TY - JOUR
AU - Huestis, Marilyn, A
AB - Abstract With advances in analytical technology and new research informing result interpretation, oral fluid (OF) testing has gained acceptance over the past decades as an alternative biological matrix for detecting drugs in forensic and clinical settings. OF testing offers simple, rapid, non-invasive, observed specimen collection. This article offers a review of the scientific literature covering analytical methods and interpretation published over the past two decades for amphetamines, cannabis, cocaine, opioids, and benzodiazepines. Several analytical methods have been published for individual drug classes and, increasingly, for multiple drug classes. The method of OF collection can have a significant impact on the resultant drug concentration. Drug concentrations for amphetamines, cannabis, cocaine, opioids, and benzodiazepines are reviewed in the context of the dosing condition and the collection method. Time of last detection is evaluated against several agencies' cutoffs, including the proposed Substance Abuse and Mental Health Services Administration, European Workplace Drug Testing Society and Driving Under the Influence of Drugs, Alcohol and Medicines cutoffs. A significant correlation was frequently observed between matrices (i.e., between OF and plasma or blood concentrations); however, high intra-subject and inter-subject variability precludes prediction of blood concentrations from OF concentrations. This article will assist individuals in understanding the relative merits and limitations of various methods of OF collection, analysis and interpretation. Introduction With advances in analytical technology and new research informing result interpretation, oral fluid (OF) testing has gained acceptance over the past decades as an alternative biological matrix for detecting drugs in forensic and clinical settings. OF testing offers simple, rapid, non-invasive, observed specimen collection, making sample adulteration more difficult and eliminating the need for specialized bathroom collection facilities or same-sex collectors, as may be the case with urine. Other advantages include potential for on-site collection and screening, lower biohazard risk during collection (compared with collection of blood) and ease of multiple sample collections. Also, as compared with urine, OF is both more likely to contain parent drugs, which may reflect more recent drug use, and more likely to have drug findings that correlate better with blood. OF testing, including on-site testing and laboratory confirmation, utilizes well-validated and accepted analytical methods and instrumentation; and published scientific studies have documented its reliability in drug treatment, workplace, pain management and driving under the influence of drugs (DUID) programs. Distribution of Drugs into Oral Fluid Secretions from the salivary glands are termed saliva, while OF consists of saliva and other debris and food products in the oral cavity. Healthy adults produce ~0.5–1.5 L of saliva per day (1). OF pH ranges from 6.2 to 7.4, becoming more alkaline when saliva secretion is stimulated due to increased bicarbonate excretion and possibly a loss of dissolved carbon dioxide (1–5). OF composition and flow rate are influenced by circadian rhythm, sensory stimuli, hormonal changes, mechanical stimulation, psychological state (e.g., anger, fear and depression), genetics, oral hygiene, sympathomimetic and parasympatholytic (anticholinergic) drugs and systemic diseases (e.g., diabetes, kidney dysfunction, anorexia and cystic fibrosis) (6, 7). In turn, drug transfer from blood into OF is affected by OF composition, flow rate and pH, as well as the drug's pKa, protein binding, lipophilicity, spatial configuration and molecular weight (4, 8). Free, unbound drugs can enter OF by passive diffusion from blood. Glucuronidated metabolites also can be detected, albeit in lower concentrations than free metabolites (9, 10). Following smoking, insufflation, sublingual and/or oral drug administration, the oral mucosa is directly exposed to drug(s) resulting in relatively high OF drug concentrations (9, 11–13). In contrast, capsule ingestion typically does not contaminate the oral mucosa unless the capsule is adulterated or chewed (14, 15). The process of ion trapping basic drugs in OF from blood produces higher drug concentrations in OF. This phenomenon occurs due to the lower OF pH compared with that of blood (~7.4); once in the blood, free, uncharged bases diffuse across membranes into OF and ionize at the lower pH, reducing diffusion back into blood and yielding higher OF than blood concentrations. There is no consensus on an appropriate biomarker to normalize OF drug concentrations. OF creatinine concentrations showed large intra-subject and inter-subject variation; coefficient of variation (%CV) over 10 weeks was 141% (range 39–225%) (16, 17). OF immunoglobulin G (IgG) concentrations ≥0.1–1.0 mg/L were suggested to identify undiluted OF, but even after a second rinse of the mouth with tap water, IgG concentrations still exceeded this criterion, indicating that diluted OF samples still had IgG concentrations that would be considered `undiluted’ (4). One suggested approach normalized hydrocodone OF concentrations to dose, body mass index, lean body weight, body surface area and calculated blood volume; however, in workplace, pain management and forensic situations, these data would be unavailable (18). As noted above, variations between drugs, their route of administration and between individuals can influence drug detectability in OF. It is important to recognize and account for these variations when evaluating OF drug findings. Oral Fluid Collection OF can be collected by a variety of techniques including passive drool and expectoration (with or without stimulation) and via a wide variety of collection devices. Each collection method has advantages and disadvantages (e.g., passive drool is unpleasant for donors and collectors, and stimulated OF samples are diluted due to increased salivary flow). Common collection techniques are characterized below. Passive drool Passive drool requires individuals to drool into a collection tube. This collection method best reflects drug concentrations in excreted saliva, as opposed to expectoration or even putting a collection device in the mouth, which can increase salivation and alter OF composition. However, passive drool OF collection is slow and unpleasant for donors and collectors. Few studies utilize this method, making interpretation of results difficult (19). Expectoration and salivary stimulation Expectoration or spitting provides neat OF without buffer dilution, increasing assay sensitivity. Although this collection method is less expensive than other collection methods, it is also unpleasant for donors and collectors. Expectorated OF is viscous and contains mucus, food particles and/or other mouth debris. Samples need to be centrifuged prior to laboratory analysis to remove precipitant material, which may yield lower drug concentrations due to drug loss in the pellet or adsorption to the tube. For example, in Δ9-tetrahydrocannabinol (THC) fortified expectorated OF centrifuged for 10 min, only 28.8% THC was recovered in the supernate, 51.7% was recovered from the protein pellet and 14.7% from the polypropylene tube after addition of the surfactant Triton® X-100 (20). Mucus in OF, which can be variable in composition, can also interfere with proper interaction with solid-phase extraction sorbent, reducing drug concentrations and increasing imprecision (21). Furthermore, the absence of stabilizing buffer may lead to lower drug stability in expectorated samples, as was demonstrated with cannabinoids (22). Finally, many drugs can produce dry mouth, yielding difficulty in expectorating OF and resulting in low sample volumes. Earlier research utilized stimulated salivary flow to facilitate OF collection. OF stimulation, for example, occurs when chewing paraffin or sucking on an acidic candy. Although the initial OF pH of specimens collected with citric acid-treated cotton swabs (mean 2.8, range 2.4–3.6) was lower than OF pH collected with a neutral cotton swab (mean 6.0, range 4.2–7.2) (14, 23), salivary flow stimulation increased saliva volume and pH, due to increased bicarbonate excretion (1, 3, 4). Stimulation increased saliva excretion and lowered, rather than increased, drug concentrations, complicating result interpretation. Lower concentrations following citric acid candy-stimulated expectoration was documented for multiple drugs, including methamphetamine and amphetamine (14) and codeine (24). Via collection device Commercial OF collection devices generally include a pad or sponge to absorb the OF and a buffer to better stabilize drugs and extract them from the collection pad. Collection time varies by device and amount of OF collected but is generally completed within a few minutes (Table I). The absorbent pad filters the OF, reducing extraneous material collection (e.g., food). Buffers reduce OF viscosity, improving measurement accuracy and increasing stability, but also dilute analyte concentrations. The collection pad must remain in the buffer for the manufacturer-specified period of time (from 4 h to overnight) in order to ensure adequate drug recovery. This is not an issue in most cases, as OF samples are generally shipped overnight or brought to a laboratory for analysis. Buffers and surfactants also interfere with liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis if injected directly or after dilution, producing ion suppression or enhancement (matrix effects); therefore, sample cleanup is suggested (31). Table I Volume of Oral Fluid Collected with Various Oral Fluid Collection Devices Device Volume Adequacy Indicator? Collection Volume as Stated by Manufacturer Collection Method Mean ± RSD Volume Collected (mL) Range (mL) Reference Quantisal (Immunalysis) Yes 1 mL ± 10% In vitro 1.009 ± 0.99% — 25 Volunteers 1.086 ± 7.27% — 25 Volunteers 0.84 ± 6.7% — 26 In vitro 1.15 ± 7.68% 1.04–1.22 27 Saliva Sampler (StatSure Diagnostic Systems) Yes 1 mL In vitro 1.176 ± 1.87% — 25 Volunteers 0.952 ± 11.97% — 25 Intercept (OraSure Technologies) No 1 mL max In vitro 0.863 ± 1.97% — 25 Volunteers 0.790 ± 29.87% — 25 Volunteers 0.224 <0.05–0.795 28 Volunteers 0.89 (median) 0.38–1.54 29 Salivette (Sarstedt AG & Co.) No Unknown In vitro 1.968 ± 1.07% — 25 Volunteers 1.905 ± 20.94% — 25 Cozart (Cozart Bioscience) Yes 1 mL In vitro 1.294 ± 12.60% — 25 Volunteers 0.967 ± 33.51% — 25 Certus (Concateno) Yes 1 mL Volunteers 0.94 ± 19% — 26 DCD 5000 (Draeger) Yes 0.38 mL In vitro 0.487 ± 1.3% — 30 Volunteers 0.467 ± 13.5% 0.387–0.535 30 Device Volume Adequacy Indicator? Collection Volume as Stated by Manufacturer Collection Method Mean ± RSD Volume Collected (mL) Range (mL) Reference Quantisal (Immunalysis) Yes 1 mL ± 10% In vitro 1.009 ± 0.99% — 25 Volunteers 1.086 ± 7.27% — 25 Volunteers 0.84 ± 6.7% — 26 In vitro 1.15 ± 7.68% 1.04–1.22 27 Saliva Sampler (StatSure Diagnostic Systems) Yes 1 mL In vitro 1.176 ± 1.87% — 25 Volunteers 0.952 ± 11.97% — 25 Intercept (OraSure Technologies) No 1 mL max In vitro 0.863 ± 1.97% — 25 Volunteers 0.790 ± 29.87% — 25 Volunteers 0.224 <0.05–0.795 28 Volunteers 0.89 (median) 0.38–1.54 29 Salivette (Sarstedt AG & Co.) No Unknown In vitro 1.968 ± 1.07% — 25 Volunteers 1.905 ± 20.94% — 25 Cozart (Cozart Bioscience) Yes 1 mL In vitro 1.294 ± 12.60% — 25 Volunteers 0.967 ± 33.51% — 25 Certus (Concateno) Yes 1 mL Volunteers 0.94 ± 19% — 26 DCD 5000 (Draeger) Yes 0.38 mL In vitro 0.487 ± 1.3% — 30 Volunteers 0.467 ± 13.5% 0.387–0.535 30 Data reported in weights were converted to volumes employing a 0.993 g/mL OF density. View Large Table I Volume of Oral Fluid Collected with Various Oral Fluid Collection Devices Device Volume Adequacy Indicator? Collection Volume as Stated by Manufacturer Collection Method Mean ± RSD Volume Collected (mL) Range (mL) Reference Quantisal (Immunalysis) Yes 1 mL ± 10% In vitro 1.009 ± 0.99% — 25 Volunteers 1.086 ± 7.27% — 25 Volunteers 0.84 ± 6.7% — 26 In vitro 1.15 ± 7.68% 1.04–1.22 27 Saliva Sampler (StatSure Diagnostic Systems) Yes 1 mL In vitro 1.176 ± 1.87% — 25 Volunteers 0.952 ± 11.97% — 25 Intercept (OraSure Technologies) No 1 mL max In vitro 0.863 ± 1.97% — 25 Volunteers 0.790 ± 29.87% — 25 Volunteers 0.224 <0.05–0.795 28 Volunteers 0.89 (median) 0.38–1.54 29 Salivette (Sarstedt AG & Co.) No Unknown In vitro 1.968 ± 1.07% — 25 Volunteers 1.905 ± 20.94% — 25 Cozart (Cozart Bioscience) Yes 1 mL In vitro 1.294 ± 12.60% — 25 Volunteers 0.967 ± 33.51% — 25 Certus (Concateno) Yes 1 mL Volunteers 0.94 ± 19% — 26 DCD 5000 (Draeger) Yes 0.38 mL In vitro 0.487 ± 1.3% — 30 Volunteers 0.467 ± 13.5% 0.387–0.535 30 Device Volume Adequacy Indicator? Collection Volume as Stated by Manufacturer Collection Method Mean ± RSD Volume Collected (mL) Range (mL) Reference Quantisal (Immunalysis) Yes 1 mL ± 10% In vitro 1.009 ± 0.99% — 25 Volunteers 1.086 ± 7.27% — 25 Volunteers 0.84 ± 6.7% — 26 In vitro 1.15 ± 7.68% 1.04–1.22 27 Saliva Sampler (StatSure Diagnostic Systems) Yes 1 mL In vitro 1.176 ± 1.87% — 25 Volunteers 0.952 ± 11.97% — 25 Intercept (OraSure Technologies) No 1 mL max In vitro 0.863 ± 1.97% — 25 Volunteers 0.790 ± 29.87% — 25 Volunteers 0.224 <0.05–0.795 28 Volunteers 0.89 (median) 0.38–1.54 29 Salivette (Sarstedt AG & Co.) No Unknown In vitro 1.968 ± 1.07% — 25 Volunteers 1.905 ± 20.94% — 25 Cozart (Cozart Bioscience) Yes 1 mL In vitro 1.294 ± 12.60% — 25 Volunteers 0.967 ± 33.51% — 25 Certus (Concateno) Yes 1 mL Volunteers 0.94 ± 19% — 26 DCD 5000 (Draeger) Yes 0.38 mL In vitro 0.487 ± 1.3% — 30 Volunteers 0.467 ± 13.5% 0.387–0.535 30 Data reported in weights were converted to volumes employing a 0.993 g/mL OF density. View Large A serum separator tube or filter separates the OF–buffer mixture from the pad (e.g., Quantisal, StatSure and Oral-Eze) or a specially designed tube for centrifugation that performs separation for other devices (e.g., Intercept and DCD 5000). Alternately, devices may not include a buffer (e.g., Salivette), instead using centrifugation with a tube insert to separate OF from the pad. As the buffer volume can be variable, OF drug concentrations are reported as neat (undiluted, that is, considering the ratio of OF volume compared with the elution buffer volume) OF concentrations, as it allows a direct comparison of drug concentrations from different collection devices. Early collection devices did not include a volume adequacy indicator, leading to large variability in OF volume collected and resultant measured drug concentrations (Table I). Higher cocaine and cannabinoid concentrations were documented in samples collected with one collection device compared with a second type of collection device, leading authors to postulate that different volumes of OF are collected with the two devices or that recovery may differ between the two devices (32, 33). On the other hand, duplicate OF samples collected with the Quantisal collection devices did not have significant differences in THC and 11-nor-9-carboxy-THC (THCCOOH) concentrations (34). To protect against such variability in collected volume, the European Workplace Drug Testing Society (EWDTS) requires a device to have a volume adequacy indicator, and a precise quantity of collected OF must be determined gravimetrically or spectrophotometrically (35). The Substance Abuse and Mental Health Services Administration (SAMHSA) proposed mandatory guidelines for OF testing that requires collection of a minimum of 1.0 ± 0.1 mL of OF (36). Oral Fluid Screening On-site OF testing An advantage of OF testing is the ease of collection and amenability to rapid on-site (point-of-collection) testing. On-site devices include an OF collector and a built-in detection system (lateral flow immunoassay on a test strip) for screening multiple drugs or drug classes. The presence or absence of a drug or drug class can be determined visually (e.g., by the appearance of a line) or through a more objective reading of drug intensity along the test strip. Several commercial devices were developed before much of the basic science on OF drug disposition was known. Many devices had poor sensitivity and specificity and are no longer available or were substantially modified. Earlier studies compared positivity rates on the on-site devices with confirmatory urine, blood or plasma tests rather than with confirmatory laboratory OF analysis, leading to poor accuracy and agreement between screening and confirmation results due to inherent differences between the matrices. Newer devices demonstrate better sensitivity, specificity and efficiency. For example, as manufacturers lowered detection limits and increased assay run times to achieve better THC sensitivity, detection of toxicologically relevant THC concentrations was achieved. Cannabis is the most common drug, other than ethanol, identified in DUID cases and drivers (37). Therefore, an on-site OF testing device should have high efficiency for detecting cannabis to be considered for inclusion in workplace, pain management and DUID programs. Several studies assessed the time course of specific drugs in OF and the detection rates of on-site devices, improving result interpretation (32, 38–44). Other studies had low drug positive rates, making it difficult to accurately assess sensitivity, specificity and efficiency of the device (45). Furthermore, several others demonstrated that sensitivity, specificity and efficiency are highly dependent on the analytes and confirmation cutoffs employed and the matrix (OF and blood) used for confirmation (32, 38–40). These factors are important considerations when selecting commercially available on-site OF testing devices. A complete review of on-site OF testing devices is beyond the scope