Sample Preparation and Analytical Methodologies

The Federal Government has proposed the determination of 5 drug classes in oral fluid: cocaine, opiates, amphetamines, phencyclidine, and cannabinoids for the purpose of workplace drug testing in the United States. The analytical cutoff concentrations for both initial screening and confirmatory procedures are shown in Table 7-1.

Two separate analytical procedures are required for workplace testing. Which are, initial screening using immunoassay followed by confirmation using mass spectroscopy. No further manipulation is carried out for the sample with immunochemical assay the confirmation requires sample clean-up using liquid- liquid or, more often, solid-phase extraction, because of the low concentra­tions of drug present in the matrix (compared to urine), the viscosity of the specimen, and the requirement for highly efficient recovery.

Since the instrumentation are required to detect low concentrations of drug becomes routinely integrated into laboratories, however the criteria for definitive identification needs to be addressed. Various professional organizations and agencies, such as the World Anti-Doping Association (WADA), and Society of Forensic Toxicologists (MS/MS guideline committee) have or are currently prepar­ing mass spectral guidelines for the newer technologies such as chemical ionization (gas chromatography/mass spectrometry; GC-MS) and tandem mass spectrometry (MS/MS).

In order to apply these concepts to oral fluid drug analysis raises some key issues in each drug class specifically to the metabolic profile, drug deposition and concentration in the sample.

Cocaine (COC)

Workplace guidelines proposed cocaine and its major metabolite benzoylecgonine (BE) as analytes for detection. Research shows that following drug use, cocaine is the major analyte detected in oral fluid because of a high saliva/plasma (S/P) ratio (Table 7-2). But it may also be noted that it has a short half-life (1-1.5 h), so depending on the time after inges­tion, both COC and BE may be present in oral fluid (in contrast to urine, which contains predominantly BE)

Therefore, any initial immunoassay must be targeted towards both analytes, and true- positives containing only the parent cocaine will be missed at the initial screening phase. Several manufacturers now have the availability for immunological reagents for screening at the cutoff concentration in the form of liquid as well as microplate. Both cocaine and its analyte i.e benzoylecgonine are relatively easy to analyze from saliva, and many other analytical procedures for the detection of both com­pounds have also been in development and reported,  which may also be used with other metabolites and associated compounds, including cocaethylene, norcocaine, and anhydroecgonine methyl ester (15). Analytical testing procedures that are adequately sensitive for the detection of these drugs include immunoassay and gas or liquid chromatography with mass spectral detection (16).

Saliva/Plasma Ratio of Commonly Analyzed Drugs

Concentrations of Drug

Jufer et al. did some controlled studies in which he followed the elimination profile of cocaine and several other metabolites in saliva (BE, ecgonine methyl ester, ben- zoylnorecgonine, norcocaine, meta and para-hydroxycocaine; meta and para-hydroxybenzoylecgonine) after multiple oral cocaine administration. And it was reported that peak drug concentrations occurred imme­diately following smoking, likely due to contamination of the oral cavity with cocaine. Its metabolite, BE, showed peak concentrations of over 5,000 ng/mL, 1-3 hours after intake, and the BE was quantifiable for as long as 12 h.

In other studies with chronic cocaine users, Moolchan et al. reported that cocaine has a  half-life of 3.8 h in plasma compared to 7.9 h in saliva; similarly for BE, the half-life for plasma was 6.6 h compared to 9.2 in saliva. Their findings appeared to show that regular use of cocaine altered the disposi­tion and elimination of the drug compared to occasional use.

Cone et al. studied the disposition of cocaine following intravenous, intranasal, and smoked administration. The parent drug appeared rapidly in saliva regardless of the route of administration. One of the other drawbacks of urinalysis for morphine is the likelihood of poppy seed ingestion, rather than illicit drug use, causing a positive result. Morphine in saliva due to the acute poppy seed ingestion is reportedly below the limit of detection 1 h after intake; however, the study did not have any idea of chronic poppy seed ingestion. Another scientist Cone reports S/P ratio fluid from all participants, averaging 14 ng/mL.


