromatographic screening and gas chromatographic/mass spectrometric confirmation in the analysis of abused drugs. In: Adamovics JA, ed. Analyzing Abused Drugs. New York, Basel: Marcel Dekker Inc; 1995:221–265. 31. Smith ML, Swawn PV, Holler JM, et al. Modern instrumental methods in forensic toxicology. J Anal Toxicol. 2007;31:237–253. 32. Gunnar T, Ariniemi K, Lillsunde P. Fast gas chromatography–negative-ion chemical ionization mass spectrometry with microscale volume sample preparation for the determination of benzodiazepines and a-hydroxy metabolites, zaleplon and zopiclone in whole blood. J Mass Spectrom. 2006;41:741–754. 33. Gunnar T, Eskola T, Lillsunde P. Fast gas chromatography/mass spectrometric assay for the validated quantitative determination of methadone and the primary metabolite EDDP in whole blood. Rapid Commun Mass Spectrom. 2006;20:673–679. 34. Niedbada RS, Kardos K, Salamone S, et al. Passive cannabis smoke exposure and oral fluid testing. J Anal Toxicol. 204;28:546–552.

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