of this review, and comparisons of devices are available (44–47). Laboratory-based immunoassay screening In addition to roadside immunoassay screening devices, multiple validated laboratory-based immunoassays are available for screening drugs in OF (48–55). Because parent drugs are present in higher prevalence in OF than metabolites, blood immunoassays should be adapted for OF screening rather than urine immunoassays, as these typically target metabolites. The drug concentration range should be considered when converting immunoassays for OF, as many drugs have higher OF concentrations compared with blood, especially shortly after the time of use after drug smoking, vaporization, insufflation, sublingual or oral intake, when concentrations >1,000 μg/L can occur due to direct exposure of the oral mucosa to drug(s). Potential interferences were investigated by studying the effects of food, mouthwash, toothpaste and vinegar on various OF assays; only vinegar produced false positives in one assay (56). On the Immunalysis THC cannabinoid OF enzyme-linked immunosorbent assay, no interferences were produced following mouthwash, orange juice, toothpaste, coffee and soy milk intake (54). One disadvantage of screening with immunoassays is the poor cross-reactivity with novel psychoactive substances (NPSs). For example, synthetic cathinones have poor cross-reactivity with most amphetamine immunoassays (57, 58), and the synthetic cannabinoid immunoassays tend to rapidly become outdated in their ability to identify the latest synthetic cannabinoids on the market. Chromatographic mass spectrometric screening methods offer improved identification of NPSs. Oral Fluid Confirmation Quantitative OF methods utilize similar analytical methods and sample volumes as blood and urine analyses. With instrumentation improvements, there is a trend towards developing chromatographic methods capable of detecting and quantifying multiple classes of drugs in OF in a single assay (Table II). Sample volume required for analysis is dependent upon the number of drugs included in the method, the sensitivity of the analytical method and whether the sample is collected by expectoration or with a collection device that includes a buffer dilution. Several validated methods are available for expectorated OF; however, if these methods are used with OF collected with collection devices, it is important to validate the method with matrix-matched samples. Manufacturers include different components in their proprietary buffers such as surfactants that can produce matrix effects (83, 84). Interferences with the mass spectrometer, such as quadrupole charging, were documented with OF samples collected with some collection devices (85). Furthermore, authentic rather than synthetic OF is recommended for method validation despite the difficulty in obtaining it, unless synthetic OF is validated as equivalent, as potential endogenous interferences can be better evaluated. Table II Chromatographic Method for the Detection of Drugs in Oral Fluid Instrumental Method Sample Volume and Type Sample Preparation Derivatization Column Run Time (min) Analytes AMPS Opioids Cocaine and Metabolites Benzodiazepines Methadone BUP Reference LC-QTOF-MS 200 μL of expectorated oral fluid SPE N/A Hypersil™ BDS phenyl column 28 ✓ ✓ ✓ 59 LC–MS/MS 200 μL of expectorated oral fluid Protein precipitation N/A Synergi Polar RP 25.5 ✓ ✓ ✓ 60 GC-MS 250 μL of expectorated oral fluid SPE MTBSTFA, MSTFA and HFBA DB-35ms and DB-5ms 9.5, 10.63 and 10.67 ✓ ✓ ✓ ✓ ✓ ✓ 61 LC–MS/MS 250 μL of Intercept oral fluid SPE N/A XTerra® MS C18 20 ✓ ✓ ✓ 67 LC–MS/MS 1 mL of Omni-Sal® oral fluid SPE N/A Luna C18 36 ✓ ✓ ✓ ✓ ✓ ✓ 62 LC–MS/MS 100 μL of expectorated oral fluid SPE N/A Atlantis dC18 17 ✓ ✓ ✓ 63 GC-MS Oral fluid collected with Salivette SPE MSTFA Unspecified methyl-silicone column 13.1 ✓ ✓ ✓ ✓ 64 LC–MS/MS 1 mL of StatSure oral fluid SPE N/A Atlantis dC18 12 ✓ ✓ ✓ ✓ ✓ 65 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Pinnacle II C18 and Allure PFP Propyl column 5 and 6 ✓ ✓ ✓ 66 LC–MS/MS 200 mg of StatSure oral fluid SPE N/A Acquity HSS T3 C18 16.2 ✓ ✓ ✓ ✓ ✓ ✓ 68 LC–MS/MS 200 μL of Intercept oral fluid SPE N/A Allure PFP Propyl column 12 ✓ ✓ ✓ 69 LC–MS/MS 250 μL of expectorated oral fluid Isolute PPT+ protein precipitation N/A Synergi Polar RP 16 ✓ ✓ ✓ ✓ 70 LC–MS/MS 100 μL of expectorated oral fluid Dilute and shoot N/A Acquity HSS T3 C18 8 ✓ ✓ ✓ ✓ 71 GC-MS 1 mL of StatSure oral fluid Liquid–liquid extraction and SPE MSTFA and MTBSTFA DB-5ms and DB-5ht 9.73, 4.5 and 5.75 ✓ ✓ ✓ ✓ ✓ ✓ 72 LC–MS/MS 250 μL of RapidSTAT or Concateno DDS Dilute and shoot N/A Kinetex C18 8 ✓ ✓ ✓ ✓ 73 LC–MS/MS 500 μL of Quantisal oral fluid Liquid–liquid extraction N/A Luna C18 22 ✓ ✓ ✓ ✓ ✓ 74 LC–MS/MS 10 μL of expectorated oral fluid Direct injection N/A Zorbax SB-Aq 18 ✓ ✓ ✓ ✓ 75 LC–MS/MS 120 μL of passive drool MEPS N/A PFP Kinetex 12.5 ✓ ✓ ✓ ✓ ✓ 76 LC–MS/MS 200 μL of Intercept or Quantisal oral fluid SLE N/A Acquity BEH C18 7.1 ✓ ✓ ✓ ✓ ✓ ✓ 77 LC–MS/MS 200 μL of oral fluid/StatSure buffer or 300 μL of Quantisal/Certus buffer SPE N/A Acquity BEH C18 8.5 ✓ ✓ ✓ 78 LC–MS/MS 100 μL of Intercept oral fluid Direct injection with online extraction column N/A Ascentis Phenyl 16 ✓ ✓ 79 LC–MS/MS 150 μL of Oral-Eze oral fluid Dilute and shoot N/A Acquity BEH C18 5 ✓ ✓ ✓ ✓ ✓ ✓ 80 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Acquity BEH C18 8.5 + wash ✓ ✓ ✓ ✓ ✓ 81 LC–MS/MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A Kinetex C18 5.5 ✓ ✓ ✓ ✓ ✓ ✓ 82 Instrumental Method Sample Volume and Type Sample Preparation Derivatization Column Run Time (min) Analytes AMPS Opioids Cocaine and Metabolites Benzodiazepines Methadone BUP Reference LC-QTOF-MS 200 μL of expectorated oral fluid SPE N/A Hypersil™ BDS phenyl column 28 ✓ ✓ ✓ 59 LC–MS/MS 200 μL of expectorated oral fluid Protein precipitation N/A Synergi Polar RP 25.5 ✓ ✓ ✓ 60 GC-MS 250 μL of expectorated oral fluid SPE MTBSTFA, MSTFA and HFBA DB-35ms and DB-5ms 9.5, 10.63 and 10.67 ✓ ✓ ✓ ✓ ✓ ✓ 61 LC–MS/MS 250 μL of Intercept oral fluid SPE N/A XTerra® MS C18 20 ✓ ✓ ✓ 67 LC–MS/MS 1 mL of Omni-Sal® oral fluid SPE N/A Luna C18 36 ✓ ✓ ✓ ✓ ✓ ✓ 62 LC–MS/MS 100 μL of expectorated oral fluid SPE N/A Atlantis dC18 17 ✓ ✓ ✓ 63 GC-MS Oral fluid collected with Salivette SPE MSTFA Unspecified methyl-silicone column 13.1 ✓ ✓ ✓ ✓ 64 LC–MS/MS 1 mL of StatSure oral fluid SPE N/A Atlantis dC18 12 ✓ ✓ ✓ ✓ ✓ 65 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Pinnacle II C18 and Allure PFP Propyl column 5 and 6 ✓ ✓ ✓ 66 LC–MS/MS 200 mg of StatSure oral fluid SPE N/A Acquity HSS T3 C18 16.2 ✓ ✓ ✓ ✓ ✓ ✓ 68 LC–MS/MS 200 μL of Intercept oral fluid SPE N/A Allure PFP Propyl column 12 ✓ ✓ ✓ 69 LC–MS/MS 250 μL of expectorated oral fluid Isolute PPT+ protein precipitation N/A Synergi Polar RP 16 ✓ ✓ ✓ ✓ 70 LC–MS/MS 100 μL of expectorated oral fluid Dilute and shoot N/A Acquity HSS T3 C18 8 ✓ ✓ ✓ ✓ 71 GC-MS 1 mL of StatSure oral fluid Liquid–liquid extraction and SPE MSTFA and MTBSTFA DB-5ms and DB-5ht 9.73, 4.5 and 5.75 ✓ ✓ ✓ ✓ ✓ ✓ 72 LC–MS/MS 250 μL of RapidSTAT or Concateno DDS Dilute and shoot N/A Kinetex C18 8 ✓ ✓ ✓ ✓ 73 LC–MS/MS 500 μL of Quantisal oral fluid Liquid–liquid extraction N/A Luna C18 22 ✓ ✓ ✓ ✓ ✓ 74 LC–MS/MS 10 μL of expectorated oral fluid Direct injection N/A Zorbax SB-Aq 18 ✓ ✓ ✓ ✓ 75 LC–MS/MS 120 μL of passive drool MEPS N/A PFP Kinetex 12.5 ✓ ✓ ✓ ✓ ✓ 76 LC–MS/MS 200 μL of Intercept or Quantisal oral fluid SLE N/A Acquity BEH C18 7.1 ✓ ✓ ✓ ✓ ✓ ✓ 77 LC–MS/MS 200 μL of oral fluid/StatSure buffer or 300 μL of Quantisal/Certus buffer SPE N/A Acquity BEH C18 8.5 ✓ ✓ ✓ 78 LC–MS/MS 100 μL of Intercept oral fluid Direct injection with online extraction column N/A Ascentis Phenyl 16 ✓ ✓ 79 LC–MS/MS 150 μL of Oral-Eze oral fluid Dilute and shoot N/A Acquity BEH C18 5 ✓ ✓ ✓ ✓ ✓ ✓ 80 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Acquity BEH C18 8.5 + wash ✓ ✓ ✓ ✓ ✓ 81 LC–MS/MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A Kinetex C18 5.5 ✓ ✓ ✓ ✓ ✓ ✓ 82 AMPS: amphetamines; BUP: buprenorphine; GC-MS: gas chromatography mass spectrometry; HFBA: heptafluorobutyric anhydride; LC–MS/MS: liquid chromatography–tandem mass spectrometry; LC-QTOF-MS: liquid chromatography–quadrupole time-of-flight mass spectrometry; MEPS: microextraction by packed sorbent; MSTFA: N-methyl-N-(trimethylsilyl)-trifluoroacetamide; MTBSTFA: N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide; SLE: supported liquid extraction. View Large Table II Chromatographic Method for the Detection of Drugs in Oral Fluid Instrumental Method Sample Volume and Type Sample Preparation Derivatization Column Run Time (min) Analytes AMPS Opioids Cocaine and Metabolites Benzodiazepines Methadone BUP Reference LC-QTOF-MS 200 μL of expectorated oral fluid SPE N/A Hypersil™ BDS phenyl column 28 ✓ ✓ ✓ 59 LC–MS/MS 200 μL of expectorated oral fluid Protein precipitation N/A Synergi Polar RP 25.5 ✓ ✓ ✓ 60 GC-MS 250 μL of expectorated oral fluid SPE MTBSTFA, MSTFA and HFBA DB-35ms and DB-5ms 9.5, 10.63 and 10.67 ✓ ✓ ✓ ✓ ✓ ✓ 61 LC–MS/MS 250 μL of Intercept oral fluid SPE N/A XTerra® MS C18 20 ✓ ✓ ✓ 67 LC–MS/MS 1 mL of Omni-Sal® oral fluid SPE N/A Luna C18 36 ✓ ✓ ✓ ✓ ✓ ✓ 62 LC–MS/MS 100 μL of expectorated oral fluid SPE N/A Atlantis dC18 17 ✓ ✓ ✓ 63 GC-MS Oral fluid collected with Salivette SPE MSTFA Unspecified methyl-silicone column 13.1 ✓ ✓ ✓ ✓ 64 LC–MS/MS 1 mL of StatSure oral fluid SPE N/A Atlantis dC18 12 ✓ ✓ ✓ ✓ ✓ 65 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Pinnacle II C18 and Allure PFP Propyl column 5 and 6 ✓ ✓ ✓ 66 LC–MS/MS 200 mg of StatSure oral fluid SPE N/A Acquity HSS T3 C18 16.2 ✓ ✓ ✓ ✓ ✓ ✓ 68 LC–MS/MS 200 μL of Intercept oral fluid SPE N/A Allure PFP Propyl column 12 ✓ ✓ ✓ 69 LC–MS/MS 250 μL of expectorated oral fluid Isolute PPT+ protein precipitation N/A Synergi Polar RP 16 ✓ ✓ ✓ ✓ 70 LC–MS/MS 100 μL of expectorated oral fluid Dilute and shoot N/A Acquity HSS T3 C18 8 ✓ ✓ ✓ ✓ 71 GC-MS 1 mL of StatSure oral fluid Liquid–liquid extraction and SPE MSTFA and MTBSTFA DB-5ms and DB-5ht 9.73, 4.5 and 5.75 ✓ ✓ ✓ ✓ ✓ ✓ 72 LC–MS/MS 250 μL of RapidSTAT or Concateno DDS Dilute and shoot N/A Kinetex C18 8 ✓ ✓ ✓ ✓ 73 LC–MS/MS 500 μL of Quantisal oral fluid Liquid–liquid extraction N/A Luna C18 22 ✓ ✓ ✓ ✓ ✓ 74 LC–MS/MS 10 μL of expectorated oral fluid Direct injection N/A Zorbax SB-Aq 18 ✓ ✓ ✓ ✓ 75 LC–MS/MS 120 μL of passive drool MEPS N/A PFP Kinetex 12.5 ✓ ✓ ✓ ✓ ✓ 76 LC–MS/MS 200 μL of Intercept or Quantisal oral fluid SLE N/A Acquity BEH C18 7.1 ✓ ✓ ✓ ✓ ✓ ✓ 77 LC–MS/MS 200 μL of oral fluid/StatSure buffer or 300 μL of Quantisal/Certus buffer SPE N/A Acquity BEH C18 8.5 ✓ ✓ ✓ 78 LC–MS/MS 100 μL of Intercept oral fluid Direct injection with online extraction column N/A Ascentis Phenyl 16 ✓ ✓ 79 LC–MS/MS 150 μL of Oral-Eze oral fluid Dilute and shoot N/A Acquity BEH C18 5 ✓ ✓ ✓ ✓ ✓ ✓ 80 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Acquity BEH C18 8.5 + wash ✓ ✓ ✓ ✓ ✓ 81 LC–MS/MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A Kinetex C18 5.5 ✓ ✓ ✓ ✓ ✓ ✓ 82 Instrumental Method Sample Volume and Type Sample Preparation Derivatization Column Run Time (min) Analytes AMPS Opioids Cocaine and Metabolites Benzodiazepines Methadone BUP Reference LC-QTOF-MS 200 μL of expectorated oral fluid SPE N/A Hypersil™ BDS phenyl column 28 ✓ ✓ ✓ 59 LC–MS/MS 200 μL of expectorated oral fluid Protein precipitation N/A Synergi Polar RP 25.5 ✓ ✓ ✓ 60 GC-MS 250 μL of expectorated oral fluid SPE MTBSTFA, MSTFA and HFBA DB-35ms and DB-5ms 9.5, 10.63 and 10.67 ✓ ✓ ✓ ✓ ✓ ✓ 61 LC–MS/MS 250 μL of Intercept oral fluid SPE N/A XTerra® MS C18 20 ✓ ✓ ✓ 67 LC–MS/MS 1 mL of Omni-Sal® oral fluid SPE N/A Luna C18 36 ✓ ✓ ✓ ✓ ✓ ✓ 62 LC–MS/MS 100 μL of expectorated oral fluid SPE N/A Atlantis dC18 17 ✓ ✓ ✓ 63 GC-MS Oral fluid collected with Salivette SPE MSTFA Unspecified methyl-silicone column 13.1 ✓ ✓ ✓ ✓ 64 LC–MS/MS 1 mL of StatSure oral fluid SPE N/A Atlantis dC18 12 ✓ ✓ ✓ ✓ ✓ 65 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Pinnacle II C18 and Allure PFP Propyl column 5 and 6 ✓ ✓ ✓ 66 LC–MS/MS 200 mg of StatSure oral fluid SPE N/A Acquity HSS T3 C18 16.2 ✓ ✓ ✓ ✓ ✓ ✓ 68 LC–MS/MS 200 μL of Intercept oral fluid SPE N/A Allure PFP Propyl column 12 ✓ ✓ ✓ 69 LC–MS/MS 250 μL of expectorated oral fluid Isolute PPT+ protein precipitation N/A Synergi Polar RP 16 ✓ ✓ ✓ ✓ 70 LC–MS/MS 100 μL of expectorated oral fluid Dilute and shoot N/A Acquity HSS T3 C18 8 ✓ ✓ ✓ ✓ 71 GC-MS 1 mL of StatSure oral fluid Liquid–liquid extraction and SPE MSTFA and MTBSTFA DB-5ms and DB-5ht 9.73, 4.5 and 5.75 ✓ ✓ ✓ ✓ ✓ ✓ 72 LC–MS/MS 250 μL of RapidSTAT or Concateno DDS Dilute and shoot N/A Kinetex C18 8 ✓ ✓ ✓ ✓ 73 LC–MS/MS 500 μL of Quantisal oral fluid Liquid–liquid extraction N/A Luna C18 22 ✓ ✓ ✓ ✓ ✓ 74 LC–MS/MS 10 μL of expectorated oral fluid Direct injection N/A Zorbax SB-Aq 18 ✓ ✓ ✓ ✓ 75 LC–MS/MS 120 μL of passive drool MEPS N/A PFP Kinetex 12.5 ✓ ✓ ✓ ✓ ✓ 76 LC–MS/MS 200 μL of Intercept or Quantisal oral fluid SLE N/A Acquity BEH C18 7.1 ✓ ✓ ✓ ✓ ✓ ✓ 77 LC–MS/MS 200 μL of oral fluid/StatSure buffer or 300 μL of Quantisal/Certus buffer SPE N/A Acquity BEH C18 8.5 ✓ ✓ ✓ 78 LC–MS/MS 100 μL of Intercept oral fluid Direct injection with online extraction column N/A Ascentis Phenyl 16 ✓ ✓ 79 LC–MS/MS 150 μL of Oral-Eze oral fluid Dilute and shoot N/A Acquity BEH C18 5 ✓ ✓ ✓ ✓ ✓ ✓ 80 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Acquity BEH C18 8.5 + wash ✓ ✓ ✓ ✓ ✓ 81 LC–MS/MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A Kinetex C18 5.5 ✓ ✓ ✓ ✓ ✓ ✓ 82 AMPS: amphetamines; BUP: buprenorphine; GC-MS: gas chromatography mass spectrometry; HFBA: heptafluorobutyric anhydride; LC–MS/MS: liquid chromatography–tandem mass spectrometry; LC-QTOF-MS: liquid chromatography–quadrupole time-of-flight mass spectrometry; MEPS: microextraction by packed sorbent; MSTFA: N-methyl-N-(trimethylsilyl)-trifluoroacetamide; MTBSTFA: N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide; SLE: supported liquid extraction. View Large Amphetamines Gas chromatography–mass spectrometry (GC-MS) and LC–MS/MS quantification methods for OF amphetamines typically include amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxyethylamphetamine, with approximate linear ranges of 20–250 μg/L (Table III). Derivatization, required for GC-MS analysis, is generally achieved with heptafluorobutyric anhydride. When enantioselective quantification of d- and l-amphetamines is required, derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (Marfey's reagent) produces diastereomers for LC–MS/MS analysis and that with S-heptafluorobutyrylprolyl chloride produces diastereomers for negative-ion chemical ionization analysis by GC-MS (90, 92). Table III Chromatographic Methods for the Detection of Amphetamines in Oral Fluid Instrumental Method Sample Volume and Type Sample Preparation Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) AMP MAMP MDMA MDA MDEA Other Analytes Reference LC–MS/MS 10 μL of expectorated oral fluid Protein precipitation and centrifugation N/A Hypersil™ BDS C18 ~ 4 0.5, 0.5 0.5–500 0.2 0.5 0.5–500 0.2, 0.5 0.5–500 0.5, 1.0 1–500 0.15, 0.5 0.5–500 N/A 86 GC-MS 100 μL of expectorated oral fluid Alkaline liquid extraction HFBA DB-5ms 18 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 Pseudoephedrine Norephedrine Ephedrine BDB, MBDB 87 GC-MS 400 μL of expectorated oral fluid SPE HFBA HP-5ms 19 2.5, 5 5–250 1, 5 5–250 2.5, 5.0 5–250 2.5, 5 5–500 2.5, 5.0 5–250 HMA HMMA 88 GC-MS 250 μL of Quantisal sample SPE HFBA DB-5 8 25 25–200 25 25–200 25 25–200 25 25–200 25 25–200 N/A 89 GC-MS-NICI 50 μL of expectorated oral fluid Liquid extraction S-HFBPCl HP-5ms 16 25 25–1,250 25 25–1,250 25 25–1,250 5 5–250 25 25–1,250 N/A 90 GC-MS 1.5 mL Quantisal sample SPME Propylchloroformate HP-5ms 20 2, 2 2–256 0.5, 2 2–256 N/A N/A N/A Fenproporex Diethylpropionmethylphenidate 91 LC–MS/MS 250 μL of Oral-Eze or Quantisal sample SPE Marfey's reagent N(α)-(2,4-dinitro-5-fluorophenyl)-1-alaninamide Kinetex® C18 20 0.5, 1 1–500 0.5, 1 1–500 N/A N/A N/A N/A 92 LC–MS/MS 100 μL of Quantisal sample SPE Marfey's reagent Zorbax Eclipse Plus C18 3.4 N/A 25–10,000 N/A N/A N/A N/A 93 Instrumental Method Sample Volume and Type Sample Preparation Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) AMP MAMP MDMA MDA MDEA Other Analytes Reference LC–MS/MS 10 μL of expectorated oral fluid Protein precipitation and centrifugation N/A Hypersil™ BDS C18 ~ 4 0.5, 0.5 0.5–500 0.2 0.5 0.5–500 0.2, 0.5 0.5–500 0.5, 1.0 1–500 0.15, 0.5 0.5–500 N/A 86 GC-MS 100 μL of expectorated oral fluid Alkaline liquid extraction HFBA DB-5ms 18 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 Pseudoephedrine Norephedrine Ephedrine BDB, MBDB 87 GC-MS 400 μL of expectorated oral fluid SPE HFBA HP-5ms 19 2.5, 5 5–250 1, 5 5–250 2.5, 5.0 5–250 2.5, 5 5–500 2.5, 5.0 5–250 HMA HMMA 88 GC-MS 250 μL of Quantisal sample SPE HFBA DB-5 8 25 25–200 25 25–200 25 25–200 25 25–200 25 25–200 N/A 89 GC-MS-NICI 50 μL of expectorated oral fluid Liquid extraction S-HFBPCl HP-5ms 16 25 25–1,250 25 25–1,250 25 25–1,250 5 5–250 25 25–1,250 N/A 90 GC-MS 1.5 mL Quantisal sample SPME Propylchloroformate HP-5ms 20 2, 2 2–256 0.5, 2 2–256 N/A N/A N/A Fenproporex Diethylpropionmethylphenidate 91 LC–MS/MS 250 μL of Oral-Eze or Quantisal sample SPE Marfey's reagent N(α)-(2,4-dinitro-5-fluorophenyl)-1-alaninamide Kinetex® C18 20 0.5, 1 1–500 0.5, 1 1–500 N/A N/A N/A N/A 92 LC–MS/MS 100 μL of Quantisal sample SPE Marfey's reagent Zorbax Eclipse Plus C18 3.4 N/A 25–10,000 N/A N/A N/A N/A 93 AMP: amphetamine; BDB: 1,3-benzodioxolylbutanamine; GC-MS: gas chromatography mass spectrometry; GC-MS-NICI: gas chromatography mass spectrometry negative-ion chemical ionization; HFBA: heptafluorobutyric anhydride; HMA: 4-hydroxy-3-methoxyamphetamine; HMMA: 4-hydroxy-3-methoxymethamphetamine; LC–MS/MS: liquid chromatography–tandem mass spectrometry; MAMP: