Oral Fluid As a Matrix for Drugsof Abuse Testing

The results of substance abuse deeply affect individuals, families, and society at large. One solution has been to test individuals for the presence of drugs of abuse (DOA). Such tests may be administered to job applicants and parolees, as well as immediately after accidents and along the roadside by police.

Many countries have legislation in place or pending for drug testing. This legislation defines when, where, and how drug testing should be performed

From: Forensic Science and Medicine: Drugs of Abuse: Body Fluid Testing Edited by R. C. Wong and H. Y. Tse © Humana Press Inc., Totowa, NJ

Fig. 1. Major glands of the mouth. Breakdown of materials contributed by various saliva glands: 65% submandibular, 23% parotid, 4% sublingual, and 8% minor glands.

(1-3). The first country to enact such legislation was the United States. This model allowed the use of urine as the primary testing matrix. Urine testing, however, has limitations, including the need for special facilities for collection and a witness to prevent the samples from being adulterated. Furthermore, urine testing does not reflect recent drug use, which therefore limits its value toward judging impairment.

With the advance of technology, newer, more sensitive analytical techniques have allowed the use of alternative body fluids such as saliva—or oral fluid—in DOA testing. "Oral fluid" has become the more common term for a sample collected from the mouth for diagnostic purposes (1). It is the combination of fluids excreted by the glands of the mouth along with other debris.

The mouth is composed of many glands, the primary ones being the parotid and submandibular glands (4-7). Figure 1 shows a diagram of the major glands of the mouth. The parotid ducts are located in the upper bucal cavity and produce fluids that are primarily low in viscosity. The submandibular glands are located in the lower bucal cavity and produce a mucous mixture. These fluids and their components have several purposes, including wetting of food matter to facilitate swallowing; infection control; maintenance of healthy teeth; and wetting of the oral mucosa. Given all of these functions, the mouth is a complex entry into the body with a diverse set of mechanisms. Therefore, as one considers DOA testing using oral fluids, one should consider these dynamics

Fig. 2. The Intercept® device.

and anticipate them when collecting samples to be used for substance-abuse analysis.

2. Method of Collecting and Testing Using the Intercept® Device

This chapter is primarily focused on describing the use of a new method to collect and test oral fluids for drugs of abuse. The Intercept® device (Orasure Technologies) (Fig. 2) has been tested and used for a variety of abused drugs. The collection device consists of an absorbent cotton fiber pad impregnated with a salt and affixed to a nylon stick, and a preservation solution (0.8 mL) in a plastic container. The collection device pad is placed between the lower gum and cheek for 2-5 min. While resident in the oral cavity, the pad will absorb a passive sample of oral mucosal transudate (OMT). The OMT is composed of collected fluids resident in the oral cavity as well as a small amount of blood components drawn into the pad transmucosally. The result is an enriched sample that allows analysis of small molecules such as drugs or large proteins such as antibodies. With this device, an average of 0.4 mL of oral fluid is collected. The collection device pad is then placed in the preservative solution. The resulting total volume is approx 1.2 mL (0.4 mL specimen and 0.8 mL

Positive

Fig. 3. Qualitative assay testing algorithm.

Negative

Positive

Negative

Fig. 3. Qualitative assay testing algorithm.

preservative solution). Consequently, the oral fluid specimen is diluted by a factor of 3. All testing is performed on the dilute specimen, and concentrations are reported based on the final diluted specimen.

3. Screening Tests for Intercept

Collected oral fluid specimens have routinely been analyzed using algorithms that are similar to those of urine testing. Figure 3 shows a diagram for the qualitative determination of drugs using the Intercept collector. After field collection, a sample is shipped to a primary testing laboratory using chain-of-custody procedures. An initial screen is completed using microtiter-based immunoassay for each target drug. An initial presumptive positive is then followed by confirmation testing using a combination of gas chromatography (GC) and mass spectrometry (MS) (GC-MS or GC-MS-MS). This same approach is currently used for urine testing by laboratories following US federal guidelines for performing DOA analysis. This algorithm is technically and legally defensible because initial screening tests that rely solely on immuno-assay are subject to the varying levels of cross-reactivity of the antibodies used in such tests. The combination of an immunoassay that can broadly identify the potential presence of an abused substance followed by a highly specific and sensitive mass spectrometric confirmation technique provides assurance of correct identification of positive samples. Thus, the testing of oral fluids can mirror the existing algorithm for testing urine, providing a similar logic to ensure accuracy.