Being slightly basic drug is expected to be detected in saliva with an S/P ratio >1. It shall be highlighted again, liquid reagents for screening and microplate assays are offered for screening oral fluid. Few reports in the literature regarding administration to humans, although in 1984 McCarron et al. reported the occurrence of 2-600 ng/mL of PCP to be in saliva using radioimmunoassay as a detection technique. He concluded that the concentration of PCP in the samples shall not be correlated with the severity of PCP intoxication. Saliva was found to be reliable as serum as a specimen for PCP analysis.

In recent times, another procedure for the determination of phencycli­dine in oral fluid was developed and later validated using LC-MS-MS, follow­ing early screening with enzyme-linked immunosorbent assay. The specimens were collected using the Quantisal device, then quantified by mixed mode solid-phase extraction followed by mass spectrometric detection with positive atmospheric pressure chemical ionization mode. For its confirmation, 2 transitions and 1 ratio were determined which had to be within 20% of the known calibration standard. The limit of quantitation was 2.5 ng/mL and the percentage recovery was 81.7 % (n = 6) from the collection pad. A specimen taken a good and authentic subject was found to contain 38 ng/mL.


Cannabiniods a.k.a Marijuana is the most common illicit drug used in the United States. The active constituent, THC, is speedily oxidized to ll-hydroxy-A9-tetra- hydrocannabinol (11-OH-THC) and then to its inactive metabolite, nor-A9-tetrahydrocannabinol-9-carboxylic acid (THCA). At times it has also been reported that these undergo further metabolism and conjugation to form glucuronides, which are more polar as compared to their precursors, which also shows that it can be elimi­nated from the body more rapidly. The identification of marijuana intake is done by the detection of THC, cannabinol, and cannabidiol, and or its pre­cursor 2-carboxy-THC, in oral fluid has been reported by the author (28). During the smoking process, the THC is deposited into the oral cavity, and fluid from this depository is the main source of the THC collected and measured in oral fluid analysis, which is more than drug that has been circulated through the body. The proposed Federal regulations addressed this issue with the following suggestion for the simultaneous collection of a urine specimen. But this probable disadvantage to oral fluid analysis was largely detached by the recent iden­tification of the metabolite THCA in saliva by 2 separate research groups. Its detection minimizes the argument of passive exposure to mari­juana being responsible for a positive THC result.

Drug Concentrations

THC is easily detectable for 14-16 h after smoking. Two scientist Huestis and Cone reported that THC concentrations with the passage of time generally dropped below 1 ng/mL approximately 12 h after smoking. Another scientist, Niedbala et al. reported a slightly longer detection time for THC in oral fluid as compared in plasma, with an regular detection time for consecutive positives of 13 h. It was also nothed that some subjects also appeared to be positive after 72 h, having previously fallen below the limit of quantitation. The scientists conclude that sequestration in the oral cavity causes fast and slow release of THC in saliva.

In this modern era one of the more interesting advances in oral fluid testing was the characterization of glucuronide bound THCA in oral fluid. In order to extend the margin of detection of marijuana in oral fluid, and removing both sequestration and passive exposure concerns, the study further investigated whether any amount of THCA was conjugated in saliva. Oral fluid specimens collected from a marijuana user just after smoking were analyzed for THC and free THCA, then hydrolyzed using 3 procedures, and were also sent for screening by 2 different immunoassays.

The parent compound, THC, was found to be not significantly conjugated, but the metabolite THCA was approximately 64% conjugated in oral fluid. Although these concentration are low, 2-dimensional GC with mass spectral detection was characteristically able to measure the concentrations present. Detection of metabolite in saliva effectively eliminates passive exposure claims, and extends the window of detection to at least 48 h.


Ethanol is distributed and or circulated throughout the body following ingestion, including saliva. In some workplace situations it is necessary and compulsory that analysis of ethanol (e.g., post-accident, for cause) is carried out. The relation between blood alcohol concentrations and saliva alcohol levels was first reported in the late 1930s. The S/P ratio for alcohol has been addressed between 0.88 and 1.36. Oral fluid and the blood drug test is analyzed for alcohol in the same way via standard procedures such as enzymatic screen, gas chromatography, etc.

The workplace testing in the United States, the Department of Transportation has a standard cutoff concentration of 20 mg/dL of oral fluid. If a subject has a detected saliva alcohol concentration between 20 mg/dL and 40 mg/dL, they are not allowed to work for a defined period of time; above 40 mg/dL, the subject is considered to be in violation of the rules. For saliva testing to be carried out, an approved device (e.g., QED) must be used.