methamphetamine; MBDB: 1,3-benzodioxolyl-N-methylbutanamine; MDA: 3,4-methylenedioxyamphetamine; MDEA: 3,4-methylenedioxyethylamphetamine; MDMA: 3,4-methylenedioxymethamphetamine; S-HFBPCl: S-heptafluorobutyrylprolyl chloride; SPE: solid-phase extraction; SPME: solid-phase microextraction. View Large Table III Chromatographic Methods for the Detection of Amphetamines in Oral Fluid Instrumental Method Sample Volume and Type Sample Preparation Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) AMP MAMP MDMA MDA MDEA Other Analytes Reference LC–MS/MS 10 μL of expectorated oral fluid Protein precipitation and centrifugation N/A Hypersil™ BDS C18 ~ 4 0.5, 0.5 0.5–500 0.2 0.5 0.5–500 0.2, 0.5 0.5–500 0.5, 1.0 1–500 0.15, 0.5 0.5–500 N/A 86 GC-MS 100 μL of expectorated oral fluid Alkaline liquid extraction HFBA DB-5ms 18 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 Pseudoephedrine Norephedrine Ephedrine BDB, MBDB 87 GC-MS 400 μL of expectorated oral fluid SPE HFBA HP-5ms 19 2.5, 5 5–250 1, 5 5–250 2.5, 5.0 5–250 2.5, 5 5–500 2.5, 5.0 5–250 HMA HMMA 88 GC-MS 250 μL of Quantisal sample SPE HFBA DB-5 8 25 25–200 25 25–200 25 25–200 25 25–200 25 25–200 N/A 89 GC-MS-NICI 50 μL of expectorated oral fluid Liquid extraction S-HFBPCl HP-5ms 16 25 25–1,250 25 25–1,250 25 25–1,250 5 5–250 25 25–1,250 N/A 90 GC-MS 1.5 mL Quantisal sample SPME Propylchloroformate HP-5ms 20 2, 2 2–256 0.5, 2 2–256 N/A N/A N/A Fenproporex Diethylpropionmethylphenidate 91 LC–MS/MS 250 μL of Oral-Eze or Quantisal sample SPE Marfey's reagent N(α)-(2,4-dinitro-5-fluorophenyl)-1-alaninamide Kinetex® C18 20 0.5, 1 1–500 0.5, 1 1–500 N/A N/A N/A N/A 92 LC–MS/MS 100 μL of Quantisal sample SPE Marfey's reagent Zorbax Eclipse Plus C18 3.4 N/A 25–10,000 N/A N/A N/A N/A 93 Instrumental Method Sample Volume and Type Sample Preparation Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) AMP MAMP MDMA MDA MDEA Other Analytes Reference LC–MS/MS 10 μL of expectorated oral fluid Protein precipitation and centrifugation N/A Hypersil™ BDS C18 ~ 4 0.5, 0.5 0.5–500 0.2 0.5 0.5–500 0.2, 0.5 0.5–500 0.5, 1.0 1–500 0.15, 0.5 0.5–500 N/A 86 GC-MS 100 μL of expectorated oral fluid Alkaline liquid extraction HFBA DB-5ms 18 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 5, 20 20–5,000 Pseudoephedrine Norephedrine Ephedrine BDB, MBDB 87 GC-MS 400 μL of expectorated oral fluid SPE HFBA HP-5ms 19 2.5, 5 5–250 1, 5 5–250 2.5, 5.0 5–250 2.5, 5 5–500 2.5, 5.0 5–250 HMA HMMA 88 GC-MS 250 μL of Quantisal sample SPE HFBA DB-5 8 25 25–200 25 25–200 25 25–200 25 25–200 25 25–200 N/A 89 GC-MS-NICI 50 μL of expectorated oral fluid Liquid extraction S-HFBPCl HP-5ms 16 25 25–1,250 25 25–1,250 25 25–1,250 5 5–250 25 25–1,250 N/A 90 GC-MS 1.5 mL Quantisal sample SPME Propylchloroformate HP-5ms 20 2, 2 2–256 0.5, 2 2–256 N/A N/A N/A Fenproporex Diethylpropionmethylphenidate 91 LC–MS/MS 250 μL of Oral-Eze or Quantisal sample SPE Marfey's reagent N(α)-(2,4-dinitro-5-fluorophenyl)-1-alaninamide Kinetex® C18 20 0.5, 1 1–500 0.5, 1 1–500 N/A N/A N/A N/A 92 LC–MS/MS 100 μL of Quantisal sample SPE Marfey's reagent Zorbax Eclipse Plus C18 3.4 N/A 25–10,000 N/A N/A N/A N/A 93 AMP: amphetamine; BDB: 1,3-benzodioxolylbutanamine; GC-MS: gas chromatography mass spectrometry; GC-MS-NICI: gas chromatography mass spectrometry negative-ion chemical ionization; HFBA: heptafluorobutyric anhydride; HMA: 4-hydroxy-3-methoxyamphetamine; HMMA: 4-hydroxy-3-methoxymethamphetamine; LC–MS/MS: liquid chromatography–tandem mass spectrometry; MAMP: methamphetamine; MBDB: 1,3-benzodioxolyl-N-methylbutanamine; MDA: 3,4-methylenedioxyamphetamine; MDEA: 3,4-methylenedioxyethylamphetamine; MDMA: 3,4-methylenedioxymethamphetamine; S-HFBPCl: S-heptafluorobutyrylprolyl chloride; SPE: solid-phase extraction; SPME: solid-phase microextraction. View Large Cannabis Analytical methods quantifying multiple cannabinoids in OF can improve interpretation of results. THC is the primary and sole analyte included in most published OF cannabinoid methods (Table IV). Published methods include GC-MS, 2D-GC-MS, LC-MS, LC–MS/MS and LC–high-resolution mass spectrometry methods; and analytes included in published methods are THC, 11-hydroxy-THC (11-OH-THC), THCCOOH, cannabidiol (CBD), and cannabinol (CBN). Due to contamination of the oral mucosa during cannabis inhalation or ingestion, a large dynamic range or validated dilution integrity is needed for OF THC (e.g., 0.5–>1,000 μg/L). However, THC is also detected in OF following passive exposure to intense environmental cannabis smoke (114–118). THC's inactive metabolite, THCCOOH, may be monitored to confirm cannabis intake, as it is not present in cannabis smoke (118). Furthermore, THCCOOH in OF extends the detection window of cannabis intake in chronic frequent cannabis smokers and also identifies intake of oral synthetic THC or Marinol, while THC does not (34, 119). Thus, despite its presence in low ng/L concentrations, monitoring THCCOOH in OF may be warranted, as it may provide additional information. Highly sensitive analytical methods achieving the required low ng/L limits of quantification (LOQs) were published for various cannabinoids (Table IV). It should be noted that some cannabis smoke components may create interferences that mask the low ng/L THCCOOH concentrations; authentic samples should be evaluated to ensure that any interferences are adequately resolved (85). Table IV Chromatographic Methods for the Detection of Cannabinoids in Oral Fluid Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) THC 11-OH-THC THC-COOH (pg/L) CBD CBN Other Analytes Reference LC-MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A XTerra® MS C18 5 2 2–250 N/A N/A N/A N/A N/A 94 LC-MS 500 μL of Salivette oral fluid SPE N/A XTerra® MS C18 6 2 2–100 N/A N/A N/A N/A N/A 95 LC–MS/MS 100 μL of or 500 μL of Intercept oral fluid Liquid–liquid extraction N/A XTerra® MS C18 8 0.5–100 (with 100 μL of sample) 0.1–10 (with 500 μL of sample) N/A N/A N/A N/A N/A 96 GC-MS/MS 100 μL of Intercept oral fluid SPE HFIP and PFAA DB-5 6 N/A N/A 10 10–240 N/A N/A N/A 97 2D-GC-MS 1 mL of Quantisal oral fluid SPE HFIP and TFAA DB-35ms and DB-1 12.5 N/A N/A 2 2–160 N/A N/A N/A 98 GC-MS Unspecified volume of Quantisal oral fluid SPE BSTFA DB-5ms 6 0.5 1–16 N/A N/A 0.5 1–16 1 1–16 THCAA 99 LC-QTOF-MS 500 μL of synthetic oral fluid Liquid–liquid extraction N/A XTerra MS C18 19 0.05, 0.1 0.1–100 N/A 0.2, 0.1 0.1–100 N/A N/A N/A 100 LC-MS 500 μL of expectorated oral fluid SPE N/A Symmetry® C18 15 2, 5 5–2,000 N/A N/A N/A N/A N/A 101 2D-GC-MS (NICI for THCCCOOH) 1 mL of Quantisal oral fluid SPE BSTFA TFAA (for THCCOOH) ZB-50 and DB-1ms DB-1ms and ZB-50 (for THCCOOH) 21 and 13 0.5, 0.5 0.5–50 0.4, 0.5 0.5–50 6, 7.5 7.5–500 0.5, 0.5 0.5–50 1, 1 1–50 N/A 102 LC–MS/MS 400 μL of Intercept oral fluid SPE N/A XTerra MS C18 12 0.2 0.25–8 N/A 0.2 0.25–8 N/A N/A N/A 103 LC–MS/MS 1 mL of Quantisal oral fluid SPE Triphenylphosphine, 2-picolylamine and 2,2′-dipyridyl disulfide Stable Bond-C18 8.5 0.6, 1 1–100 N/A 6, 10 10–1,000 N/A N/A N/A 104 LC–MS/MS (quadrupole/Orbitrap) 400 μL of oral fluid in preservation buffer Liquid–liquid extraction and SPE N/A Hypersil GOLD aQ 12.5 2 (one-point calibration) N/A 7.5 7.5–300 N/A N/A N/A 105 LC–MS/MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A Zorbax® Eclipse XDB C18 3.5 1 1–500 N/A N/A N/A N/A N/A 106 LC–MS/MS 250 μL of expectorated oral fluid Dilute and shoot Dansyl chloride Gemini C18 4 0.005, 0.025 0.2–20 N/A 2, 5 5–500 N/A N/A N/A 107 LC–MS/MS 250 μL of expectorated oral fluid SPE N/A Kinetex® C18 12.5 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 N/A 108 LC-HRMS 250 μL of Oral-Eze and 500 μL of Quantisal oral fluid SPE N/A Hypersil GOLD aQ 10 0.5 0.5–50 N/A 15 15–500 0.5 0.5–50 0.5 0.5–50 N/A 109 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Kinetex C18 9 N/A N/A 9, 12 12–1,020 N/A N/A N/A 110 LC–MS/MS 225 μL of expectorated oral fluid MEPS N/A Kinetex C18-XB 10 0.08, 0.25 0.25–250 0.12, 0.4 0.4–250 8, 20 20–1,000 0.1, 0.3 0.3–250 0.12, 0.3 0.3–250 N/A 76 LC–MS/MS 100 μL of StatSure, 200 μL of Quantisal or Certus oral fluid Liquid–liquid extraction N/A Acquity UPLC BEH C18 5 5 5–320 N/A N/A N/A N/A N/A 111 LC–MS/MS 1.5 μL of combined Quantisal sample/methanol extract SPE N/A Kinetex C18 11 0.3, 0.5 0.5–75 0.2, 0.5 0.5–75 50, 80 50–500 0.3, 0.5 0.5–75 0.3, 0.5 0.5–75 THCAA 112 GC-MS/MS 1 mL of Quantisal or 750 μL of Oral-Eze oral fluid SPE HFIP and TFAA 2 HP-5ms 5.2 N/A N/A 7.5, 10 10–1,000 N/A N/A N/A 113 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Selectra PFPP 14 0.1, 0.2 0.2–100 0.1, 0.2 0.2–50 15, 15 15–3,750 0.1, 0.2 0.2–50 N/A THCV CBG 85 Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) THC 11-OH-THC THC-COOH (pg/L) CBD CBN Other Analytes Reference LC-MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A XTerra® MS C18 5 2 2–250 N/A N/A N/A N/A N/A 94 LC-MS 500 μL of Salivette oral fluid SPE N/A XTerra® MS C18 6 2 2–100 N/A N/A N/A N/A N/A 95 LC–MS/MS 100 μL of or 500 μL of Intercept oral fluid Liquid–liquid extraction N/A XTerra® MS C18 8 0.5–100 (with 100 μL of sample) 0.1–10 (with 500 μL of sample) N/A N/A N/A N/A N/A 96 GC-MS/MS 100 μL of Intercept oral fluid SPE HFIP and PFAA DB-5 6 N/A N/A 10 10–240 N/A N/A N/A 97 2D-GC-MS 1 mL of Quantisal oral fluid SPE HFIP and TFAA DB-35ms and DB-1 12.5 N/A N/A 2 2–160 N/A N/A N/A 98 GC-MS Unspecified volume of Quantisal oral fluid SPE BSTFA DB-5ms 6 0.5 1–16 N/A N/A 0.5 1–16 1 1–16 THCAA 99 LC-QTOF-MS 500 μL of synthetic oral fluid Liquid–liquid extraction N/A XTerra MS C18 19 0.05, 0.1 0.1–100 N/A 0.2, 0.1 0.1–100 N/A N/A N/A 100 LC-MS 500 μL of expectorated oral fluid SPE N/A Symmetry® C18 15 2, 5 5–2,000 N/A N/A N/A N/A N/A 101 2D-GC-MS (NICI for THCCCOOH) 1 mL of Quantisal oral fluid SPE BSTFA TFAA (for THCCOOH) ZB-50 and DB-1ms DB-1ms and ZB-50 (for THCCOOH) 21 and 13 0.5, 0.5 0.5–50 0.4, 0.5 0.5–50 6, 7.5 7.5–500 0.5, 0.5 0.5–50 1, 1 1–50 N/A 102 LC–MS/MS 400 μL of Intercept oral fluid SPE N/A XTerra MS C18 12 0.2 0.25–8 N/A 0.2 0.25–8 N/A N/A N/A 103 LC–MS/MS 1 mL of Quantisal oral fluid SPE Triphenylphosphine, 2-picolylamine and 2,2′-dipyridyl disulfide Stable Bond-C18 8.5 0.6, 1 1–100 N/A 6, 10 10–1,000 N/A N/A N/A 104 LC–MS/MS (quadrupole/Orbitrap) 400 μL of oral fluid in preservation buffer Liquid–liquid extraction and SPE N/A Hypersil GOLD aQ 12.5 2 (one-point calibration) N/A 7.5 7.5–300 N/A N/A N/A 105 LC–MS/MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A Zorbax® Eclipse XDB C18 3.5 1 1–500 N/A N/A N/A N/A N/A 106 LC–MS/MS 250 μL of expectorated oral fluid Dilute and shoot Dansyl chloride Gemini C18 4 0.005, 0.025 0.2–20 N/A 2, 5 5–500 N/A N/A N/A 107 LC–MS/MS 250 μL of expectorated oral fluid SPE N/A Kinetex® C18 12.5 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 N/A 108 LC-HRMS 250 μL of Oral-Eze and 500 μL of Quantisal oral fluid SPE N/A Hypersil GOLD aQ 10 0.5 0.5–50 N/A 15 15–500 0.5 0.5–50 0.5 0.5–50 N/A 109 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Kinetex C18 9 N/A N/A 9, 12 12–1,020 N/A N/A N/A 110 LC–MS/MS 225 μL of expectorated oral fluid MEPS N/A Kinetex C18-XB 10 0.08, 0.25 0.25–250 0.12, 0.4 0.4–250 8, 20 20–1,000 0.1, 0.3 0.3–250 0.12, 0.3 0.3–250 N/A 76 LC–MS/MS 100 μL of StatSure, 200 μL of Quantisal or Certus oral fluid Liquid–liquid extraction N/A Acquity UPLC BEH C18 5 5 5–320 N/A N/A N/A N/A N/A 111 LC–MS/MS 1.5 μL of combined Quantisal sample/methanol extract SPE N/A Kinetex C18 11 0.3, 0.5 0.5–75 0.2, 0.5 0.5–75 50, 80 50–500 0.3, 0.5 0.5–75 0.3, 0.5 0.5–75 THCAA 112 GC-MS/MS 1 mL of Quantisal or 750 μL of Oral-Eze oral fluid SPE HFIP and TFAA 2 HP-5ms 5.2 N/A N/A 7.5, 10 10–1,000 N/A N/A N/A 113 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Selectra PFPP 14 0.1, 0.2 0.2–100 0.1, 0.2 0.2–50 15, 15 15–3,750 0.1, 0.2 0.2–50 N/A THCV CBG 85 11-OH-THC: 11-hydroxy-THC; 2D-GC-MS: 2D GC-MS; BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide; CBD: cannabidiol; CBG: cannabigerol; CBN: cannabinol; GC-MS/MS: gas chromatography–tandem mass spectrometry; GC-MS: gas chromatography–mass spectrometry; HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol; LC-HRMS: liquid chromatography–high-resolution mass spectrometry; LC–MS/MS: liquid chromatography–tandem mass spectrometry; LC-MS: liquid chromatography–mass spectrometry; LC-QTOF-MS: liquid chromatography–quadrupole time-of-flight mass spectrometry; MEPS: microextraction by packed sorbent; PFAA: pentafluoropropionic anhydride; SPE: solid-phase extraction; TFAA: trifluoroacetic anhydride; THC: Δ9-tetrahydrocannabinol; THCAA: Δ9-tetrahydrocannabinolic acid A; THCCOOH: 11-nor-9-carboxy-THC; THCV: tetrahydrocannabivarin. View Large Table IV Chromatographic Methods for the Detection of Cannabinoids in Oral Fluid Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) THC 11-OH-THC THC-COOH (pg/L) CBD CBN Other Analytes Reference LC-MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A XTerra® MS C18 5 2 2–250 N/A N/A N/A N/A N/A 94 LC-MS 500 μL of Salivette oral fluid SPE N/A XTerra® MS C18 6 2 2–100 N/A N/A N/A N/A N/A 95 LC–MS/MS 100 μL of or 500 μL of Intercept oral fluid Liquid–liquid extraction N/A XTerra® MS C18 8 0.5–100 (with 100 μL of sample) 0.1–10 (with 500 μL of sample) N/A N/A N/A N/A N/A 96 GC-MS/MS 100 μL of Intercept oral fluid SPE HFIP and PFAA DB-5 6 N/A N/A 10 10–240 N/A N/A N/A 97 2D-GC-MS 1 mL of Quantisal oral fluid SPE HFIP and TFAA DB-35ms and DB-1 12.5 N/A N/A 2 2–160 N/A N/A N/A 98 GC-MS Unspecified volume of Quantisal oral fluid SPE BSTFA DB-5ms 6 0.5 1–16 N/A N/A 0.5 1–16 1 1–16 THCAA 99 LC-QTOF-MS 500 μL of synthetic oral fluid Liquid–liquid extraction N/A XTerra MS C18 19 0.05, 0.1 0.1–100 N/A 0.2, 0.1 0.1–100 N/A N/A N/A 100 LC-MS 500 μL of expectorated oral fluid SPE N/A Symmetry® C18 15 2, 5 5–2,000 N/A N/A N/A N/A N/A 101 2D-GC-MS (NICI for THCCCOOH) 1 mL of Quantisal oral fluid SPE BSTFA TFAA (for THCCOOH) ZB-50 and DB-1ms DB-1ms and ZB-50 (for THCCOOH) 21 and 13 0.5, 0.5 0.5–50 0.4, 0.5 0.5–50 6, 7.5 7.5–500 0.5, 0.5 0.5–50 1, 1 1–50 N/A 102 LC–MS/MS 400 μL of Intercept oral fluid SPE N/A XTerra MS C18 12 0.2 0.25–8 N/A 0.2 0.25–8 N/A N/A N/A 103 LC–MS/MS 1 mL of Quantisal oral fluid SPE Triphenylphosphine, 2-picolylamine and 2,2′-dipyridyl disulfide Stable Bond-C18 8.5 0.6, 1 1–100 N/A 6, 10 10–1,000 N/A N/A N/A 104 LC–MS/MS (quadrupole/Orbitrap) 400 μL of oral fluid in preservation buffer Liquid–liquid extraction and SPE N/A Hypersil GOLD aQ 12.5 2 (one-point calibration) N/A 7.5 7.5–300 N/A N/A N/A 105 LC–MS/MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A Zorbax® Eclipse XDB C18 3.5 1 1–500 N/A N/A N/A N/A N/A 106 LC–MS/MS 250 μL of expectorated oral fluid Dilute and shoot Dansyl chloride Gemini C18 4 0.005, 0.025 0.2–20 N/A 2, 5 5–500 N/A N/A N/A 107 LC–MS/MS 250 μL of expectorated oral fluid SPE N/A Kinetex® C18 12.5 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 N/A 108 LC-HRMS 250 μL of Oral-Eze and 500 μL of Quantisal oral fluid SPE N/A Hypersil GOLD aQ 10 0.5 0.5–50 N/A 15 15–500 0.5 0.5–50 0.5 0.5–50 N/A 109 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Kinetex C18 9 N/A N/A 9, 12 12–1,020 N/A N/A N/A 110 LC–MS/MS 225 μL of expectorated oral fluid MEPS N/A Kinetex C18-XB 10 0.08, 0.25 0.25–250 0.12, 0.4 0.4–250 8, 20 20–1,000 0.1, 0.3 0.3–250 0.12, 0.3 0.3–250 N/A 76 LC–MS/MS 100 μL of StatSure, 200 μL of Quantisal or Certus oral fluid Liquid–liquid extraction N/A Acquity UPLC BEH C18 5 5 5–320 N/A N/A N/A N/A N/A 111 LC–MS/MS 1.5 μL of combined Quantisal sample/methanol extract SPE N/A Kinetex C18 11 0.3, 0.5 0.5–75 0.2, 0.5 0.5–75 50, 80 50–500 0.3, 0.5 0.5–75 0.3, 0.5 0.5–75 THCAA 112 GC-MS/MS 1 mL of Quantisal or 750 μL of Oral-Eze oral fluid SPE HFIP and TFAA 2 HP-5ms 5.2 N/A N/A 7.5, 10 10–1,000 N/A N/A N/A 113 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Selectra PFPP 14 0.1, 0.2 0.2–100 0.1, 0.2 0.2–50 15, 15 15–3,750 0.1, 0.2 0.2–50 N/A THCV CBG 85 Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) THC 11-OH-THC THC-COOH (pg/L) CBD CBN Other Analytes Reference LC-MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A XTerra® MS C18 5 2 2–250 N/A N/A N/A N/A N/A 94 LC-MS 500 μL of Salivette oral fluid SPE N/A XTerra® MS C18 6 2 2–100 N/A N/A N/A N/A N/A 95 LC–MS/MS 100 μL of or 500 μL of Intercept oral fluid Liquid–liquid extraction N/A XTerra® MS C18 8 0.5–100 (with 100 μL of sample) 0.1–10 (with 500 μL of sample) N/A N/A N/A N/A N/A 96 GC-MS/MS 100 μL of Intercept oral fluid SPE HFIP and PFAA DB-5 6 N/A N/A 10 10–240 N/A N/A N/A 97 2D-GC-MS 1 mL of Quantisal oral fluid SPE HFIP and TFAA DB-35ms and DB-1 12.5 N/A N/A 2 2–160 N/A N/A N/A 98 GC-MS Unspecified volume of Quantisal oral fluid SPE BSTFA DB-5ms 6 0.5 1–16 N/A N/A 0.5 1–16 1 1–16 THCAA 99 LC-QTOF-MS 500 μL of synthetic oral fluid Liquid–liquid extraction N/A XTerra MS C18 19 0.05, 0.1 0.1–100 N/A 0.2, 0.1 0.1–100 N/A N/A N/A 100 LC-MS 500 μL of expectorated oral fluid SPE N/A Symmetry® C18 15 2, 5 5–2,000 N/A N/A N/A N/A N/A 101 2D-GC-MS (NICI for THCCCOOH) 1 mL of Quantisal oral fluid SPE BSTFA TFAA (for THCCOOH) ZB-50 and DB-1ms DB-1ms and ZB-50 (for THCCOOH) 21 and 13 0.5, 0.5 0.5–50 0.4, 0.5 0.5–50 6, 7.5 7.5–500 0.5, 0.5 0.5–50 1, 1 1–50 N/A 102 LC–MS/MS 400 μL of Intercept oral fluid SPE N/A XTerra MS C18 12 0.2 0.25–8 N/A 0.2 0.25–8 N/A N/A N/A 103 LC–MS/MS 1 mL of Quantisal oral fluid SPE Triphenylphosphine, 2-picolylamine and 2,2′-dipyridyl disulfide Stable Bond-C18 8.5 0.6, 1 1–100 N/A 6, 10 10–1,000 N/A N/A N/A 104 LC–MS/MS (quadrupole/Orbitrap) 400 μL of oral fluid in preservation buffer Liquid–liquid extraction and SPE N/A Hypersil GOLD aQ 12.5 2 (one-point calibration) N/A 7.5 7.5–300 N/A N/A N/A 105 LC–MS/MS 200 μL of expectorated oral fluid Liquid–liquid extraction N/A Zorbax® Eclipse XDB C18 3.5 1 1–500 N/A N/A N/A N/A N/A 106 LC–MS/MS 250 μL of expectorated oral fluid Dilute and shoot Dansyl chloride Gemini