The following procedures are typical of a microplate-based enzyme immu-noassay (EIA) using tetrahydrocannabinol (THC) as an example (Fig. 4). Briefly, 25 ^L of specimen, calibrator, or control is added to each well of an anti-THC-coated plate (immobilized sheep anti-cannabinoids polyclonal antibody) followed by addition of 25 ^L of buffer and incubation for 60 min at

Fig. 4. Enzyme immunoassay.

room temperature (RT). After incubation, 50 ^L of THC enzyme conjugate (horseradish peroxidase labeled with THC derivative) is added and the plate is incubated for an additional 30 min at RT. The plate is then washed six times with 0.3 mL of distilled water, followed by addition of 0.1 mL substrate reagent (tetramethylbenzidine) and incubation for 30 min at RT. After incubation, 0.1 mL of stopping reagent (2 N sulfuric acid) is added. Absorbance is measured at 450 nm and 630 nm within 15 min of stopping the reaction. The specific signal is measured at 450 nm while the 630 nm measurement is used to blank the sample. The final color signal developed is inversely proportional to the amount of drug present in a sample. Mean values of specimens are compared to the mean value of the calibrator (1 ng/mL, N = 4). Specimens with absorbance less than or equal to the calibrator were considered positive and specimens with responses greater than the calibrator were considered negative (8-10).

EIA technologies are inexpensive and provide sufficient analytical sensitivity for routine analysis of oral fluid specimens. Future technological enhancements are expected to introduce new homogeneous immunoassay techniques that require no wash or separation steps, which will further simplify the screening process. Once such techniques are available, oral-fluid screening may be automated on large-scale analyzers.

4. Characteristics of Screening Tests

Each of the microplate immunoassays has specific performance characteristics that are critical to the effectiveness of the overall Intercept system of collection and testing. Some of the most critical analytical parameters deserve more detailed discussions.

Table 1

Limit of Detection (LOD), Range of Calibrators, and Cutoff for Each Intercept® Assay

Table 1

Limit of Detection (LOD), Range of Calibrators, and Cutoff for Each Intercept® Assay

Assay*

LOD (ng/mL)

Assay calibrator range (ng/mL)

Cutoff (ng/mL)

Amphetamine

25.5

0-200

100

Barbiturate

8.2

0-40

20

Methamphetamine

8.0

0-80

40

Cannabinoid (THC)

0.37

0-2.0

1.0

Benzodiazepine

0.2

0-2.0

1.0

Cocaine Metabolite

1.5

0-10

5.0

PCP

0.49

0-10

5.0

Methadone

0.50

0-10

5.0

Opiates

1.4

0-20

10

*Note: All values shown as calculated for Intercept and not whole oral fluids. Multiply the values by 3 to obtain whole oral fluid values. TCH, tetrahydrocannabinol.

*Note: All values shown as calculated for Intercept and not whole oral fluids. Multiply the values by 3 to obtain whole oral fluid values. TCH, tetrahydrocannabinol.

4.1. Analytical Sensitivity/Limit of Detection

The limit of detection (LOD) is defined from the signal-to-noise ratio at the zero-drug concentration as the mean zero absorbance (A0) minus three times the noise level (LOD = A0 - 3SD). The LOD was determined by obtaining the average absorbance value for 80 readings of blank oral-fluid diluent and calculating the standard deviation (SD) and three times the standard deviation (3SD) of the absorbance. The absorbance value minus 3SD was then extrapolated from the curve and represents the sensitivity of the assay. The LOD range of calibrations and cutoff for each Intercept assay are listed in Table 1. The assay cutoffs are separately determined through clinical testing. It is important to note the separation of the cutoffs used from the LOD. It would not be appropriate for a routine screening technique to use the LOD also as its cutoff, because other performance characteristics such as precision would most likely not be acceptable at the LOD.