Other Drugs

The five main drug classes have been the chief focus of Federal workplace drug testing for many years; whereas other companies require using expanded drug test panels for their employees. There are many prescription medications, such as benzodiazepines and antidepressants, can be severely abused and can cause fatal impairment particularly when combined with alcohol.


Benzodiazepines are sedative class of drugs which have a low S/P ratio (0.03-0.08), mainly because of the extent of protein binding and low pKa values. However various benzodiazepines have been found in saliva, the concentrations are less, which require widely cross-reactive screening tests and sensitive confirmatory methods. Link et al. studied the analysis of the short-acting drug, midazolam, and its hydroxymetabolites in plasma and oral fluid. The procedure used for the detection of the parent drug and its metabolites in oral fluid following administration of single 2-mg dose of midazolam. Diazepam, and its metabolites nordiazepam and oxazepam, has been reported to be present in saliva in the range of 2-5 ng/mL following therapeutic chronic dosing. The stability parameters of some benzodiazepines in saliva have also been discussed. In the late 1980s, Hart et al. addressed that although diazepam and clonazepam were stable in saliva, nitrazepam was converted to 7-aminoni- trazepam at room temperature. The conversion rate however was strongly dependent on the composition of the subject’s saliva. Similarly, Samyn et al. noted that flunitrazepam wasnot stable in oral fluid, even if treated with preservatives and/or refrigerated. Some recent publications on the determination of benzodiazepines in oral fluid have used tandem mass spectrometry in multiple reaction-monitoring (MRM) mode to detect even the lowest concentration of benzodiazepines present. The presence of oxazepam glucuronide in oral fluid was addressed at the International Association of Forensic Toxicologists annual meeting of 2007. This is a very interesting observation which may provide scientists with enhanced ways to detect benzodiazepines in saliva.


The recommended analytical profile for the amphetamine class includes the determination of the following compounds amphetamine (AMP), methamphetamine (METH), methyl- enedioxymethamphetamine (MDMA, Ecstasy), methylenedioxyethamphet- amine (MDEA), and methylenedioxyamphetamine (MDA). Liquid reagent and microplate forms immunoassay screening kits are available for these drugs as usual laboratory assays. Chromatographic procedures have also been widely used.

The presence of a good measurable drug levels in the oral fluids has engaged the number of laboratories in the analysis of oral fluid, and as the overall number of analysis increase, the quick, reproducible extraction of drugs from the collection buffer is possible. Since a some limited degree of sample volume is generally collected, procedures using reduced sample volume testing (62 pL of neat oral fluid), competent recovery from the collection pad (>84%), and enhanced analytical capability (LOD 25 ng/mL) have been reported, with detailed reference to the amphetamine class.

Drug Concentrations

Amphetamines are one of the easy compound that is detected in oral fluid several hours after use, since its concentrations are higher than plasma, with reported S/P rations >2.0, but this  S/P ratio is highly variable between individuals. The pharmacokinetics of methamphetamine in saliva following oral administration has been described by many scientists. Schepers et al. studied the presence of methamphetamine in the saliva 0.08-2 hours after its intake, with a peak concentration of 75-322 ng/mL, occurring from 2-12 hours after a 20-mg dose (25). Whereas after a 10-mg dose, amphetamine was only detected in the oral fluid of 5 of the 8 subjects with an average concentration of 8 ng/mL; With a 20 mg dose, amphetamine was detected in oral fluid from all participants, approximating 14 ng/mL. One limitation for the workplace proposal notes that if methamphetamine is detected above 50 ng/mL, then amphetamine should also be present above the limit of detection of the method for a specimen to be reported as “positive.” The scientists applied this criterion to their results following low and high repeated methamphetamine dosing. After administering four doses of 10 mg, only 33% of the subjects were positive at this level; after administering four 20-mg doses, 80% of the subjects were positive 24 h after administration. Forty-eight hours after the last of four 10 or 20-mg doses, both methamphetamine and amphetamine concentra­tions were lower than the proposed cutoffs standards. The accumulation of drug in oral fluid can occur from more than one (multiple) dosing and therefore increased than normal concentrations can be observed in saliva.