C18 4 0.005, 0.025 0.2–20 N/A 2, 5 5–500 N/A N/A N/A 107 LC–MS/MS 250 μL of expectorated oral fluid SPE N/A Kinetex® C18 12.5 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 0.1 0.1–50 N/A 108 LC-HRMS 250 μL of Oral-Eze and 500 μL of Quantisal oral fluid SPE N/A Hypersil GOLD aQ 10 0.5 0.5–50 N/A 15 15–500 0.5 0.5–50 0.5 0.5–50 N/A 109 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Kinetex C18 9 N/A N/A 9, 12 12–1,020 N/A N/A N/A 110 LC–MS/MS 225 μL of expectorated oral fluid MEPS N/A Kinetex C18-XB 10 0.08, 0.25 0.25–250 0.12, 0.4 0.4–250 8, 20 20–1,000 0.1, 0.3 0.3–250 0.12, 0.3 0.3–250 N/A 76 LC–MS/MS 100 μL of StatSure, 200 μL of Quantisal or Certus oral fluid Liquid–liquid extraction N/A Acquity UPLC BEH C18 5 5 5–320 N/A N/A N/A N/A N/A 111 LC–MS/MS 1.5 μL of combined Quantisal sample/methanol extract SPE N/A Kinetex C18 11 0.3, 0.5 0.5–75 0.2, 0.5 0.5–75 50, 80 50–500 0.3, 0.5 0.5–75 0.3, 0.5 0.5–75 THCAA 112 GC-MS/MS 1 mL of Quantisal or 750 μL of Oral-Eze oral fluid SPE HFIP and TFAA 2 HP-5ms 5.2 N/A N/A 7.5, 10 10–1,000 N/A N/A N/A 113 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Selectra PFPP 14 0.1, 0.2 0.2–100 0.1, 0.2 0.2–50 15, 15 15–3,750 0.1, 0.2 0.2–50 N/A THCV CBG 85 11-OH-THC: 11-hydroxy-THC; 2D-GC-MS: 2D GC-MS; BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide; CBD: cannabidiol; CBG: cannabigerol; CBN: cannabinol; GC-MS/MS: gas chromatography–tandem mass spectrometry; GC-MS: gas chromatography–mass spectrometry; HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol; LC-HRMS: liquid chromatography–high-resolution mass spectrometry; LC–MS/MS: liquid chromatography–tandem mass spectrometry; LC-MS: liquid chromatography–mass spectrometry; LC-QTOF-MS: liquid chromatography–quadrupole time-of-flight mass spectrometry; MEPS: microextraction by packed sorbent; PFAA: pentafluoropropionic anhydride; SPE: solid-phase extraction; TFAA: trifluoroacetic anhydride; THC: Δ9-tetrahydrocannabinol; THCAA: Δ9-tetrahydrocannabinolic acid A; THCCOOH: 11-nor-9-carboxy-THC; THCV: tetrahydrocannabivarin. View Large Identifying minor cannabinoids CBN, tetrahydrocannabivarin (THCV) and cannabigerol (CBG) in OF was suggested to document past-day intake in occasional and chronic frequent cannabis smokers, although various cannabis strains were not evaluated (85, 102, 109). These cannabinoids are easily included in OF analytical methods, although their concentrations are generally lower. Inclusion of CBD, CBG, CBN and THCV can improve interpretation of cannabis OF results. Cocaine Cocaine and its metabolite benzoylecgonine (BE) are relatively easy to detect in OF due to their high concentrations, frequently by LC separation on a C18 column. Cocaine and BE can also be detected by GC-MS. Several analytical methods are available for quantification of cocaine and metabolites alone, with BE extending cocaine's detection window (Table V), or in combination with other drugs (Table II). Typical concentration ranges for cocaine and BE are from 1 or 10 to 100 or 1,000 μg/L. Other OF assays also include cocaethylene and ecgonine methyl ester. Cocaine and BE concentrations >1,000 μg/L were reported due to ion trapping and contamination of the oral mucosa during smoking or insufflation; therefore, large dynamic ranges are required for quantifying cocaine markers or dilution may be necessary. Table V Chromatographic Methods for the Detection of Cocaine in Oral Fluid Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Cocaine Benzoylecgonine Ecgonine Methyl Ester Cocaethylene Other Analytes Reference GC-MS 1 mL of expectorated oral fluid SPE BSTFA HP-1 N/A 1 1.1–500 1 1.1–500 1 1.1–500 N/A N/A 13 LC-QTOF-MS 100 μL of oral fluid SPE N/A Hypersil™ BDS C18 33 1, 10 10–1,000 1, 10 10–1,000 N/A 1, 10 10–1,000 N/A 120 2D-GC-MS 750 μL of Oral-Eze or 500 μL of StatSure oral fluid SPE MTBSTFA DB-1ms and ZB-50 18 1, 1 1–100 0.5, 1 1–100 N/A N/A N/A 33 LC-MS 100 μL of Multi-Drugs Multi-Line Twist Screen Test Devices oral fluid Filtration N/A Kinetex® HILIC ~12 4.25–544 4.25–544 N/A 4.25–544 AEME AEC 121 Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Cocaine Benzoylecgonine Ecgonine Methyl Ester Cocaethylene Other Analytes Reference GC-MS 1 mL of expectorated oral fluid SPE BSTFA HP-1 N/A 1 1.1–500 1 1.1–500 1 1.1–500 N/A N/A 13 LC-QTOF-MS 100 μL of oral fluid SPE N/A Hypersil™ BDS C18 33 1, 10 10–1,000 1, 10 10–1,000 N/A 1, 10 10–1,000 N/A 120 2D-GC-MS 750 μL of Oral-Eze or 500 μL of StatSure oral fluid SPE MTBSTFA DB-1ms and ZB-50 18 1, 1 1–100 0.5, 1 1–100 N/A N/A N/A 33 LC-MS 100 μL of Multi-Drugs Multi-Line Twist Screen Test Devices oral fluid Filtration N/A Kinetex® HILIC ~12 4.25–544 4.25–544 N/A 4.25–544 AEME AEC 121 2D-GC-MS: 2D GC-MS; AEC: anhydroecgonine; AEMA: anhydroecgonine methyl ester; BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide; GC-MS: gas chromatography–mass spectrometry; LC-QTOF-MS: liquid chromatography–quadrupole time-of-flight mass spectrometry; MTBSTFA: N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide; SPE: solid-phase extraction. View Large Table V Chromatographic Methods for the Detection of Cocaine in Oral Fluid Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Cocaine Benzoylecgonine Ecgonine Methyl Ester Cocaethylene Other Analytes Reference GC-MS 1 mL of expectorated oral fluid SPE BSTFA HP-1 N/A 1 1.1–500 1 1.1–500 1 1.1–500 N/A N/A 13 LC-QTOF-MS 100 μL of oral fluid SPE N/A Hypersil™ BDS C18 33 1, 10 10–1,000 1, 10 10–1,000 N/A 1, 10 10–1,000 N/A 120 2D-GC-MS 750 μL of Oral-Eze or 500 μL of StatSure oral fluid SPE MTBSTFA DB-1ms and ZB-50 18 1, 1 1–100 0.5, 1 1–100 N/A N/A N/A 33 LC-MS 100 μL of Multi-Drugs Multi-Line Twist Screen Test Devices oral fluid Filtration N/A Kinetex® HILIC ~12 4.25–544 4.25–544 N/A 4.25–544 AEME AEC 121 Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Cocaine Benzoylecgonine Ecgonine Methyl Ester Cocaethylene Other Analytes Reference GC-MS 1 mL of expectorated oral fluid SPE BSTFA HP-1 N/A 1 1.1–500 1 1.1–500 1 1.1–500 N/A N/A 13 LC-QTOF-MS 100 μL of oral fluid SPE N/A Hypersil™ BDS C18 33 1, 10 10–1,000 1, 10 10–1,000 N/A 1, 10 10–1,000 N/A 120 2D-GC-MS 750 μL of Oral-Eze or 500 μL of StatSure oral fluid SPE MTBSTFA DB-1ms and ZB-50 18 1, 1 1–100 0.5, 1 1–100 N/A N/A N/A 33 LC-MS 100 μL of Multi-Drugs Multi-Line Twist Screen Test Devices oral fluid Filtration N/A Kinetex® HILIC ~12 4.25–544 4.25–544 N/A 4.25–544 AEME AEC 121 2D-GC-MS: 2D GC-MS; AEC: anhydroecgonine; AEMA: anhydroecgonine methyl ester; BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide; GC-MS: gas chromatography–mass spectrometry; LC-QTOF-MS: liquid chromatography–quadrupole time-of-flight mass spectrometry; MTBSTFA: N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide; SPE: solid-phase extraction. View Large Opioids OF opioid methods are available for GC-MS with N,O-bis(trimethylsilyl)trifluoroacetamide derivatization and by LC–MS/MS (Table VI); however, due to the structural similarities between opioids, chromatographic separation and characteristic transition selection are imperative (125). Opioids such as codeine, morphine, 6-acetylmorphine (6-AM), heroin, hydrocodone, hydromorphone, oxycodone and oxymorphone are the most common analytes, with 6-AM presence identifying heroin administration. Concentration ranges are quite variable due to the differing potencies of opioids. Typical 6-AM OF concentrations are 2–500 μg/L, although higher 6-AM concentrations are common and may require dilutions following some routes of administration (e.g., smoking). Table VI Chromatographic Methods for the Detection of Opioids in Oral Fluid Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Morphine Codeine 6-AM Hydrocodone/Hydromorphone Oxycodone/Oxymorphone Other Analytes Reference GC-MS 1 mL of unspecified oral fluid SPE BSTFA DB-5 15 2, 2 2–50 2, 2 2–50 2, 3 3–50 3, 10/2, 3 10–50/3–50 2, 3 3–50 (oxycodone only) N/A 122 GC-MS-PICI 1 mL of Salivette oral fluid Toxitubes A® BSTFA Unspecified 5% phenyl-methyl-siloxane column 13.5 2, 6.7 30–500 0.7, 2.3 30–500 0.6 30–500 N/A N/A N/A 123 GC-MS 1 mL of Quantisal oral fluid SPE BSTFA or MSTFA DB-5ms 7, 8 and 17.5 N/A N/A N/A N/A 10 10–80 (oxycodone only) Meperidine Tramadol 124 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Zorbax® Eclipse XDB C18 8 1 1–160 1 1–160 1 1–160 1 1–160 1 1–160 6-Acetylcodeine Dihydrocodeine Norhydrocodone Norcodeine Noroxycodone 125 LC–MS/MS 100 μL of Salivette oral fluid Protein Precipitation N/A Linearly bridged dual columns of Xbridge HILIC and Poroshell 120 EC-C18 10 N/A N/A N/A N/A N/A Tramadol and metabolites 126 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Zorbax Eclipse Plus C18 5.5 2.5–1,000 N/A N/A 2.5–1,000 2.5–1,000 Fentanyl Tramadol 127 Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Morphine Codeine 6-AM Hydrocodone/Hydromorphone Oxycodone/Oxymorphone Other Analytes Reference GC-MS 1 mL of unspecified oral fluid SPE BSTFA DB-5 15 2, 2 2–50 2, 2 2–50 2, 3 3–50 3, 10/2, 3 10–50/3–50 2, 3 3–50 (oxycodone only) N/A 122 GC-MS-PICI 1 mL of Salivette oral fluid Toxitubes A® BSTFA Unspecified 5% phenyl-methyl-siloxane column 13.5 2, 6.7 30–500 0.7, 2.3 30–500 0.6 30–500 N/A N/A N/A 123 GC-MS 1 mL of Quantisal oral fluid SPE BSTFA or MSTFA DB-5ms 7, 8 and 17.5 N/A N/A N/A N/A 10 10–80 (oxycodone only) Meperidine Tramadol 124 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Zorbax® Eclipse XDB C18 8 1 1–160 1 1–160 1 1–160 1 1–160 1 1–160 6-Acetylcodeine Dihydrocodeine Norhydrocodone Norcodeine Noroxycodone 125 LC–MS/MS 100 μL of Salivette oral fluid Protein Precipitation N/A Linearly bridged dual columns of Xbridge HILIC and Poroshell 120 EC-C18 10 N/A N/A N/A N/A N/A Tramadol and metabolites 126 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Zorbax Eclipse Plus C18 5.5 2.5–1,000 N/A N/A 2.5–1,000 2.5–1,000 Fentanyl Tramadol 127 BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide; GC-MS: gas chromatography–mass spectrometry; GC-MS-PICI: gas chromatography–mass spectrometry–positive-ion chemical ionization; LC–MS/MS: liquid chromatography–tandem mass spectrometry; LC-QTOF-MS: liquid chromatography–quadrupole time-of-flight mass spectrometry; MSTFA: N-methyl-N-(trimethylsilyl)-trifluoroacetamide; SPE: solid-phase extraction. View Large Table VI Chromatographic Methods for the Detection of Opioids in Oral Fluid Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Morphine Codeine 6-AM Hydrocodone/Hydromorphone Oxycodone/Oxymorphone Other Analytes Reference GC-MS 1 mL of unspecified oral fluid SPE BSTFA DB-5 15 2, 2 2–50 2, 2 2–50 2, 3 3–50 3, 10/2, 3 10–50/3–50 2, 3 3–50 (oxycodone only) N/A 122 GC-MS-PICI 1 mL of Salivette oral fluid Toxitubes A® BSTFA Unspecified 5% phenyl-methyl-siloxane column 13.5 2, 6.7 30–500 0.7, 2.3 30–500 0.6 30–500 N/A N/A N/A 123 GC-MS 1 mL of Quantisal oral fluid SPE BSTFA or MSTFA DB-5ms 7, 8 and 17.5 N/A N/A N/A N/A 10 10–80 (oxycodone only) Meperidine Tramadol 124 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Zorbax® Eclipse XDB C18 8 1 1–160 1 1–160 1 1–160 1 1–160 1 1–160 6-Acetylcodeine Dihydrocodeine Norhydrocodone Norcodeine Noroxycodone 125 LC–MS/MS 100 μL of Salivette oral fluid Protein Precipitation N/A Linearly bridged dual columns of Xbridge HILIC and Poroshell 120 EC-C18 10 N/A N/A N/A N/A N/A Tramadol and metabolites 126 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Zorbax Eclipse Plus C18 5.5 2.5–1,000 N/A N/A 2.5–1,000 2.5–1,000 Fentanyl Tramadol 127 Instrumental Method Sample Volume and Type Sample Prep Derivatization Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Morphine Codeine 6-AM Hydrocodone/Hydromorphone Oxycodone/Oxymorphone Other Analytes Reference GC-MS 1 mL of unspecified oral fluid SPE BSTFA DB-5 15 2, 2 2–50 2, 2 2–50 2, 3 3–50 3, 10/2, 3 10–50/3–50 2, 3 3–50 (oxycodone only) N/A 122 GC-MS-PICI 1 mL of Salivette oral fluid Toxitubes A® BSTFA Unspecified 5% phenyl-methyl-siloxane column 13.5 2, 6.7 30–500 0.7, 2.3 30–500 0.6 30–500 N/A N/A N/A 123 GC-MS 1 mL of Quantisal oral fluid SPE BSTFA or MSTFA DB-5ms 7, 8 and 17.5 N/A N/A N/A N/A 10 10–80 (oxycodone only) Meperidine Tramadol 124 LC–MS/MS 1 mL of Quantisal oral fluid SPE N/A Zorbax® Eclipse XDB C18 8 1 1–160 1 1–160 1 1–160 1 1–160 1 1–160 6-Acetylcodeine Dihydrocodeine Norhydrocodone Norcodeine Noroxycodone 125 LC–MS/MS 100 μL of Salivette oral fluid Protein Precipitation N/A Linearly bridged dual columns of Xbridge HILIC and Poroshell 120 EC-C18 10 N/A N/A N/A N/A N/A Tramadol and metabolites 126 LC–MS/MS 500 μL of Quantisal oral fluid SPE N/A Zorbax Eclipse Plus C18 5.5 2.5–1,000 N/A N/A 2.5–1,000 2.5–1,000 Fentanyl Tramadol 127 BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide; GC-MS: gas chromatography–mass spectrometry; GC-MS-PICI: gas chromatography–mass spectrometry–positive-ion chemical ionization; LC–MS/MS: liquid chromatography–tandem mass spectrometry; LC-QTOF-MS: liquid chromatography–quadrupole time-of-flight mass spectrometry; MSTFA: N-methyl-N-(trimethylsilyl)-trifluoroacetamide; SPE: solid-phase extraction. View Large LC–MS/MS analysis of OF methadone and its metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) achieved linear ranges of 1–500 μg/L (128–132), with some separating methadone enantiomers (128, 130, 131). In addition, methadone and EDDP are frequently included in methods with multiple analytes (Table II). Although methadone is basic, with expected high OF/blood ratios, it is highly protein-bound; thus, OF/blood ratios were found to vary from 0.13 to 1.97 (median 0.76) depending upon time after dosing (133). Delays in sample analysis may affect methadone results; in one study, which used the Cozart RapiScan, although methadone OF concentrations were stable for 2 months at 4°C, EDDP concentrations decreased (134). Few methods were published for the detection of buprenorphine and metabolites in OF, possibly due to low prevalence of buprenorphine relative to other opioids at the time earlier methods were published. Nevertheless, newer methods include quantification of buprenorphine and its metabolites by LC–MS/MS with reported linear ranges of 0.1–500 μg/L (9). Buprenorphine markers are typically included in more comprehensive LC–MS/MS methods, especially for pain management testing (Table II). Benzodiazepines Benzodiazepines are highly protein-bound in blood, leading to relatively low OF (0.1–50 μg/L) concentrations (Table VII) (135–140). Published methods involve LC–MS/MS analysis and typically include alprazolam, diazepam, lorazepam, midazolam and nordiazepam, with the possibility of including zolpidem, zopiclone and zaleplon in the same method. Concentration ranges of ~0.5–50 μg/L are achieved. In the few multi-analyte methods that include benzodiazepines, a two-step elution is frequently necessary for their recovery. Table VII Chromatographic Methods for the Detection of Benzodiazepines in Oral Fluid Instrumental Method Sample Volume and Type Sample Prep Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Alprazolam Diazepam Lorazepam Midazolam Nordiazepam Triazolam Other Analytes Reference LC–MS/MS 500 μL of Intercept Liquid extraction XTerra® MS C18 10 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 7-Aminoclonazepam 7-Aminoflunitrazepam Bromazepam Clobazam Lormetazepam Oxazepam Temazepam Tetrazepam Zaleplon Zopiclone Zolpidem 135 LC–MS/MS 500 μL of Salivette oral fluid Liquid Extraction XTerra® RP18 16 0.2, 0.5 0.5–50 0.1, 0.2 0.2–50 0.2, 0.5 0.5–50 0.1, 0.2 0.2–25 N/A 0.1, 0.2 0.2–50 Bromazepam Flunitrazepam Lormetazepam Tetrazepam 136 LC–MS/MS 1 mL of expectorated oral fluid Liquid extraction Luna® C18 8 N/A N/A N/A 0.025, 0.05 0.05–20 N/A N/A 1′-Hydroxymidazolam 4-Hydroxymidazolam 137 LC–MS/MS 1 mL of Quantisal oral fluid SPE Zorbax® Eclipse XDB C18 14.5 0.5 0.5–40 1 1–40 5 5–40 0.5 0.5–40 0.5 0.5–40 0.5 0.5–40 Bromazepam Chlordiazepoxide Clonazepam Flunitrazepam Flurazepam Nitrazepam Oxazepam Temazepam 138 LC–MS/MS 400 μL of Intercept oral fluid SPE Zorbax® Bonus-RP 8 0.02, 0.1 0.1–20 0.05, 0.1 0.1–20 0.05, 0.1 0.1–20 N/A 0.05, 0.1 0.1–20 N/A 7-Aminoclonazepam 7-Aminoflunitrazepam α-Hydroxyalprazolam Chlordiazepoxide Clonazepam Desalkylflurazepam Flunitrazepam Hydroxyethylflurazepam Oxazepam Temazepam 139 LC–MS/MS 1 mL of Quantisal sample Liquid extraction Zorbax® Eclipse XDB C18 15 0.05, 0.1 0.1–5 0.1, 0.1 0.1–5 0.25, 0.5 0.5–5 0.05, 0.1 0.1–5 0.25, 0.25 0.25–5 0.1, 0.1 0.1–5 7-Aminoclonazepam 7-Aminoflunitrazepam 7-Aminonitrazepam Bromazeam Chlordiazepoxide Clobazam Clonazepam Desalkylflurazepam Estazolam Flunitrazepam Flurazepam Hydroxyalprazolam Hydroxymidazolam Hydroxytriazolam Lormetazepam Nitrazepam Oxazepam Prazepam Temazepam Zolpidem 140 Instrumental Method Sample Volume and Type Sample Prep Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Alprazolam Diazepam Lorazepam Midazolam Nordiazepam Triazolam Other Analytes Reference LC–MS/MS 500 μL of Intercept Liquid extraction XTerra® MS C18 10 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 7-Aminoclonazepam 7-Aminoflunitrazepam Bromazepam Clobazam Lormetazepam Oxazepam Temazepam Tetrazepam Zaleplon Zopiclone Zolpidem 135 LC–MS/MS 500 μL of Salivette oral fluid Liquid Extraction XTerra® RP18 16 0.2, 0.5 0.5–50 0.1, 0.2 0.2–50 0.2, 0.5 0.5–50 0.1, 0.2 0.2–25 N/A 0.1, 0.2 0.2–50 Bromazepam Flunitrazepam Lormetazepam Tetrazepam 136 LC–MS/MS 1 mL of expectorated oral fluid Liquid extraction Luna® C18 8 N/A N/A N/A 0.025, 0.05 0.05–20 N/A N/A 1′-Hydroxymidazolam 4-Hydroxymidazolam 137 LC–MS/MS 1 mL of Quantisal oral fluid SPE Zorbax® Eclipse XDB C18 14.5 0.5 0.5–40 1 1–40 5 5–40 0.5 0.5–40 0.5 0.5–40 0.5 0.5–40 Bromazepam Chlordiazepoxide Clonazepam Flunitrazepam Flurazepam Nitrazepam Oxazepam Temazepam 138 LC–MS/MS 400 μL of Intercept oral fluid SPE Zorbax® Bonus-RP 8 0.02, 0.1 0.1–20 0.05, 0.1 0.1–20 0.05, 0.1 0.1–20 N/A 0.05, 0.1 0.1–20 N/A 7-Aminoclonazepam 7-Aminoflunitrazepam