4.2. Precision

The precision of the OraSure Technologies Inc. (OTI ) Intercept MicroPlate EIAs was assessed by testing oral-fluid diluent containing various concentrations of the target drug. The intra-assay precision was determined by analyzing each level 16 times per run for four runs. The inter-assay precision was determined by analyzing two samples at each level twice per day for 5-20 d, depending on the assay. The oral-fluid diluent used for these tests is carried in a phosphate buffer, which adjusts collected oral-fluid specimens to neutral pH. All tests were performed at room temperature. It should be noted that absolute absorbance values for microplates will be affected by room temperature; this should be considered when performing inter-assay or inter-day precision analysis. The results of this testing are shown in Table 2.

4.3. Cross-Reactivity

The cross-reactivity was determined for each assay for analogous and ubiquitous compounds. Analogous compounds that were cross-reactive in each of the Intercept assays are shown in Fig. 5. Cross-reactivity was determined by spiking various concentrations of each tested compound into the Intercept diluent fluid. A sample that showed a response was compared with each assay standard curve in order to calculate the percent cross-reactivity. For example, a test compound that showed equal immunoassay response to the cutoff concentration in a particular assay would be judged as showing 100% cross-reactivity. Some compounds shown have calculated cross reactivities that are very low. These values are included to show that extraordinarily large concentrations of such drugs would be required to elicit a response in the immunoassay. Thus, those performing a secondary confirmation by GC-MS-MS would not target confirming such compounds in presumptively positive clinical specimens.

4.4. Interferents

The effect of interfering substances or adulterants was examined in the Intercept Micro-Plate EIAs. Testing interferents by spiking them into buffer or some other artificial matrix is not relevant. Therefore, samples from volunteers were used after they consumed a potential interferent. In this experiment, five subjects consumed 1 oz of each adulterant, and oral-fluid samples were collected from each volunteer using the Intercept oral-fluid collection device after a 5-min and 10-min period following consumption. Samples were processed and pooled for each interferent and collection time. Aliquots from each sample pool were spiked with various concentrations of target drug and tested in the assay. The signals obtained for samples containing only the adulterants were used to assess any effects that may lead to false-positive results. The signals of samples containing drug in the presence of each adulterant were used to assess the overall effects of the adulterant. The substance was considered not to interfere if, after the 10-min waiting period, the samples containing 0 or the cutoff level of drug produced absorbance readings greater than the cutoff and if the samples containing 1.5 and 2.0 times the cutoff produced absorbance readings less than the cutoff. Data generated for an Intercept secobarbital assay are

Table 2

Precision of Intercept® Micro-Plate Enzyme Immunoassays

Concentrations Intra-assay Inter-assay ng/mL %CV (n = 64) %CV (n = 4/d, 20 d)

Amphetamine

0

3.9

6.7

50

3.5

6.7

100

4.0

7.5

150

4.5

7.7

200

6.4

7.9

Secobarbital

0

4.1

8.5

10

4.4

8.9

20

3.8

8.9

30

7.1

8.9

40

4.9

9.4

Methamphetamine

0

7.8

7.5

20

7.0

7.7

40

6.2

7.9

60

7.8

7.5

80

6.4

8.4

A9-THC

0

4.7

8.7

0.5

4.5

9.3

1.0

5.2

11.0

1.5

5.5

11.6

2.0

4.6

10.8

Nordiazepam

0

5.1

7.6

0.5

6.1

10.4

1.0

6.5

11.0

1.5

4.9

11.4

Benzoylecgonine

0

3.7

8.0

2.5

3.4

9.0

5.0

4.3

9.6

7.5

7.6

10.5

PCP

0

7.2

10.7*

0.5

6.1

11.8*

1

7.1

14.0*

1.5

8.8

18.5*

Methadone

0

6.3

9.9

2.5

6.6

12.3

5.0

6.7

12.9

7.5

6.8

13.6

Morphine

0

3.6**

7.5***

5

6.4**

8.9***

10

6.6**

9.5***

20

6.9**

8.7***

* %CV (n = 4/d, 14 d). ** %CV (n = 20). *** %CV (n = 20/d, 5 d).