Pain Medications

Propoxyphene has been found to be detected in oral fluid taken from a subject who has been prescribed propoxyphene after surgery. Usually two oral fluid specimens were collected using the Quantisal device after 1 h, and one sample was collected 8 h after  ingestion of propoxyphene (100 mg) The two sam­ples taken after 1 h showed propoxyphene at concentrations of 223 ng/mL and 215 ng/mL respectively. Whereas a significant amount of the drug was still present  in the oral fluid collected after 8 h (37 ng/mL).

Moore et al. gave sufficient proof for the determination of tramadol, oxyco­done, and meperidine in oral fluid in 2006. During the analysis of corresponding blood and saliva specimens, tramadol was easily detectable in concentrations up to 12 times in the oral fluid higher than blood.

Multi-Drug Panels

Many authors have published procedures and supporting documentations for the concurrent analy­sis of a wide range of drugs in oral fluid. Wylie et al. was a intelligent scientist who developed and validated a qualitative and quantitative analytical method for the determination of 49 drugs in oral fluid. In this procedure, saliva was collected using a modified Omni-Sal device and the analytes were separated from an oral fluid/buffer mixture using a solid-phase process. LC-MS-MS and GC-MS were used in corresponding to analyze the extracts for the targeted drugs. Since sample volume is very less, Another scientist Concheiro et al. used only 100 pL of oral fluid for the analysis of morphine, 6-acetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, cocaine, and benzoylecgonine using solid-phase extraction and LC-MS.

One of the most famous scientist of all time Gunnar et al. studied the analysis of 30 drugs in oral fluid using long-column fast GC with mass spectral detection. In a comparable procedure, Pujadas et al. described a simple and reliable GC-MS method for detecting and quantifying a wide range of drugs in oral fluid. The drugs in their method were  amphetamine, methamphetamine, MDMA, MDA, MDEA, phen- termine, cocaine, benzoylecgonine, cocaethylene, and ecgonine methyl ester, THC, THCA, 11-OH-TIIC, carinabinol, cannabidiol, 6-AM, morphine, codeine, flurazepam, flunitrazepam, dipotassium chlorazepate, alprazolam, diazepam and oxazepam, amitryptiline, paroxetine, sertraline, haloperidol, chlorpromazine, fluphenazine, chlormethiazole, loratidine, hydroxyzine, diphenhydramine, valproic acid, and gabapentin. The compounds were concurrently extracted from oral fluid by solid-phase extraction method and ana­lyzed using GC-MS.

The most important workplace testing shall include a use of 2 assays, which operate on different chemical principles and methodologies, which shall be required for the forensic acceptability of the result. Even though these methods offer acceptable reliability to screen a wide range of drugs (including those suggested for Federal workplace analysis) however it should also be noted that the secondary confirmation should be carried out using a different technique. The current drug test panel from a single oral fluid specimen in one of the research laboratory incorporates several drug classes and alcohol (showed in Table 7-3), and the methodology used include the immunochemical screening of the drug classes followed by mass spectral detection. Any Improvements in sensitivity however will allow the panel to be further increased as necessary. This profile was also presented at the International Association of Forensic Toxicologists annual meeting.


While only morphine, codeine, and 6-AM are recommended as workplace target analytes, many other opioids have also been detected in oral fluid, which also includes the commonly abused pain medications hydrocodone and oxycodone. Immunoassay-based screening kits in liquid reagent and microplate forms are available at the suggested cut-off concentration for detection of opiates. Many procedures using GC-MS and LC-MS for opiate identification have been also reported.

Drug Concentrations

The S/P ratio of >1 is found to be for heroin, appearing in saliva rapidly, and lasting detectable for at least 24 h. Following heroin intake 6-AM and morphine were also detectable in saliva, with 6-AM concentrations approximating 0.5 and 8 h after smoking. If the heroin was given intravenously, the peak concentration of 6-AM was reached after 4 h. Furthermore, an average S/P ratio of 0.67 (0.1-1.82) was concluded for morphine after intravenous administration of heroin.

Whereas the administration of codeine is a simpler outlook than injection of heroin, therefore it’s not surprising that more documents are based on the codeine detection in saliva.  In single-dose studies, codeine detection in saliva lasted for 7 h following dosing of 60 and 120 mg, when the proposed cut-off concentration of 40ng/mL was applied. Another scientist O’Neal et al. indicated a middling S/P ratio of 3.7 for codeine following oral dosing of 30 mg, which remained steady for 2-12 h.