α-Hydroxyalprazolam Chlordiazepoxide Clonazepam Desalkylflurazepam Flunitrazepam Hydroxyethylflurazepam Oxazepam Temazepam 139 LC–MS/MS 1 mL of Quantisal sample Liquid extraction Zorbax® Eclipse XDB C18 15 0.05, 0.1 0.1–5 0.1, 0.1 0.1–5 0.25, 0.5 0.5–5 0.05, 0.1 0.1–5 0.25, 0.25 0.25–5 0.1, 0.1 0.1–5 7-Aminoclonazepam 7-Aminoflunitrazepam 7-Aminonitrazepam Bromazeam Chlordiazepoxide Clobazam Clonazepam Desalkylflurazepam Estazolam Flunitrazepam Flurazepam Hydroxyalprazolam Hydroxymidazolam Hydroxytriazolam Lormetazepam Nitrazepam Oxazepam Prazepam Temazepam Zolpidem 140 LC–MS/MS: liquid chromatography–tandem mass spectrometry; SPE: solid-phase extraction. View Large Table VII Chromatographic Methods for the Detection of Benzodiazepines in Oral Fluid Instrumental Method Sample Volume and Type Sample Prep Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Alprazolam Diazepam Lorazepam Midazolam Nordiazepam Triazolam Other Analytes Reference LC–MS/MS 500 μL of Intercept Liquid extraction XTerra® MS C18 10 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 7-Aminoclonazepam 7-Aminoflunitrazepam Bromazepam Clobazam Lormetazepam Oxazepam Temazepam Tetrazepam Zaleplon Zopiclone Zolpidem 135 LC–MS/MS 500 μL of Salivette oral fluid Liquid Extraction XTerra® RP18 16 0.2, 0.5 0.5–50 0.1, 0.2 0.2–50 0.2, 0.5 0.5–50 0.1, 0.2 0.2–25 N/A 0.1, 0.2 0.2–50 Bromazepam Flunitrazepam Lormetazepam Tetrazepam 136 LC–MS/MS 1 mL of expectorated oral fluid Liquid extraction Luna® C18 8 N/A N/A N/A 0.025, 0.05 0.05–20 N/A N/A 1′-Hydroxymidazolam 4-Hydroxymidazolam 137 LC–MS/MS 1 mL of Quantisal oral fluid SPE Zorbax® Eclipse XDB C18 14.5 0.5 0.5–40 1 1–40 5 5–40 0.5 0.5–40 0.5 0.5–40 0.5 0.5–40 Bromazepam Chlordiazepoxide Clonazepam Flunitrazepam Flurazepam Nitrazepam Oxazepam Temazepam 138 LC–MS/MS 400 μL of Intercept oral fluid SPE Zorbax® Bonus-RP 8 0.02, 0.1 0.1–20 0.05, 0.1 0.1–20 0.05, 0.1 0.1–20 N/A 0.05, 0.1 0.1–20 N/A 7-Aminoclonazepam 7-Aminoflunitrazepam α-Hydroxyalprazolam Chlordiazepoxide Clonazepam Desalkylflurazepam Flunitrazepam Hydroxyethylflurazepam Oxazepam Temazepam 139 LC–MS/MS 1 mL of Quantisal sample Liquid extraction Zorbax® Eclipse XDB C18 15 0.05, 0.1 0.1–5 0.1, 0.1 0.1–5 0.25, 0.5 0.5–5 0.05, 0.1 0.1–5 0.25, 0.25 0.25–5 0.1, 0.1 0.1–5 7-Aminoclonazepam 7-Aminoflunitrazepam 7-Aminonitrazepam Bromazeam Chlordiazepoxide Clobazam Clonazepam Desalkylflurazepam Estazolam Flunitrazepam Flurazepam Hydroxyalprazolam Hydroxymidazolam Hydroxytriazolam Lormetazepam Nitrazepam Oxazepam Prazepam Temazepam Zolpidem 140 Instrumental Method Sample Volume and Type Sample Prep Column Run Time (min) Analytes, LOD, LOQ and Linear Range (μg/L) Alprazolam Diazepam Lorazepam Midazolam Nordiazepam Triazolam Other Analytes Reference LC–MS/MS 500 μL of Intercept Liquid extraction XTerra® MS C18 10 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 0.1 0.1–20 7-Aminoclonazepam 7-Aminoflunitrazepam Bromazepam Clobazam Lormetazepam Oxazepam Temazepam Tetrazepam Zaleplon Zopiclone Zolpidem 135 LC–MS/MS 500 μL of Salivette oral fluid Liquid Extraction XTerra® RP18 16 0.2, 0.5 0.5–50 0.1, 0.2 0.2–50 0.2, 0.5 0.5–50 0.1, 0.2 0.2–25 N/A 0.1, 0.2 0.2–50 Bromazepam Flunitrazepam Lormetazepam Tetrazepam 136 LC–MS/MS 1 mL of expectorated oral fluid Liquid extraction Luna® C18 8 N/A N/A N/A 0.025, 0.05 0.05–20 N/A N/A 1′-Hydroxymidazolam 4-Hydroxymidazolam 137 LC–MS/MS 1 mL of Quantisal oral fluid SPE Zorbax® Eclipse XDB C18 14.5 0.5 0.5–40 1 1–40 5 5–40 0.5 0.5–40 0.5 0.5–40 0.5 0.5–40 Bromazepam Chlordiazepoxide Clonazepam Flunitrazepam Flurazepam Nitrazepam Oxazepam Temazepam 138 LC–MS/MS 400 μL of Intercept oral fluid SPE Zorbax® Bonus-RP 8 0.02, 0.1 0.1–20 0.05, 0.1 0.1–20 0.05, 0.1 0.1–20 N/A 0.05, 0.1 0.1–20 N/A 7-Aminoclonazepam 7-Aminoflunitrazepam α-Hydroxyalprazolam Chlordiazepoxide Clonazepam Desalkylflurazepam Flunitrazepam Hydroxyethylflurazepam Oxazepam Temazepam 139 LC–MS/MS 1 mL of Quantisal sample Liquid extraction Zorbax® Eclipse XDB C18 15 0.05, 0.1 0.1–5 0.1, 0.1 0.1–5 0.25, 0.5 0.5–5 0.05, 0.1 0.1–5 0.25, 0.25 0.25–5 0.1, 0.1 0.1–5 7-Aminoclonazepam 7-Aminoflunitrazepam 7-Aminonitrazepam Bromazeam Chlordiazepoxide Clobazam Clonazepam Desalkylflurazepam Estazolam Flunitrazepam Flurazepam Hydroxyalprazolam Hydroxymidazolam Hydroxytriazolam Lormetazepam Nitrazepam Oxazepam Prazepam Temazepam Zolpidem 140 LC–MS/MS: liquid chromatography–tandem mass spectrometry; SPE: solid-phase extraction. View Large Novel psychoactive substances OF analysis offers advantages over testing urine for the presence of NPSs because of the presence of parent drugs. When an NPS is first introduced onto the market, urinary metabolites are typically unknown, making it difficult for urine analysis to identify the drug producing the toxicity. It is critical from a public health perspective to know which drug is responsible for overdoses and deaths, so officials can notify first responders, emergency department personnel and most importantly the public about the new drug. OF may be advantageous in prevalence testing, as parent drugs can be identified, while urine testing may not be feasible until urinary metabolites are identified. Analytical methods require constant updating as NPSs emerge. Several methods, including GC-MS and LC–MS/MS methods, were published for the detection or quantification of NPSs in OF, including synthetic cannabinoids (73, 141–143), synthetic cathinones (143–146), and piperazines (143, 144, 146). Other Considerations Recovery of drugs from collection devices Another consideration in confirmation analysis is that, in addition to variability in collected OF volume and buffer volume, drug recovery differs by collection device. Drugs can adsorb onto the collection device, and good recovery of the drugs from the pad is key to obtaining accurate results. Cannabinoids are particularly susceptible to drug adsorption and, as a result, exhibited poor recovery from first-generation OF collection devices. Early efforts revealed that some low drug recoveries were caused by insufficient time in the buffer to allow drugs to elute off the pad (25). Therefore, it is imperative to allow sufficient time (4–24 h depending upon the manufacturer's recommendation) for the pad to interact with the buffer and allow maximal drug recovery. In general, OF–buffer mixes are shipped overnight to laboratories for analysis, providing sufficient time for maximal recovery from the pad. Table VIII identifies drug recoveries with common OF collection devices. Recoverable volume of OF–buffer mixture may be lower than the total volume (OF volume plus buffer volume), leaving ~1–3 mL of OF–buffer mixture for analysis. Table VIII Recovery of Drugs from Various Oral Fluid Collection Devices Analyte Device Concentration (μg/L) Extraction Technique Recovery (%) Reference Codeine Quantisal 20, 40, 80 Buffer–oral fluid mixture separated with serum separator tube 95.6–104.1 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 99.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 81.3 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 116.0 25 Salivette 10, 25, 100, 200 Centrifugation with tube insert 91.7 24 1,000 Centrifugation with tube insert 39.0 25 Cozart 1,000 N/A 87.1 25 Certus 454 Pad placed in buffer for 24 h at 4°C 118 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 92.5, 93.7 30 Finger Collector 10, 25, 100, 200 Milking 53.3 24 Morphine Quantisal 20, 40, 80 Buffer–oral fluid mixture separated with serum separator tube 91.9–98.7 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 82.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 88.5 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 92.4 25 Salivette 10, 25, 100, 200 Centrifugation with tube insert 93.2 24 50, 250, 400 Centrifugation with tube insert 73.9–78.3 148 1,000 Centrifugation with tube insert 35.2 25 Cozart 1,000 N/A 80.8 25 Certus 454 Pad placed in buffer for 24 h at 4°C 113 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 98.4–98.5 30 Finger Collector 10, 25, 100, 200 Milking 60.9 24 Heroin Salivette 50, 250, 400 Centrifugation with tube insert 79.2–85.2 148 6-Acetylmorphine Quantisal 2, 4, 8 Buffer–oral fluid mixture separated with serum separator tube 92.2–99.5 147 454 Pad placed in buffer for 24 h at 4°C 98 26 Salivette 50, 250, 400 Centrifugation with tube insert 86.9–92.6 148 Certus 454 Pad placed in buffer for 24 h at 4°C 112 26 DCD 5000 20, 50 Placed in isopropanol for 1 h and centrifuged 92.3–96.3 30 Oxycodone Quantisal 10 Buffer–oral fluid mixture extracted from pad the next day with serum separator 96 149 Cocaine Quantisal 10, 20, 40 Buffer–oral fluid mixture separated with serum separator tube 91.2–95.7 147 454 Pad placed in buffer for 24 h at 4°C 97 26 1,000 N/A 81.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad using filter 85.6 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 96.9 25 Salivette 50, 250, 400 Centrifugation with tube insert 81.7–91.4 148 1,000 Centrifugation with tube insert 33.3 25 Cozart 1,000 N/A 76.3 25 Certus 454 Pad placed in buffer for 24 h at 4°C 106 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 95.2–95.6 30 Benzoylecgonine Quantisal 10, 20, 40 Buffer–oral fluid mixture separated with serum separator tube 82.7–91.2 147 454 Pad placed in buffer for 24 h at 4°C 88 26 Salivette 50, 250, 400 Centrifugation with tube insert 90.7–98.2 148 Certus 454 Pad placed in buffer for 24 h at 4°C 107 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 86.8–92.2 30 Amphetamine Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 94.2–96.9 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 89.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 88.7 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 103.1 25 Salivette 1,000 Centrifugation with tube insert 51.8 25 Cozart 1,000 N/A 75.4 25 Certus 454 Pad placed in buffer for 24 h at 4°C 78 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 92.1–92.3 30 MDMA Quantisal 1,000 N/A 82.3 25 454 Pad placed in buffer for 24 h at 4°C 98 26 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 86.3 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 101.1 25 Salivette 1,000 Centrifugation with tube insert 26.5 25 Cozart 1,000 N/A 76.0 25 Certus 454 Pad placed in buffer for 24 h at 4°C 102 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 89.9–93.9 30 Methamphetamine Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 93.1–103.8 147 454 Pad placed in buffer for 24 h at 4°C 100 26 Cozart 1,5,10 Eluted with proprietary buffer 96 150 Certus 454 Pad placed in buffer for 24 h at 4°C 102 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 91.1–92.5 30 THC Quantisal 2, 4, 8 Buffer–oral fluid mixture separated with serum separator tube 81.3–91.4 147 454 Pad placed in buffer for 24 h at 4°C 94 26 1,000 N/A 55.8 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 85.4 25 1.5, 7.5, 30 Buffer–oral fluid mixture extracted from pad using a filter 65.5–68.1 32 Oral-Eze 1.5, 7.5, 30 Buffer–oral fluid mixture extracted from pad using a filter 42.5–48.8 151 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 37.6 25 10, 100 Centrifuged to recover buffer–oral fluid mixture 31.2–57.2 29 10, 100 Centrifuge, add 2 mL methanol to stabilization buffer and pad, incubate and shake 15 min, centrifuge Additional 19.2–34.4 29 Salivette 1,000 Centrifugation with tube insert <12.5 25 Cozart 200, 500, 1,000 Elute with proprietary buffer 96 150 1,000 N/A 75.9 25 Certus 454 Pad placed in buffer for 24 h at 4°C 54 26 DCD 5000 20, 50 Placed in isopropanol for 1 h and centrifuged 89.8–93.9 30 Diazepam Quantisal 1,000 N/A 81.1 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 87.4 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 88.9 25 Salivette 1,000 Centrifugation with tube insert 15.9 25 Cozart 1,000 N/A 91.6 25 Alprazolam Quantisal 1,000 N/A 111.0 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 91.1 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 91.2 25 Salivette 1,000 Centrifugation with tube insert 27.3 25 Cozart 1,000 N/A 66.0 25 Oxazepam Quantisal 10, 20, 50 Buffer–oral fluid mixture separated with serum separator tube 97.9–109.4 147 Methadone Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 99.7–106.5 147 454 Pad placed in buffer for 24 h at 4°C 98 26 Certus 454 Pad placed in buffer for 24 h at 4°C 84 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 96.1–98.1 30 Buprenorphine Quantisal 454 Pad placed in buffer for 24 h at 4°C 97 26 Certus 454 Pad placed in buffer for 24 h at 4°C 84 26 DCD 5000 10, 20 Placed in isopropanol for 1 h and centrifuged 94.7–103 30 Analyte Device Concentration (μg/L) Extraction Technique Recovery (%) Reference Codeine Quantisal 20, 40, 80 Buffer–oral fluid mixture separated with serum separator tube 95.6–104.1 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 99.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 81.3 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 116.0 25 Salivette 10, 25, 100, 200 Centrifugation with tube insert 91.7 24 1,000 Centrifugation with tube insert 39.0 25 Cozart 1,000 N/A 87.1 25 Certus 454 Pad placed in buffer for 24 h at 4°C 118 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 92.5, 93.7 30 Finger Collector 10, 25, 100, 200 Milking 53.3 24 Morphine Quantisal 20, 40, 80 Buffer–oral fluid mixture separated with serum separator tube 91.9–98.7 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 82.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 88.5 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 92.4 25 Salivette 10, 25, 100, 200 Centrifugation with tube insert 93.2 24 50, 250, 400 Centrifugation with tube insert 73.9–78.3 148 1,000 Centrifugation with tube insert 35.2 25 Cozart 1,000 N/A 80.8 25 Certus 454 Pad placed in buffer for 24 h at 4°C 113 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 98.4–98.5 30 Finger Collector 10, 25, 100, 200 Milking 60.9 24 Heroin Salivette 50, 250, 400 Centrifugation with tube insert 79.2–85.2 148 6-Acetylmorphine Quantisal 2, 4, 8 Buffer–oral fluid mixture separated with serum separator tube 92.2–99.5 147 454 Pad placed in buffer for 24 h at 4°C 98 26 Salivette 50, 250, 400 Centrifugation with tube insert 86.9–92.6 148 Certus 454 Pad placed in buffer for 24 h at 4°C 112 26 DCD 5000 20, 50 Placed in isopropanol for 1 h and centrifuged 92.3–96.3 30 Oxycodone Quantisal 10 Buffer–oral fluid mixture extracted from pad the next day with serum separator 96 149 Cocaine Quantisal 10, 20, 40 Buffer–oral fluid mixture separated with serum separator tube 91.2–95.7 147 454 Pad placed in buffer for 24 h at 4°C 97 26 1,000 N/A 81.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad using filter 85.6 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 96.9 25 Salivette 50, 250, 400 Centrifugation with tube insert 81.7–91.4 148 1,000 Centrifugation with tube insert 33.3 25 Cozart 1,000 N/A 76.3 25 Certus 454 Pad placed in buffer for 24 h at 4°C 106 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 95.2–95.6 30 Benzoylecgonine Quantisal 10, 20, 40 Buffer–oral fluid mixture separated with serum separator tube 82.7–91.2 147 454 Pad placed in buffer for 24 h at 4°C 88 26 Salivette 50, 250, 400 Centrifugation with tube insert 90.7–98.2 148 Certus 454 Pad placed in buffer for 24 h at 4°C 107 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 86.8–92.2 30 Amphetamine Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 94.2–96.9 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 89.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 88.7 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 103.1 25 Salivette 1,000 Centrifugation with tube insert 51.8 25 Cozart 1,000 N/A 75.4 25 Certus 454 Pad placed in buffer for 24 h at 4°C 78 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 92.1–92.3 30 MDMA Quantisal 1,000 N/A 82.3 25 454 Pad placed in buffer for 24 h at 4°C 98 26 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 86.3 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 101.1 25 Salivette 1,000 Centrifugation with tube insert 26.5 25 Cozart 1,000 N/A 76.0 25 Certus 454 Pad placed in buffer for 24 h at 4°C 102 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 89.9–93.9 30 Methamphetamine Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 93.1–103.8 147 454 Pad placed in buffer for 24 h at 4°C 100 26 Cozart 1,5,10 Eluted with proprietary buffer 96 150 Certus 454 Pad placed in buffer for 24 h at 4°C 102 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 91.1–92.5 30 THC Quantisal 2, 4, 8 Buffer–oral fluid mixture separated with serum separator tube 81.3–91.4 147 454 Pad placed in buffer for 24 h at 4°C 94 26 1,000 N/A 55.8 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 85.4 25 1.5, 7.5, 30 Buffer–oral fluid mixture extracted from pad using a filter 65.5–68.1 32 Oral-Eze 1.5, 7.5, 30 Buffer–oral fluid mixture extracted from pad using a filter 42.5–48.8 151 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 37.6 25 10, 100 Centrifuged to recover buffer–oral fluid mixture 31.2–57.2 29 10, 100 Centrifuge, add 2 mL methanol to stabilization buffer and pad, incubate and shake 15 min, centrifuge Additional 19.2–34.4 29 Salivette 1,000 Centrifugation with tube insert <12.5 25 Cozart 200, 500, 1,000 Elute with proprietary buffer 96 150 1,000 N/A 75.9 25 Certus 454 Pad placed in buffer for 24 h at 4°C 54 26 DCD 5000 20, 50 Placed in isopropanol for 1 h and centrifuged 89.8–93.9 30 Diazepam Quantisal 1,000 N/A 81.1 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 87.4 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 88.9 25 Salivette 1,000 Centrifugation with tube insert 15.9 25 Cozart 1,000 N/A 91.6 25 Alprazolam Quantisal 1,000 N/A 111.0 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 91.1 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 91.2 25 Salivette 1,000 Centrifugation with tube insert 27.3 25 Cozart 1,000 N/A 66.0 25 Oxazepam Quantisal 10, 20, 50 Buffer–oral fluid mixture separated with serum separator tube 97.9–109.4 147 Methadone Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 99.7–106.5 147 454 Pad placed in buffer for 24 h at 4°C 98 26 Certus 454 Pad placed in buffer for 24 h at 4°C 84 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 96.1–98.1 30 Buprenorphine Quantisal 454 Pad placed in buffer for 24 h at 4°C 97 26 Certus 454 Pad placed in buffer for 24 h at 4°C 84 26 DCD 5000 10, 20 Placed in isopropanol for 1 h and centrifuged 94.7–103 30 View Large Table VIII Recovery of Drugs from Various Oral Fluid Collection Devices Analyte Device Concentration (μg/L) Extraction Technique Recovery (%) Reference Codeine Quantisal 20, 40, 80 Buffer–oral fluid mixture separated with serum separator tube 95.6–104.1 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 99.