THC, tetrahydrocannabinol; PCP, phencyclidine.

* %CV (n = 4/d, 14 d). ** %CV (n = 20). *** %CV (n = 20/d, 5 d).

THC, tetrahydrocannabinol; PCP, phencyclidine.

Fig. 5. Drugs-of-abuse cross-reactivity results for structurally related compounds tested using Intercept® immunoassays.
Fig. 5. (continued)

ro 200

i 150

fi 100

50 0

Benzoylecgonine - Structurally Related Compounds Cross-Reactivity

50 0

ro 200

i 150

fi 100

■ ,

Nordiazepam- Structurally Related Compounds Cross-

Reactivity

160 140 120 100 80 60 40 20 0

/ / // x x./ / / / / * / / </ ^ y s # ¡f </ y

_

■ n

■ ■

- . d .

PCP ■ Structurally Related Compounds Cross-Reactivity jr

Table 3

Effects of Adulterants on Intercept® Enzyme Immunoassays

Table 3

Effects of Adulterants on Intercept® Enzyme Immunoassays

Substance

EIA Result at 10 min. (adulterant only)

EIA Result at 10 min. (adulterant + secobarbital)

Sugar

No effect

No effect

Toothpaste

No effect

No effect

Cranberry Juice

No effect

No effect

TUMS® '

No effect

No effect

Orange Juice

No effect

False negative

Cola

No effect

No effect

Cough Syrup

No effect

False negative

Antiseptic

No effect

No effect

Water

No effect

No effect

shown in Table 3. The exact mechanism for some adulterants affecting the EIA assays is not known; but most likely this is an effect of pH. It should be noted that particular attention should be given to waiting 5-10 min prior to collection, which will remedy these effects.

5. Intercept Oral-Fluid Sample GC-MS Confirmation Method

In the algorithm used for Intercept, a presumptive positive specimen requires a GC-MS-MS analysis to assure true positivity. However, the levels to be determined require improved procedures compared with those commonly used for urine testing. Although numerous instruments are available that are capable of performing Intercept confirmations, the general steps and target ions would be the same. Therefore, this section presents a sample procedure used for the most difficult analyte, THC (8), followed by critical factors used to identify other typical DOA targets. Ultimately, each laboratory will validate its equipment and adopted procedures.

Quantitative analysis of THC in oral-fluid specimens can be performed by GC MS MS on a Finnegan TSQ 7000 Triple Stage Quadrupole (ThermoQuest, San Jose, CA) equipped with a 5% phenyl methyl silicone capillary column (15-m x 0.25-mm i.d.). The capillary inlet system can be operated in the split-less mode. Instrumental conditions were as follows: injection port, 275°C; GC temperature program, 100°C for 0.5 min, ramp 45°C/min to 235°C, hold 1.5 min, ramp 45°C/min to 310°C, and hold 1.5 min; transfer line, 250°C; source, 200°C; manifold, 90°C. A total of 200 ^L of each oral-fluid specimen was used in the extraction procedure. Initially, internal standard (D3-THC) at a concentration of 0.5 ng/mL was added to each specimen, calibrator, and control sample. Each sample was treated with 2 mL of 0.2 M NaOH and 3 mL of hexane: ethyl acetate (9: 1 v/v). The tubes were rocked for 30 min and then cen-trifuged. The upper organic layer was removed, acidified with 3 mL of 0.1 M HCl, and rocked an additional 15 min. Following centrifugation, the upper organic layer was removed and evaporated to dryness at 40°C. The residue was derivatized with 30 |L of bis(trimethylsilyl)-trifluoroacetamide (BSTFA) (1% trimethylchlorosilane [TMCS]) and 30 |L of ethyl acetate at 70°C for 30 min. A calibration standard of THC was prepared for each batch at 0.5 ng/mL concentration in artificial saliva (certified blank matrix). The following parent ions were selected for each compound to form product ions: THC, m/z 386 and D3-THC, m/z 389. The following product ions were selected for quantitation: THC, m/z 371 and D3-THC, m/z 374. For a specimen to be considered positive for THC, both the parent and product ions had to be present and within 2% of the retention time of the calibrator. In addition, the area of each ion had to be greater than the corresponding area of the ion in the calibration standard. The assay exhibited a between-run precision for THC in oral-fluid specimens of 4.3% at 0.25 ng/mL and 9.5% at 1 ng/mL. The assay limit of quantitation (LOQ)/LOD for THC was 0.2 ng/mL for a 0.2-mL extracted specimen.