The most interesting reason of opiate analysis is the examination by several research groups that norcodeine, not morphine, is identified in oral fluid after the administration of codeine. Therefore, the morphine detection in saliva can be interpreted as intake of heroin or morphine. This is in dissimilarity to urine where the presence of high morphine levels can be accredited to its metabolism from the ingestion of codeine. One of the important drawbacks of urinalysis for morphine is the likelihood of poppy seed ingestion, rather than illicit drug use, causing a positive result. Mor­phine in saliva consequential from acute poppy seed ingestion is apparently below the limit of detection 1 h after intake; however, the research did not address chronic poppy seed ingestion. Another scientist Cone reported a S/P ratio for morphine of 0.2 after the administration of morphine sulphate, with peak concentrations reached after 30 mins and the ability to maximum detectable no more than 24h.


In recent times, Cone and Huestis published a broad paper on the interpretation of oral fluid tests for drugs of abuse (47). They pointed that the interpretation of oral fluid drug results is in part reliant on the purpose for testing. Workplace drug testing is generally meant as a limited program, to identify drug abusers in the workforce.

The concentration of drugs in oral fluid reflects the concentration of free drug in the blood, providing probable oral cavity contamination issues are addressed. Therefore, saliva is an excellent source for the purposes of determining the likelihood of impairment such as in driving cases or fitness for duty testing. However the detection of the parent drug at suitable concentrations may be connected with recent drug intake.


In the workplace drug testing programs, the presence of cocaine can only be indicated within the last 8-12 h of administration, and both cocaine and benzoylecgonine within t 24 h. But Benzoylecgonine can be detected for a maximum of 2-3 days, providing a larger detection window similar to urine.

The detection of other metabolites (when used in combination with alcohol) such as norcocaine, or cocaeth- ylene, would help out in the interpretation of drug intake; however, these are not part of the planned Federal guidelines.


The detection of 6-acetylmorphine and morphine in oral fluid specimen indicates the use of heroin; the occurrence of morphine only indicates heroin or morphine use within the last 24 h, but that depends on the dosage patterns. There are no supporting data that the ingestion of codeine producing morphine in the saliva. Low levels of morphine should be related with vigilance, since chronic intake of large amounts of poppy seeds has not been studied.


The tests indicating the detection of dextro (d)-methamphetamine refers to the intake of the drug within the last few hours. The occurrence of amphetamine is required in a specimen positive for methamphetamine is projected under the guidelines. There are no controlled studies on the subject of the chronic use of the Vicks inhaler, whose active ingredient is levo (l)-methamphetamine. Most of the immunoassays are targeted at the d-methamphetamine form of the drug. Even though this restricts the detection of the legal isomer, in some cases a chiral determination may be required to make a distinction the 2 forms. It may also be pointed that there are various prescription medications, which are metabolized primarily to methamphetamine and then amphetamine including the analgesic, famprofazone (mixture of d and I isomers) and selegiline (Z-isomers), used in the treatment of Parkinson’s disease. Any of these medications suspected, chiral separation is required as well as prescription drug history. The presence of drug is investigative for intake within the last 24 h, and/or 48 h in frequent users.


Investigational and comparable data for PCP are inadequate; probably it is present in the body in a similar behavior to cocaine, with positive specimens indicating use within the last several hours.


Parent THC is detectable in the saliva for 8-16 h, but a longer window (approximating more than 48hrs) of detection can be resolute by the identification of the THCA metabolite. Furthermore it must be noted that pharmaceutical preparations of THC, including Sativex®, may cause positive results and should be measured in the prescription drug history, while it has been reported that Marinol® does not turn out a positive oral fluid result for THC.

The analytical point of view for the test, and the validity of a reported quantitative drug concentration should be carefully measured. For the authenticity of the value it is necessary that a known quantity of oral fluid be tested and, if a collection pad is used, then the pad is assessed for the recovery of drug. Quantitative values are normally not adjusted for the degree of recovery, which gives advantage to the subject being tested since administrative cutoff concentrations are used in workplace program rather than the limits of detection. Furthermore, quantitative results from specimens collected with devices, (not by expectoration), also needs to be adjusted for the dilution factor of the specific device to be accurate.

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