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 81.3 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 116.0 25 Salivette 10, 25, 100, 200 Centrifugation with tube insert 91.7 24 1,000 Centrifugation with tube insert 39.0 25 Cozart 1,000 N/A 87.1 25 Certus 454 Pad placed in buffer for 24 h at 4°C 118 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 92.5, 93.7 30 Finger Collector 10, 25, 100, 200 Milking 53.3 24 Morphine Quantisal 20, 40, 80 Buffer–oral fluid mixture separated with serum separator tube 91.9–98.7 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 82.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 88.5 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 92.4 25 Salivette 10, 25, 100, 200 Centrifugation with tube insert 93.2 24 50, 250, 400 Centrifugation with tube insert 73.9–78.3 148 1,000 Centrifugation with tube insert 35.2 25 Cozart 1,000 N/A 80.8 25 Certus 454 Pad placed in buffer for 24 h at 4°C 113 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 98.4–98.5 30 Finger Collector 10, 25, 100, 200 Milking 60.9 24 Heroin Salivette 50, 250, 400 Centrifugation with tube insert 79.2–85.2 148 6-Acetylmorphine Quantisal 2, 4, 8 Buffer–oral fluid mixture separated with serum separator tube 92.2–99.5 147 454 Pad placed in buffer for 24 h at 4°C 98 26 Salivette 50, 250, 400 Centrifugation with tube insert 86.9–92.6 148 Certus 454 Pad placed in buffer for 24 h at 4°C 112 26 DCD 5000 20, 50 Placed in isopropanol for 1 h and centrifuged 92.3–96.3 30 Oxycodone Quantisal 10 Buffer–oral fluid mixture extracted from pad the next day with serum separator 96 149 Cocaine Quantisal 10, 20, 40 Buffer–oral fluid mixture separated with serum separator tube 91.2–95.7 147 454 Pad placed in buffer for 24 h at 4°C 97 26 1,000 N/A 81.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad using filter 85.6 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 96.9 25 Salivette 50, 250, 400 Centrifugation with tube insert 81.7–91.4 148 1,000 Centrifugation with tube insert 33.3 25 Cozart 1,000 N/A 76.3 25 Certus 454 Pad placed in buffer for 24 h at 4°C 106 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 95.2–95.6 30 Benzoylecgonine Quantisal 10, 20, 40 Buffer–oral fluid mixture separated with serum separator tube 82.7–91.2 147 454 Pad placed in buffer for 24 h at 4°C 88 26 Salivette 50, 250, 400 Centrifugation with tube insert 90.7–98.2 148 Certus 454 Pad placed in buffer for 24 h at 4°C 107 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 86.8–92.2 30 Amphetamine Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 94.2–96.9 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 89.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 88.7 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 103.1 25 Salivette 1,000 Centrifugation with tube insert 51.8 25 Cozart 1,000 N/A 75.4 25 Certus 454 Pad placed in buffer for 24 h at 4°C 78 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 92.1–92.3 30 MDMA Quantisal 1,000 N/A 82.3 25 454 Pad placed in buffer for 24 h at 4°C 98 26 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 86.3 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 101.1 25 Salivette 1,000 Centrifugation with tube insert 26.5 25 Cozart 1,000 N/A 76.0 25 Certus 454 Pad placed in buffer for 24 h at 4°C 102 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 89.9–93.9 30 Methamphetamine Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 93.1–103.8 147 454 Pad placed in buffer for 24 h at 4°C 100 26 Cozart 1,5,10 Eluted with proprietary buffer 96 150 Certus 454 Pad placed in buffer for 24 h at 4°C 102 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 91.1–92.5 30 THC Quantisal 2, 4, 8 Buffer–oral fluid mixture separated with serum separator tube 81.3–91.4 147 454 Pad placed in buffer for 24 h at 4°C 94 26 1,000 N/A 55.8 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 85.4 25 1.5, 7.5, 30 Buffer–oral fluid mixture extracted from pad using a filter 65.5–68.1 32 Oral-Eze 1.5, 7.5, 30 Buffer–oral fluid mixture extracted from pad using a filter 42.5–48.8 151 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 37.6 25 10, 100 Centrifuged to recover buffer–oral fluid mixture 31.2–57.2 29 10, 100 Centrifuge, add 2 mL methanol to stabilization buffer and pad, incubate and shake 15 min, centrifuge Additional 19.2–34.4 29 Salivette 1,000 Centrifugation with tube insert <12.5 25 Cozart 200, 500, 1,000 Elute with proprietary buffer 96 150 1,000 N/A 75.9 25 Certus 454 Pad placed in buffer for 24 h at 4°C 54 26 DCD 5000 20, 50 Placed in isopropanol for 1 h and centrifuged 89.8–93.9 30 Diazepam Quantisal 1,000 N/A 81.1 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 87.4 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 88.9 25 Salivette 1,000 Centrifugation with tube insert 15.9 25 Cozart 1,000 N/A 91.6 25 Alprazolam Quantisal 1,000 N/A 111.0 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 91.1 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 91.2 25 Salivette 1,000 Centrifugation with tube insert 27.3 25 Cozart 1,000 N/A 66.0 25 Oxazepam Quantisal 10, 20, 50 Buffer–oral fluid mixture separated with serum separator tube 97.9–109.4 147 Methadone Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 99.7–106.5 147 454 Pad placed in buffer for 24 h at 4°C 98 26 Certus 454 Pad placed in buffer for 24 h at 4°C 84 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 96.1–98.1 30 Buprenorphine Quantisal 454 Pad placed in buffer for 24 h at 4°C 97 26 Certus 454 Pad placed in buffer for 24 h at 4°C 84 26 DCD 5000 10, 20 Placed in isopropanol for 1 h and centrifuged 94.7–103 30 Analyte Device Concentration (μg/L) Extraction Technique Recovery (%) Reference Codeine Quantisal 20, 40, 80 Buffer–oral fluid mixture separated with serum separator tube 95.6–104.1 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 99.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 81.3 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 116.0 25 Salivette 10, 25, 100, 200 Centrifugation with tube insert 91.7 24 1,000 Centrifugation with tube insert 39.0 25 Cozart 1,000 N/A 87.1 25 Certus 454 Pad placed in buffer for 24 h at 4°C 118 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 92.5, 93.7 30 Finger Collector 10, 25, 100, 200 Milking 53.3 24 Morphine Quantisal 20, 40, 80 Buffer–oral fluid mixture separated with serum separator tube 91.9–98.7 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 82.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 88.5 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 92.4 25 Salivette 10, 25, 100, 200 Centrifugation with tube insert 93.2 24 50, 250, 400 Centrifugation with tube insert 73.9–78.3 148 1,000 Centrifugation with tube insert 35.2 25 Cozart 1,000 N/A 80.8 25 Certus 454 Pad placed in buffer for 24 h at 4°C 113 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 98.4–98.5 30 Finger Collector 10, 25, 100, 200 Milking 60.9 24 Heroin Salivette 50, 250, 400 Centrifugation with tube insert 79.2–85.2 148 6-Acetylmorphine Quantisal 2, 4, 8 Buffer–oral fluid mixture separated with serum separator tube 92.2–99.5 147 454 Pad placed in buffer for 24 h at 4°C 98 26 Salivette 50, 250, 400 Centrifugation with tube insert 86.9–92.6 148 Certus 454 Pad placed in buffer for 24 h at 4°C 112 26 DCD 5000 20, 50 Placed in isopropanol for 1 h and centrifuged 92.3–96.3 30 Oxycodone Quantisal 10 Buffer–oral fluid mixture extracted from pad the next day with serum separator 96 149 Cocaine Quantisal 10, 20, 40 Buffer–oral fluid mixture separated with serum separator tube 91.2–95.7 147 454 Pad placed in buffer for 24 h at 4°C 97 26 1,000 N/A 81.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad using filter 85.6 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 96.9 25 Salivette 50, 250, 400 Centrifugation with tube insert 81.7–91.4 148 1,000 Centrifugation with tube insert 33.3 25 Cozart 1,000 N/A 76.3 25 Certus 454 Pad placed in buffer for 24 h at 4°C 106 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 95.2–95.6 30 Benzoylecgonine Quantisal 10, 20, 40 Buffer–oral fluid mixture separated with serum separator tube 82.7–91.2 147 454 Pad placed in buffer for 24 h at 4°C 88 26 Salivette 50, 250, 400 Centrifugation with tube insert 90.7–98.2 148 Certus 454 Pad placed in buffer for 24 h at 4°C 107 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 86.8–92.2 30 Amphetamine Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 94.2–96.9 147 454 Pad placed in buffer for 24 h at 4°C 98 26 1,000 N/A 89.7 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 88.7 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 103.1 25 Salivette 1,000 Centrifugation with tube insert 51.8 25 Cozart 1,000 N/A 75.4 25 Certus 454 Pad placed in buffer for 24 h at 4°C 78 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 92.1–92.3 30 MDMA Quantisal 1,000 N/A 82.3 25 454 Pad placed in buffer for 24 h at 4°C 98 26 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 86.3 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 101.1 25 Salivette 1,000 Centrifugation with tube insert 26.5 25 Cozart 1,000 N/A 76.0 25 Certus 454 Pad placed in buffer for 24 h at 4°C 102 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 89.9–93.9 30 Methamphetamine Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 93.1–103.8 147 454 Pad placed in buffer for 24 h at 4°C 100 26 Cozart 1,5,10 Eluted with proprietary buffer 96 150 Certus 454 Pad placed in buffer for 24 h at 4°C 102 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 91.1–92.5 30 THC Quantisal 2, 4, 8 Buffer–oral fluid mixture separated with serum separator tube 81.3–91.4 147 454 Pad placed in buffer for 24 h at 4°C 94 26 1,000 N/A 55.8 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 85.4 25 1.5, 7.5, 30 Buffer–oral fluid mixture extracted from pad using a filter 65.5–68.1 32 Oral-Eze 1.5, 7.5, 30 Buffer–oral fluid mixture extracted from pad using a filter 42.5–48.8 151 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 37.6 25 10, 100 Centrifuged to recover buffer–oral fluid mixture 31.2–57.2 29 10, 100 Centrifuge, add 2 mL methanol to stabilization buffer and pad, incubate and shake 15 min, centrifuge Additional 19.2–34.4 29 Salivette 1,000 Centrifugation with tube insert <12.5 25 Cozart 200, 500, 1,000 Elute with proprietary buffer 96 150 1,000 N/A 75.9 25 Certus 454 Pad placed in buffer for 24 h at 4°C 54 26 DCD 5000 20, 50 Placed in isopropanol for 1 h and centrifuged 89.8–93.9 30 Diazepam Quantisal 1,000 N/A 81.1 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 87.4 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 88.9 25 Salivette 1,000 Centrifugation with tube insert 15.9 25 Cozart 1,000 N/A 91.6 25 Alprazolam Quantisal 1,000 N/A 111.0 25 StatSure 1,000 Buffer–oral fluid mixture extracted from pad with filter 91.1 25 Intercept 1,000 Centrifuged to recover buffer–oral fluid mixture 91.2 25 Salivette 1,000 Centrifugation with tube insert 27.3 25 Cozart 1,000 N/A 66.0 25 Oxazepam Quantisal 10, 20, 50 Buffer–oral fluid mixture separated with serum separator tube 97.9–109.4 147 Methadone Quantisal 25, 50, 100 Buffer–oral fluid mixture separated with serum separator tube 99.7–106.5 147 454 Pad placed in buffer for 24 h at 4°C 98 26 Certus 454 Pad placed in buffer for 24 h at 4°C 84 26 DCD 5000 25, 50 Placed in isopropanol for 1 h and centrifuged 96.1–98.1 30 Buprenorphine Quantisal 454 Pad placed in buffer for 24 h at 4°C 97 26 Certus 454 Pad placed in buffer for 24 h at 4°C 84 26 DCD 5000 10, 20 Placed in isopropanol for 1 h and centrifuged 94.7–103 30 View Large Agency cutoff concentrations Various agencies proposed or standardized cutoffs for various drug-testing programs, including impaired driving drug testing and workplace drug testing. In 2004, the SAMHSA proposed the Mandatory Guidelines for Federal Workplace Drug Testing Programs (Table IX) (152). However, lack of resolution of important scientific questions delayed final approval. In 2015, revised guidelines were proposed (Table IX) (36). As of April 2019, the final SAMHSA guidelines are still not approved. The guidelines indicate that for initial immunoassay tests that have multiple target analytes (e.g., cocaine/BE), the assay should be calibrated with one of the targets and demonstrate >80% cross-reactivity with the other analyte(s). The revised SAMHSA guidelines also include new required analytes (hydrocodone, hydromorphone, oxycodone and oxymorphone) and lowered cutoffs for some analytes. Table IX Screening and Confirmation Cutoffs for Various Agencies Initial (Screening) Test Analyte Initial (Screening) Test Cutoff (μg/L Confirmatory Test Analyte Confirmatory Test Cutoff Concentration (μg/L) 2004 SAMHSA Guidelines 2015 SAMHSA Guidelines EWDTS Guidelines Australian Standard Canadian Drug Testing Committee Standards 2004 SAMHSA Guidelines 2015 SAMHSA Guidelines EWDTS Guidelines DRUID Recommendations Talloires Recommendations Cannabis (THC) 4 4 10 25 25 THC 2 2 2 1 2 Cocaine/benzoylecgonine 20 15 30 50 50 (cocaine) Cocaine 8 8 8 10 10 Benzoylecgonine 8 8 8 10 10 Codeine/morphine 40 30 40 (morphine) 50 (opiates) Codeine 40 15 15 20 20 Morphine 40 15 15 20 20 Norcodeine 2 6-Acetylcodeine 2 Dihydrocodeine 15 Hydrocodone/hydromorphone 30 Hydrocodone N/A 15 Hydromorphone N/A 15 Oxycodone/oxymorphone 30 Oxycodone N/A 15 Oxymorphone N/A 15 6-Acetylmorphine 3 4 6-Acetylmorphine N/A 2 2 5 5 l-Methadone 50 Methadone or metabolites (d + l) EDDP 20 20 20 Buprenorphine 5 Buprenorphine or metabolites 1 Propoxyphene or metabolites 40 Propoxyphene or metabolites 5 Tramadol 50 Phencyclidine 10 3 Phencyclidine 10 2 Amphetamine/methamphetamine 50 25 40 50 50 (meth) Amphetamine 50 15 15 (d + l) 25 20 Methamphetamine 50 15 15 25 20 MDMA/MDA/MDEA 50 25 MDMA 50 15 15 25 20 MDA 50 15 15 25 20 MDEA 50 15 25 20 Benzodiazepines 10 7-Aminoflunitrazepam 3 1 7-Aminoclonazepam 3 1 7-Aminonitrazepam 3 Alprazolam 3 1 Bromazepam 3 Clonazepam 3 1 Desmethyldiazepam/nordiazepam 3 1 Diazepam 3 5 Flunitrazepam 3 2 Flurazepam 3 Lorazepam 3 1 Lormetazepam 3 Midazolam 3 Nitrazepam 3 Oxazepam 3 5 Phenazepam 3 Temazepam 10 Zolpidem 10 Zopiclone 10 Initial (Screening) Test Analyte Initial (Screening) Test Cutoff (μg/L Confirmatory Test Analyte Confirmatory Test Cutoff Concentration (μg/L) 2004 SAMHSA Guidelines 2015 SAMHSA Guidelines EWDTS Guidelines Australian Standard Canadian Drug Testing Committee Standards 2004 SAMHSA Guidelines 2015 SAMHSA Guidelines EWDTS Guidelines DRUID Recommendations Talloires Recommendations Cannabis (THC) 4 4 10 25 25 THC 2 2 2 1 2 Cocaine/benzoylecgonine 20 15 30 50 50 (cocaine) Cocaine 8 8 8 10 10 Benzoylecgonine 8 8 8 10 10 Codeine/morphine 40 30 40 (morphine) 50 (opiates) Codeine 40 15 15 20 20 Morphine 40 15 15 20 20 Norcodeine 2 6-Acetylcodeine 2 Dihydrocodeine 15 Hydrocodone/hydromorphone 30 Hydrocodone N/A 15 Hydromorphone N/A 15 Oxycodone/oxymorphone 30 Oxycodone N/A 15 Oxymorphone N/A 15 6-Acetylmorphine 3 4 6-Acetylmorphine N/A 2 2 5 5 l-Methadone 50 Methadone or metabolites (d + l) EDDP 20 20 20 Buprenorphine 5 Buprenorphine or metabolites 1 Propoxyphene or metabolites 40 Propoxyphene or metabolites 5 Tramadol 50 Phencyclidine 10 3 Phencyclidine 10 2 Amphetamine/methamphetamine 50 25 40 50 50 (meth) Amphetamine 50 15 15 (d + l) 25 20 Methamphetamine 50 15 15 25 20 MDMA/MDA/MDEA 50 25 MDMA 50 15 15 25 20 MDA 50 15 15 25 20 MDEA 50 15 25 20 Benzodiazepines 10 7-Aminoflunitrazepam 3 1 7-Aminoclonazepam 3 1 7-Aminonitrazepam 3 Alprazolam 3 1 Bromazepam 3 Clonazepam 3 1 Desmethyldiazepam/nordiazepam 3 1 Diazepam 3 5 Flunitrazepam 3 2 Flurazepam 3 Lorazepam 3 1 Lormetazepam 3 Midazolam 3 Nitrazepam 3 Oxazepam 3 5 Phenazepam 3 Temazepam 10 Zolpidem 10 Zopiclone 10 View Large Table IX Screening and Confirmation Cutoffs for Various Agencies Initial (Screening) Test Analyte Initial (Screening) Test Cutoff (μg/L Confirmatory Test Analyte Confirmatory Test Cutoff Concentration (μg/L) 2004 SAMHSA Guidelines 2015 SAMHSA Guidelines EWDTS Guidelines Australian Standard Canadian Drug Testing Committee Standards 2004 SAMHSA Guidelines 2015 SAMHSA Guidelines EWDTS Guidelines DRUID Recommendations Talloires Recommendations Cannabis (THC) 4 4 10 25 25 THC 2 2 2 1 2 Cocaine/benzoylecgonine 20 15 30 50 50 (cocaine) Cocaine 8 8 8 10 10 Benzoylecgonine 8 8 8 10 10 Codeine/morphine 40 30 40 (morphine) 50 (opiates) Codeine 40 15 15 20 20 Morphine 40 15 15 20 20 Norcodeine 2 6-Acetylcodeine 2 Dihydrocodeine 15 Hydrocodone/hydromorphone 30 Hydrocodone N/A 15 Hydromorphone N/A 15 Oxycodone/oxymorphone 30 Oxycodone N/A 15 Oxymorphone N/A 15 6-Acetylmorphine 3 4 6-Acetylmorphine N/A 2 2 5 5 l-Methadone 50 Methadone or metabolites (d + l) EDDP 20 20 20 Buprenorphine 5 Buprenorphine or metabolites 1 Propoxyphene or metabolites 40 Propoxyphene or metabolites 5 Tramadol 50 Phencyclidine 10 3 Phencyclidine 10 2 Amphetamine/methamphetamine 50 25 40 50 50 (meth) Amphetamine 50 15 15 (d + l) 25 20 Methamphetamine 50 15 15 25 20 MDMA/MDA/MDEA 50 25 MDMA 50 15 15 25 20 MDA 50 15 