Other compounds can be similarly analyzed. For reference, Table 4 lists the target analytes and their target ions that may be detected in oral fluids, and Table 5 shows typical LOQ/LOD values of various drugs of abuse obtained by GC-MS-MS using Intercept diluent. The target analytes, in some cases, are similar to those in urinalysis, but in other cases, such as with THC and cocaine (benzoylecgonine [BE]), they identify the compounds specific to oral fluids. Laboratories working with Intercept samples should validate their own instruments and methods.

Confirmation of presumptively positive specimens is perhaps the single most important procedure for any testing laboratory. The confirmation result will be the focus of any contested tests in a court of law. The defensibility of the procedure will require detailed attention to all aspects of the analysis. Therefore, the above information serves to provide some insight to potential approaches and expected results for confirmation of Intercept samples.

6. Analysis of Intercept Testing Results

The Intercept device has been reviewed by the US Food and Drug Administration (FDA) for all of the assays discussed in this chapter. The Intercept system for oral-fluid analysis has been further tested in a large number of studies. The largest study to date includes an overall analysis of 77,000 specimens

Table 4

Ions Monitored for Internal Standard and Analytes

Table 4

Ions Monitored for Internal Standard and Analytes

Analyte

Description

Quant Ions

THC d-3

Internal Std

238

THC

Std

238

BE d-3

Internal Std

303

BE

Std

30G

PCP d-5

Internal Std

84 + 122

PCP

Std

84 + 117

Codeine d-3

Internal Std

285

Codeine

Std

282

Dihydrocodeine

Std

285

Morphine d-3

Internal Std

269 + 27G

Morphine

Std

266 + 267

6-MAM

Std

266 + 267

Methamphetamine d-11

Internal Std

26G

Methamphetamine

Std

254

MDMA

Std

254

MDEA

Std

268

Pseudoephedrine

Std

254

Amphetamine d-11

Internal Std

244

Amphetamine

Std

24G

MDA

Std

162

THC, tetrahydrocannabinol; BE, benzoylecgonine; PCP, phencyclidine; 6-MAM; 6-monoacetylmorphine; MDMA, 3, 4-methylenedioxymethamphetamine; MDEA, 3,4 methylenedioxyethylamphetamine; MDA, methylenedioxyamphetamine.

THC, tetrahydrocannabinol; BE, benzoylecgonine; PCP, phencyclidine; 6-MAM; 6-monoacetylmorphine; MDMA, 3, 4-methylenedioxymethamphetamine; MDEA, 3,4 methylenedioxyethylamphetamine; MDA, methylenedioxyamphetamine.

submitted to a reference laboratory over a 12-mo period (11). The results for the common five-panel DOA were compared to urine results within the same laboratory and also to the Quest laboratory data base for urine testing. Tables 6-8 show that, overall, oral-fluid results obtained by the Intercept system are comparable to those found with urine testing. This is somewhat surprising considering the shorter window of detection of drugs in oral fluid. Possible reasons for these results include broad use of urine adulterants masking many positives or the fact that many individuals abusing drugs have ingested substances close to the time of their sample collection. In either case, it suggests that oral fluid is a good alternative to urine testing for routine pre-employment testing.