15 25 20 MDEA 50 15 25 20 Benzodiazepines 10 7-Aminoflunitrazepam 3 1 7-Aminoclonazepam 3 1 7-Aminonitrazepam 3 Alprazolam 3 1 Bromazepam 3 Clonazepam 3 1 Desmethyldiazepam/nordiazepam 3 1 Diazepam 3 5 Flunitrazepam 3 2 Flurazepam 3 Lorazepam 3 1 Lormetazepam 3 Midazolam 3 Nitrazepam 3 Oxazepam 3 5 Phenazepam 3 Temazepam 10 Zolpidem 10 Zopiclone 10 Initial (Screening) Test Analyte Initial (Screening) Test Cutoff (μg/L Confirmatory Test Analyte Confirmatory Test Cutoff Concentration (μg/L) 2004 SAMHSA Guidelines 2015 SAMHSA Guidelines EWDTS Guidelines Australian Standard Canadian Drug Testing Committee Standards 2004 SAMHSA Guidelines 2015 SAMHSA Guidelines EWDTS Guidelines DRUID Recommendations Talloires Recommendations Cannabis (THC) 4 4 10 25 25 THC 2 2 2 1 2 Cocaine/benzoylecgonine 20 15 30 50 50 (cocaine) Cocaine 8 8 8 10 10 Benzoylecgonine 8 8 8 10 10 Codeine/morphine 40 30 40 (morphine) 50 (opiates) Codeine 40 15 15 20 20 Morphine 40 15 15 20 20 Norcodeine 2 6-Acetylcodeine 2 Dihydrocodeine 15 Hydrocodone/hydromorphone 30 Hydrocodone N/A 15 Hydromorphone N/A 15 Oxycodone/oxymorphone 30 Oxycodone N/A 15 Oxymorphone N/A 15 6-Acetylmorphine 3 4 6-Acetylmorphine N/A 2 2 5 5 l-Methadone 50 Methadone or metabolites (d + l) EDDP 20 20 20 Buprenorphine 5 Buprenorphine or metabolites 1 Propoxyphene or metabolites 40 Propoxyphene or metabolites 5 Tramadol 50 Phencyclidine 10 3 Phencyclidine 10 2 Amphetamine/methamphetamine 50 25 40 50 50 (meth) Amphetamine 50 15 15 (d + l) 25 20 Methamphetamine 50 15 15 25 20 MDMA/MDA/MDEA 50 25 MDMA 50 15 15 25 20 MDA 50 15 15 25 20 MDEA 50 15 25 20 Benzodiazepines 10 7-Aminoflunitrazepam 3 1 7-Aminoclonazepam 3 1 7-Aminonitrazepam 3 Alprazolam 3 1 Bromazepam 3 Clonazepam 3 1 Desmethyldiazepam/nordiazepam 3 1 Diazepam 3 5 Flunitrazepam 3 2 Flurazepam 3 Lorazepam 3 1 Lormetazepam 3 Midazolam 3 Nitrazepam 3 Oxazepam 3 5 Phenazepam 3 Temazepam 10 Zolpidem 10 Zopiclone 10 View Large In Europe, the EWDTS published the `European Guidelines for Workplace in Oral Fluid’ recommending maximum cutoffs for screening and confirmation tests (Table IX) (35). Other EWDTS screening and confirmation cutoffs are proposed for barbiturates (screen only), ketamine, lysergic acid diethylamide or metabolites, other opioids (oxycodone, hydromorphone, tramadol, tilidine, and fentanyl), phencyclidine, pregabalin, synthetic cannabinoids, synthetic cathinones (3,4-methylenedioxypyrovalerone (MDPV), etc.) and Z-drugs (zopiclone, zolpidem and zaleplon) (35). Separate guidelines exist for OF testing for DUID programs, such as Driving Under the Influence of Drugs, Alcohol and Medicines (DRUID), Talloires, Australian Standards and Canada's Drugs and Driving Committee (Table IX) (153–156). Authentic vs. synthetic oral fluid for calibrators and controls Another important consideration when analyzing OF is the matrix for calibrators and controls. Synthetic blank OF is available for calibrator and control preparation. However, due to potential differences in extraction efficiency and matrix effects, authentic OF obtained by passive drool or expectoration, centrifugation and dilution with buffer is often the preferred matrix for calibrator and control preparation. If it is not possible to prepare calibrators and controls in this manner, laboratories should validate performance of synthetic OF compared with human OF to ensure that calibrators and controls prepared in synthetic OF do not differ significantly from human OF, as differences were previously noted (68). As human OF can be variable, several sources of OF should be compared. Interpretation Drug OF disposition data are improving test interpretation by toxicologists and medical review officers. The prevalence of drugs in different populations was reported for workplace testing (157), pain management monitoring (158–160), substance abuse treatment (148, 161, 162) and drugged drivers (37, 150, 163–166). Good agreement (>85–90%) was frequently shown between matrices, especially correlations between OF and plasma or blood concentrations. Although a significant correlation between OF and blood was frequently demonstrated, high intra-subject and inter-subject variability precludes prediction of simultaneously collected blood concentrations from those in OF. When interpreting and comparing OF drug concentrations, it is crucial to consider collection method. Earlier published concentrations often utilized citric acid-stimulated collection methods that yielded lower concentrations than newer non-stimulated collection methods. Therefore, concentrations reported in a non-stimulated study may not be in the same range as another study following the same dose but with a different collection method that had a stimulated OF collection. Comparison with other biological matrices When test results from different matrices are available, there may be legitimate reasons that drug test results differ. Each biological specimen has unique physiological and chemical properties that may alter drug disposition. Renal excretion favors water-soluble metabolite elimination, whereas OF excretion favors parent drugs capable of rapid passive diffusion across membranes. OF's acidic nature favors ion trapping of drugs containing basic nitrogen moieties (14, 15). Residence times in each matrix also differ substantially, yielding wide variability in detection windows. Disparate specimen results and possible explanations are included in Table X. Table X Disparate Results from Testing Two Different Biological Matrices and Possible Explanations Scenario Matrix Possible Explanations for Disparate Results Blood Urine Oral Fluid 1 Positive Negative Highly protein-bound drugs may be poorly distributed to oral fluid, e.g., benzodiazepines; highly polar compounds and low lipophilicity reducing transfer from blood to oral fluid 2 Positive Negative Long interval after dosing; concentration effect by kidney; highly protein-bound drug; sampling time outside oral fluid detection `window’ 3 Negative Positive Insufficient time for drug absorption; `depot’ effect, ion trapping 4 Negative Positive Insufficient time for drug absorption, metabolism and excretion; `depot’ effect Scenario Matrix Possible Explanations for Disparate Results Blood Urine Oral Fluid 1 Positive Negative Highly protein-bound drugs may be poorly distributed to oral fluid, e.g., benzodiazepines; highly polar compounds and low lipophilicity reducing transfer from blood to oral fluid 2 Positive Negative Long interval after dosing; concentration effect by kidney; highly protein-bound drug; sampling time outside oral fluid detection `window’ 3 Negative Positive Insufficient time for drug absorption; `depot’ effect, ion trapping 4 Negative Positive Insufficient time for drug absorption, metabolism and excretion; `depot’ effect View Large Table X Disparate Results from Testing Two Different Biological Matrices and Possible Explanations Scenario Matrix Possible Explanations for Disparate Results Blood Urine Oral Fluid 1 Positive Negative Highly protein-bound drugs may be poorly distributed to oral fluid, e.g., benzodiazepines; highly polar compounds and low lipophilicity reducing transfer from blood to oral fluid 2 Positive Negative Long interval after dosing; concentration effect by kidney; highly protein-bound drug; sampling time outside oral fluid detection `window’ 3 Negative Positive Insufficient time for drug absorption; `depot’ effect, ion trapping 4 Negative Positive Insufficient time for drug absorption, metabolism and excretion; `depot’ effect Scenario Matrix Possible Explanations for Disparate Results Blood Urine Oral Fluid 1 Positive Negative Highly protein-bound drugs may be poorly distributed to oral fluid, e.g., benzodiazepines; highly polar compounds and low lipophilicity reducing transfer from blood to oral fluid 2 Positive Negative Long interval after dosing; concentration effect by kidney; highly protein-bound drug; sampling time outside oral fluid detection `window’ 3 Negative Positive Insufficient time for drug absorption; `depot’ effect, ion trapping 4 Negative Positive Insufficient time for drug absorption, metabolism and excretion; `depot’ effect View Large Amphetamines Amphetamines are weak bases and ion trap in OF, leading to higher OF concentrations compared with blood (14, 15). Nevertheless, dry mouth is common following amphetamine use, leading to low sample volumes and/or longer collection times (167). Stability issues are not generally a concern for amphetamines (25, 26, 168). Methamphetamine Methamphetamine generally has higher OF concentrations as compared with blood. Oral contamination may occur following smoking, and concentrations of up to ~60 mg/L were documented in a population of Norwegian drivers (165). Both methamphetamine and its metabolite amphetamine can be detected in OF. Following oral administration of four 10-mg doses of methamphetamine HCl sustained-release tablets within 1 week, methamphetamine was detected as early as 0.08–2 h after dosing, and mean (range) Cmax was 106 μg/L (24.7–312) in samples collected by expectoration after citric acid candy stimulation; after four 20-mg doses, mean Cmax was 192 μg/L (75.3–322) (14). Amphetamine was also detected but at lower concentrations than methamphetamine: mean Cmax was 8.6 (3.8–21.3) and 14.2 (2.8–20.2) μg/L after low and high doses, respectively. Maximal OF concentrations occurred 2–12 h for methamphetamine and amphetamine. At the 2004 SAMHSA 50 μg/L methamphetamine cutoff, 60% and 20% of OF samples were still positive at 11.5 and 24 h after the high dose, respectively. The revised 15 μg/L SAMHSA cutoff was not evaluated. Smoked methamphetamine would invariably be expected to contaminate the oral mucosa, leading to higher OF concentrations and possibly extending the window of detection; however, it is unclear how high these concentrations would be or how long methamphetamine would be positive, as no controlled administration studies have been published. Methamphetamine and amphetamine detection windows following long-term drug use were determined with a positive result of >8 μg/L for methamphetamine and >7 μg/L for amphetamine: positive and negative OF samples were interspersed with negative samples, with the last positive methamphetamine sample occurring after 8 days for users residing on a secure unit (169). A recent study evaluated d- and l-methamphetamine and amphetamine disposition following Vicks VapoInhaler® (l-methamphetamine) administration according to manufacturers' instructions (two inhalations in each nostril every 2 h over 10 h) (42). No d-methamphetamine or d-amphetamine was detected. l-Methamphetamine concentrations were low (median Cmax was < 20 μg/L, and only one participant was positive for l-amphetamine. At a 50 μg/L methamphetamine cutoff, 3 of 16 participants had positive methamphetamine samples, although only in 5–6% of their samples. Therefore, chiral analysis may be necessary to rule out intake of d-methamphetamine. 3,4-Methylenedioxymethamphetamine Similar to methamphetamine, MDMA is also more readily detected in OF than blood due to ion trapping. MDMA OF disposition (unstimulated expectoration) in eight participants following 1.0 and 1.6 mg/kg of oral doses yielded 1,643 (1,160–3,382) and 4,760 (2,881–11,985) μg/L median Cmax, respectively (167). Its metabolite, MDA, was never present in OF without MDMA; including MDA above the SAMHSA cutoff did not identify additional positive samples. The metabolites 4-hydroxy-3-methoxymethamphetamine and 4-hydroxy-3-methoxyamphetamine were not detected in OF. Generally, MDMA can be detected in OF for 12–48 h after a single dose. At the 2004 SAMHSA cutoff (50 μg/L of MDMA), 94.8% of positives occurred within the first 23 h. Initial MDMA-positive samples occur as early as 0.25 h, but more often at 0.5 to 0.75 h after dosing. At the 50 μg/L cutoff, median tlast occurred at 23.0 (13.0–29.0) and 29 (23.0–47.0) h after low and high doses, respectively. Although the 15 μg/L confirmation (EWDTS and updated SAMHSA) cutoff was not evaluated, the DRUID 20 μg/L was; tlast was 12.0 (3.0–29.0) and 23 (23.0–47.0) h after the low and high MDMA doses, respectively. Although OF and plasma concentrations were weakly but significantly correlated, it was not possible to predict one concentration from the other due to high variability (15). Cannabis OF cannabinoids and their metabolites' disposition following various controlled cannabis administration were investigated, although most of the scientific literature focuses on smoked cannabis (21, 34, 38, 170–179). THC in cannabis smoke or vapor rapidly contaminates the oral mucosa, leading to high OF concentrations. Oral mucosa contamination also occurs following oral consumption of edible products, but to a much lower degree than after smoking or vaporization (171). There is rapid initial drug clearance from the oral cavity, as >10,000 μg/L of THC OF concentrations 15 min after cannabis smoking fell to ~1,000–2,000 μg/L by 1 h (171, 178, 179). Rinsing the mouth with water significantly reduced OF THC concentrations (180). THC concentration after consuming a cannabis-containing brownie reached up to 938 μg/L in frequent users and 380 μg/L in occasional users (171). It is unclear why there was a difference in the two groups. OF from daily cannabis smokers abstaining from cannabis smoking but who received multiple oral Marinol® (dronabinol, synthetic THC capsules) generally had decreasing OF THC concentrations despite doses of up to 120 mg/day but increasing THCCOOH concentrations (34, 119). This suggests that THC in OF is predominately present from oral mucosa contamination rather than transfer from the blood. THCCOOH, the primary inactive THC metabolite, is present in low ng/L concentrations in OF, with increased detectability if OF samples are hydrolyzed prior to analysis (171). THCCOOH is not present in cannabis smoke, indicating that it can be utilized to differentiate active use from acute passive cannabis exposure (118). The source of THCCOOH in OF may be from the blood and/or from THC metabolism in the oral mucosa. THCCOOH concentrations are generally higher and more detectable in chronic frequent smokers than occasional smokers (151, 171, 172). At the SAMHSA and EWDTS 2 μg/L THC OF confirmation cutoffs, detection times as long as 26 h in occasional smokers and >72 h in frequent smokers were documented after smoked, vaporized and oral cannabis (171). Therefore, low THC concentrations can be detected in OF for several days, similar to blood and urine. tlast was not significantly different between the three routes of administration. Although not detected in all individuals, minor cannabinoids such as CBG, CBN and THCV in OF were suggested as potential markers of use within 26 h following oral, smoked and vaporized administration in occasional and frequent cannabis users (171). In another study of chronic daily cannabis smokers during sustained monitored abstinence, THC was generally quantifiable for 48 h, although two individuals still had intermittent positive THC samples up to 28 days after initiation of abstinence, with THC concentrations of 1.1 and 2.3 μg/L on Day 28 (181). There is some concern that some individuals may have had access to cannabis from other participants in another study of ad lib cannabis smoking. Positive THC OF samples may occur following passive exposure to cannabis. An early cannabis exposure study documented THC-positive OF samples after passive exposure; it was ultimately determined that collection devices themselves became contaminated by the smoking environment, as OF samples collected outside of the smoking environment were negative (114). More recent investigations indicated that THC-positive samples following passive exposure to cannabis smoke can occur but are generally <5 μg/L and typically dissipate within 3 h (114–118). Higher OF THC concentrations were reported in non-ventilated environments: concentrations of up to 308 μg/L were documented 15 min after exposure to cannabis smoking in a non-ventilated environment (117). In the non-ventilated environment, median tlast at a 2 μg/L cutoff was 2 h (1–12), whereas tlast in ventilated environments was 0.25 h (0–2). Cannabinoids may be particularly susceptible to concentration differences between OF collection devices or between collection and analytical methods. Expectorated OF samples can have variable concentrations due to cannabinoids adsorption to proteins, especially if samples are centrifuged. Significantly higher concentrations were measured in expectorated OF from Dutch `coffee shop’ patrons compared with samples consecutively collected from the same individuals using a collection device (182). Furthermore, OF THC concentrations in consecutively collected duplicate expectorated samples were much more variable than duplicate samples collected with collection devices. Indeed, studies involving two different OF collection devices did not detect any significant differences between simultaneously collected OF samples (34, 183). Commercial OF devices offer better stability and more reproducible results than do expectorated samples, as buffers help stabilize drugs, improve drug recovery from the collection pad, reduce OF viscosity and reduce analyte adsorption to container surfaces (22). Cannabinoid stability depends on collection method, buffer composition in commercial collection devices, the analytes, storage containers and storage temperature and duration. Cannabinoids are more stable in samples collected with OF devices than expectorated OF (22). Acceptable stability was documented with various devices (e.g., Quantisal, Intercept and Cozart) with some showing better stability at 4°C compared with −20°C (22, 25, 176, 184). On the other hand, reduced cannabinoid stability was demonstrated with other collection devices (e.g., Salivette), possibly due to adsorption to the collection pad in those devices (25). Cocaine Cocaine can easily be detected in OF, with cocaine and BE concentrations of up to 881 and 2,074 μg/L, respectively, measured in a group of chronic pain patients and ~70,000 μg/L in a population of Norwegian drivers (159, 165). Cocaine and BE OF disposition was also evaluated following various routes of administration (11, 13, 33, 148, 185, 186). Cocaine, a weak base, is ion trapped in OF yielding higher cocaine concentrations in OF compared with blood. In addition, cocaine can contaminate the oral mucosa following insufflation and inhalation. In a study of six participants receiving 25 mg IV, 32 mg intranasal and 42 mg smoked cocaine, OF Cmax in citric acid-stimulated expectorated samples were 258–1,303, 75–1,255,380 and 94–12,582 μg/L, respectively (13). Interestingly, oral mucosal contamination following intranasal and smoked cocaine did not occur in all individuals, but when it did, contamination dissipated within 0.5–1 h. Another study reported concentrations of up to 504,880 μg/L after smoking 40 mg of cocaine base (citric acid-stimulated expectorated samples) (11). Cocaine and BE disposition was evaluated in a recent study following 25 mg of IV cocaine (33). Median cocaine Cmax was 732 μg/L (83.3–1,892) with the StatSure collection device and 932 μg/L (394–1,574) with the Oral-Eze device. BE Cmax was 360 μg/L (77.2–836) with the StatSure device and 248 μg/L (96.9–1,892) with the Oral-Eze device. Therefore, cocaine and BE concentrations can be quite variable depending on the route of administration, with the highest concentrations typically observed after smoked cocaine. At SAMHSA's proposed and EWDTS's 8 μg/L confirmation cutoffs, tlast was reported as 6 to ≥12 h after intranasal cocaine and 0.5–6 h after smoking administration (13). Not surprisingly, detection times were extended following repeated cocaine dosing (186). Monitoring OF BE extended detection windows, but BE's Cmax was lower than cocaine's (33, 185). In citric acid-stimulated expectorated samples collected from participants receiving 75 mg/70 kg or 150 mg/70 kg subcutaneous cocaine, Cmax was 1,092 (406–3,006) and 2,600 (1,193–8,495) μg/L for cocaine and 134 (81.8–441) and 280 (133–757) μg/L for BE after the low and high doses, respectively (185). Median detection times with an 8 μg/L cutoff were 8.0 (4.0–24.1) h for cocaine and 28.0 (4.1–71.6) h for BE after the low dose and 9.8 (7.9–28.5) h and 32 (24.0–72.0) h after the high dose. In the study evaluating 25 mg of IV cocaine, cocaine tlast was 2–4 and 2–12.5 h with the StatSure and Oral-Eze collection devices, respectively, at an 8 μg/L cutoff; BE tlast was 6.5–21 and 9.5–28 h (33). Stimulation did not appear to affect cocaine concentrations: there were no significant differences between citric acid-stimulated expectoration, citric acid-treated Salivette and neutral Salivette OF cocaine concentrations (185). In addition, in two studies evaluating 25 mg of IV cocaine, similar Cmax was observed in citric acid-stimulated expectorated samples and OF samples collected with OF devices (13, 33). Stability of OF cocaine and BE collected with different devices was evaluated at room, refrigerated and frozen temperatures. Stability was acceptable for most devices, although an increase in BE concentration was documented after 1 week at room temperature with the StatSure device (25, 26, 168). Opioids As weak bases, opioids also have higher OF concentrations relative to blood. A growing number of reports documented opioid distribution in pain management patients, improving OF result interpretation. Opioids in OF are relatively stable, although 6-AM losses occurred in some OF samples collected with the Intercept device (25, 26, 168). Codeine In 19 volunteers administered 60 and 120 mg/70 kg codeine doses in capsules, citric acid-stimulated expectorated OF had mean codeine Cmax of 639 (184–1,289) after the low dose and 1,599 (620–3,350) μg/L after the high dose (187). Mean norcodeine Cmax was 17 (3.9–58) after the low dose and 47 (10–191) μg/L after the high dose. Morphine and normorphine were not detected. Codeine was initially detected between 0.8 and 1.0 h. tlast was not evaluated at the 15 μg/L codeine cutoff (current SAMHSA and EWDTS cutoffs), but mean tlast was 21 and 22 h after low and high codeine doses at a 2.5 μg/L cutoff and 7 h for both doses at the 40 μg/L cutoff (187). At a 2.5 μg/L cutoff for norcodeine (EWDTS cutoff is 2 μg/L), tlast was 6 and 9 h after the low and high doses, respectively. Citric acid-stimulated expectoration, Salivette with citric acid-treated cotton swab and Salivette with neutral cotton swab collection methods were compared (187). Codeine concentrations tended to be higher in samples collected after citric acid stimulated expectoration, but this difference was not significant. Two recent studies evaluated codeine disposition following poppy seed ingestion and demonstrated that codeine and morphine can be detected in OF following poppy seed ingestion, but only for a short time (41, 188). After two 45 g raw poppy seeds doses each containing 3.1 mg of codeine, Cmax was 8.6 (3.8–31.8) after the first dose and 9.5 (1.1–32.6) μg/L after the second dose in OF samples collected with the Oral-Eze collection device (41). Codeine remained above the 15 μg/L cutoff for a median of 2 (0.5–2) h and 1 (0.5–2.5) h after the first and second doses, respectively. In the second study, codeine Cmax was 18 (9–28) and 49 (16–112) μg/L after consuming a poppy seed roll containing 16 g of cooked poppy seeds (0.6 mg of codeine) and 15 g of raw poppy seeds (0.6 mg of codeine), respectively (188). In that research, tlast at the 15 μg/L cutoff was 0–0.25 h and 0.5–1.5 h, respectively, after these two dosing scenarios. Morphine To our knowledge, no clinical studies documented morphine OF disposition following therapeutic morphine administration; however, the prevalence of licit and illicit drugs in chronic pain patients' OF documented a median (range) morphine concentration of 18.1 (1.0–130,570) μg/L (158). Morphine may be detected in OF following poppy seed ingestion. After consumption of two 45 g raw poppy seed doses containing 15.7 mg of morphine, morphine Cmax was 34 (11.9–99.9) after the first dose and 9.5 (1.1–32.6) μg/L after the second dose in OF collected with the Oral-Eze collection device (41). OF remained above the 15 μg/L cutoff for a median of 1 h (0.5–2.5) after the first and second doses. Morphine Cmax of 35 (7–143) and 158 (47–284) μg/L was noted after consuming a poppy seed roll containing 16 g of cooked poppy seeds (3.2 mg of morphine) and 15 g of raw poppy seeds (3.3 mg of morphine), respectively (188). In that study, tlast at the 15 μg/L cutoff was 0.5–1.5 h for morphine after the roll and 0.5–3 h after each dosing scenario. Heroin One advantage with OF is that heroin and 6-AM are readily detected, providing better identification of heroin use than does blood or urine (11, 148, 189, 190). In a study evaluating three escalating smoked and IV heroin doses in two individuals, OF 6-AM concentrations in samples collected following citric acid-stimulated expectoration reached as high as 20,580 μg/L after smoking and 30 μg/L after IV administration, indicating that 6-AM likely contaminates the oral mucosa (11). In this study, at the SAMHSA and EWDTS 2 μg/L 6-AM cutoff, tlast was 0.5–2 h. Although there is no recommended cutoff for heroin, at a 1 μg/L cutoff, heroin was detectable 2–24 h after smoking and 5 min to 12 h after IV administration. In 77,218 Intercept-collected OF samples primarily for workplace drug testing programs, 48 were morphine positive, 32 of which (66.7%) were positive for 6-AM at a mean (range) concentration of 416 (3–4,095) μg/L (189). The authors suggested that an OF 6-AM/morphine ratio >1 was highly suggestive of heroin use within 1 h of specimen collection. Oxycodone Oxycodone is a weakly basic opioid commonly prescribed for pain but also commonly abused. Following a single 20 mg controlled-released oxycodone administration (OxyContin®) to 12 individuals, OF concentrations in expectorated OF were oxycodone > noroxycodone > oxymorphone; noroxymorphone was not detected (191). Oxycodone was initially identified within 15–30 min, with a biphasic absorption pattern. Reported OF Cmax was 133 μg/L (49.2–219) for oxycodone, 18.7 μg/L (10.3–31.8) for noroxycodone and 1.6 μg/L (1.2–2.4) for oxymorphone. Mean oxycodone detection time was 17.5 h (12–28), while oxymorphone was not detected at the 15 μg/L proposed SAMHSA cutoff. A 120–1,200 μg/L therapeutic range for oxycodone in OF was proposed to compare with a 10–1,000 μg/L blood therapeutic range (149). Hydrocodone In a pain management population, the median OF hydrocodone concentration was 122 μg/L (range 1.6–6,902) in samples collected with the Quantisal device (192). Prevalence of drugs and metabolites in OF was hydrocodone > norhydrocodone > hydromorphone. A clinical study documented similar prevalence following a 20 mg oral hydrocodone bitartrate dose (193). In OF collected by expectoration, hydrocodone mean tfirst was 0.27 h (0.25–0.5). OF concentrations were higher compared with blood, with a mean Cmax of 208 μg/L (61.7–626) of hydrocodone, 12.8 μg/L (3.6–27.0) of norhydrocodone and 6.4 μg/L (2.6–18.2) of dihydrocodeine. Reported detection times at a 15 μg/L cutoff were 10.7 h (8–14) hydrocodone and 0.7 h (0–8) dihydrocodeine. In two studies evaluating OF in patients undergoing treatment for chronic pain, median OF concentrations in samples collected with the Quantisal device was 22.6 μg/L (1.4–494) and 67.8 μg/L (1.0–3,344) (158, 159). Fentanyl Despite frequent use of fentanyl for pain and increasing fentanyl abuse in the USA, there are few OF data to guide OF fentanyl interpretation. In patients wearing patches delivering up to 200 μg/h of fentanyl, passive drool OF concentrations were ~50 μg/L (concentration estimated based on published figure) (19). In two chronic pain studies, median OF fentanyl concentrations (Quantisal device) were 1.2 μg/L (0.1–26.7) and 6.6 μg/L (102–5,341.3) (158, 159). Tramadol Although proposed SAMHSA and EWDTS guidelines do not include tramadol testing, it is being considered for inclusion by EWDTS. In 12 participants receiving 50 mg of encapsulated tramadol, median OF Cmax (Quantisal device) was 1,181 (459–3,905) and 43 (2–158) μg/L for tramadol and O-desmethyltramadol, respectively (194). Lower O-desmethyltramadol concentrations were reported in intermediate and slow metabolizers. Tramadol OF concentrations were ~10-fold higher than plasma concentrations, with tramadol OF tlast at least 48 h and O-desmethyltramadol for up to 32 h. Following two 100 mg tramadol doses daily over 2 days, median OF Cmax was 7,830 μg/L in slow metabolizers (CYP2D6*10/10* genotype) and 4,801 μg/L in the wild-type group (126). Methadone In 46 unstimulated expectorated OF samples collected ~23 h after the last methadone dose from opioid-dependent patients enrolled in methadone-assisted treatment, median OF Cmax was 105 μg/L (25–401) (133). However, participants were asked to rinse their mouth with water before providing samples, potentially diluting true OF concentrations. In another study, 16 opioid-dependent pregnant women receiving 30–110 mg/day of methadone provided unstimulated OF via the Salivette device (132). Methadone was detected in all OF samples, while the metabolites EDDP and methadol were identified in 88% and 12% of samples, respectively. No apparent dose–concentration relationship was observed. OF concentrations were 5.2–78,225, 1.0–1,791 and 5.0–281 μg/L for methadone, EDDP and methadol, respectively. Methadone was stable in OF pooled samples collected with the Intercept and StatSure devices (168). In fortified OF, methadone was not detected in the 7-day sample but was again identified in the 14-day sample, prompting the authors to suggest that methadone was trapped in the collection pad on Day 7 and had released into the OF supernatant by Day 14 (26). Buprenorphine There are few studies that document the pharmacokinetics of buprenorphine in OF. In one study, OF was collected (Salivette®) from nine pregnant women receiving 2 to 24 mg/day of sublingual buprenorphine HCl (9). Buprenorphine and norbuprenorphine were detected while buprenorphine-glucuronide was infrequently identified and at concentrations of <0.6 μg/L; norbuprenorphine-glucuronide was not detected in OF. In that study, buprenorphine OF Cmax was 672–12,300 μg/L, whereas the overall concentration range over the course of the study was 0.1–12,300 μg/L (median 15.4). High concentrations occurred due to sublingual buprenorphine contamination of the oral mucosa. A secondary peak was present after 8 h, possibly due to a buprenorphine depot in the oral mucosa, or to enterohepatic circulation. Buprenorphine fortified into OF was stable for at least 14 days with the Quantisal and Certus collection devices (26). Benzodiazepines Monitoring benzodiazepine exposure with OF is especially challenging due to the wide range of available benzodiazepines, variable potencies and their high protein-binding, leading to low OF concentrations. However, technological advances have improved sensitivity, with a growing body of literature documenting OF benzodiazepine disposition. Following 10 mg of oral diazepam, OF concentrations (Intercept device) were generally <2 μg/L; diazepam metabolites were not detected (195). In six samples collected from individuals enrolled in a detoxification center, diazepam concentrations were 1–8 μg/L (196). In eight males receiving 15 or 30 mg of oxazepam, median OF Cmax was 11 (8–24) and 19 (15–45) μg/L, respectively (10). Oxazepam-glucuronide concentrations were lower than those of oxazepam. Oxazepam was detected for at least 8.5 h at a 0.5 μg/L cutoff, with lower concentrations in OF compared with blood. Mean OF/blood ratio was 0.05 (0.03–0.07) for oxazepam and 0.004 (0.002–0.006) for oxazepam-glucuronide. In a study of chronic pain patients, OF alprazolam concentrations ranged from 0.7 to 46 μg/L (159). In another study with seven samples from detoxification patients, alprazolam OF concentrations were 2–25 μg/L; hydroxyalprazolam was not detected (196). In 11 volunteers administered a single oral dose of 0.5 mg of alprazolam, the median (range) Cmax was 0.98 (0.12–23) μg/L, with tmax occurring at 2 (2–13) h; the median (range) detection time of alprazolam in OF was 26 (4–37) h (197). In four volunteers administered 1 mg of flunitrazepam, expectorated OF concentrations were always below the 3 μg/L EWDTS cutoff (198). Flunitrazepam was below the 0.1 μg/L LOQ in one individual; stability may have been an issue, as no preservative was used in this participants' OF samples. Flunitrazepam Cmax in the other three participants were 0.29, 0.57 and 0.58 μg/L. Flunitrazepam stability was improved by addition of 2% NaF; without the preservative, concentrations decreased 23% after 48 h at 4°C, although 7-aminoflunitrazepam concentrations did not increase significantly. In OF (Intercept device) from opioid-dependent patients receiving opioid-assistance treatment, clonazepam, nitrazepam, flunitrazepam and their metabolites were detected (199). The 7-amino metabolites are more likely to be detected in OF than is the parent drug. In 1,001 clonazepam and/or 7-aminoclonazepam cases, 70.6% had both parent and metabolite in OF, 6.4% had only the parent drug and 23.0% had only the metabolite. In 211 nitrazepam cases, only the parent drug was identified in 7.6% of cases, 26.5% had only the metabolite and 65.9% had both. There were four flunitrazepam cases, one with parent and metabolite and three with only the metabolite. Benzodiazepine stability is especially important to consider when interpreting OF results. For example, there were decreases in OF diazepam collected with the Intercept device and the Salivette (25, 168). In addition, substantial benzodiazepine degradation was documented for diazepam, nordiazepam, oxazepam, temazepam, bromazepam, flurazepam, lorazepam, midazolam, chlordiazepoxide, clonazepam and flunitrazepam in fortified OF with the Draeger DCD 5000 when the collection device was stored dry in the transport tube; better stability was noted when the collection device was stored with methanol in the transport tube (200). Novel psychoactive substances In the current era of highly available and constantly changing NPSs, OF offers some advantages over urine monitoring. As parent drugs are more common in OF, metabolic studies are not required to identify which compounds should be targeted for urine analyses. Several NPSs were identified in OF including ethylone, methylone, α-PVP, dimethylone, butylone and 4-fluoroamphetamine, with mean concentrations of 582 (41–4,105), 2,445 (40–10,027), 474 (86–1,301), 611, 497 (175–905) and 329 (281–378) μg/L, respectively (81). Not surprisingly, synthetic cathinones were unstable in OF (201). Disposition data are lacking for NPSs, and NPS potencies vary considerably and are unknown when first appearing on the market, making NPS concentrations difficult to interpret in OF and blood. Conclusions The technology surrounding OF collection and data describing the distribution of drugs in OF dramatically increased over the last two decades. Furthermore, validated chromatographic methods to quantify multiple analytes in OF at low concentrations are available. Controlled drug administration studies and prevalence data improved interpretation of OF test results. 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TI - Oral Fluid Drug Testing: Analytical Approaches, Issues and Interpretation of Results
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bkz048
DA - 2019-07-24
UR - https://www.deepdyve.com/lp/oxford-university-press/oral-fluid-drug-testing-analytical-approaches-issues-and-2Bq1RtEPtK
SP - 415
VL - 43
IS - 6
DP - DeepDyve
ER -