7. Conclusions

DOA testing has become routine in many aspects of life. An established algorithm, which appears to be universally accepted, utilizes antibody-based tests

Table 5

Typical Limit of Quantitation (LOQ)/Limit of Detection (LOD) of Various Drugs of Abuse Obtained by GC-MS-MS Using Intercept® Diluent

Table 5

Typical Limit of Quantitation (LOQ)/Limit of Detection (LOD) of Various Drugs of Abuse Obtained by GC-MS-MS Using Intercept® Diluent

Target Analyte

LOQ (ng/mL)

LOD (ng/mL)

Amphetamine

10.0

1.0

Codeine

5.0

2.5

MDMA, MDEA, MDA

1.0

0.5

BE

2.5

1.25

Morphine

5.0

2.5

Methamphetamine

1.0

0.5

PCP

0.5

0.25

THC

0.5

0.25

6-MAM

1.0

0.5

GC-MS-MS, gas chromatography-tandem mass spectrometry; MDMA, 3, 4-methylenedioxymethamphetamine; MDEA, 3,4 methylenedioxyethylamphetamine; MDA, methylenedioxyamphetamine; BE, benzoylecgonine; PCP, phencyclidine; THC, tetrahydrocannabinol; 6-MAM ; 6-monoacetylmorphine.

GC-MS-MS, gas chromatography-tandem mass spectrometry; MDMA, 3, 4-methylenedioxymethamphetamine; MDEA, 3,4 methylenedioxyethylamphetamine; MDA, methylenedioxyamphetamine; BE, benzoylecgonine; PCP, phencyclidine; THC, tetrahydrocannabinol; 6-MAM ; 6-monoacetylmorphine.

Table 6

Cutoff Concentrations in Oral-Fluid Specimens Tested by Intercept® (Whole Saliva) and in GC-MS-MS Confirmation Assays

Cutoff concentrations

Assays oral fluid (ng/mL)

Initial Test (Intercept®)

THC (Parent Drug and Metabolite) 3

Cocaine Metabolites 15

Opiate Metabolites 30

Phencyclidine 3

Amphetamines 120 Confirmatory Test

THC (Parent Drug) 1.5

Benzoylecgonine 6

Morphine 30

Codeine 30

6-Acetylmorphine 3

Phencyclidine 1 . 5

Amphetamine 120

Methamphetamine 120

Table 7

Overall Confirmed Positive Rates for Oral-Fluid Specimens Tested in LabOne Over a 10-mo Period (Jan. 2QQ1-Oct. 2001)*

Table 7

Overall Confirmed Positive Rates for Oral-Fluid Specimens Tested in LabOne Over a 10-mo Period (Jan. 2QQ1-Oct. 2001)*

Oral fluid specimens

Number of

(n = 77,218)

specimens

% Positive

Confirmed Positive Tests

3,908

5.06

THC (Parent)

2,486

3.22

Cocaine

865

1.12

Opiates

175

0.23

Phencyclidine

21

0.03

Amphetamine/Methamphetamine

361

0.47

*6-Acetylmorphine was only tested for morphine positives and is not included in the overall total number of positives.

*6-Acetylmorphine was only tested for morphine positives and is not included in the overall total number of positives.

Table 8

Comparison of Positive Drug Prevalence Rate Found in Oral-Fluid Testing With Federally Mandated and General Workforce Urine Drug Testing Programs According to Quest Diagnostics' Drug Testing Index

Table 8

Comparison of Positive Drug Prevalence Rate Found in Oral-Fluid Testing With Federally Mandated and General Workforce Urine Drug Testing Programs According to Quest Diagnostics' Drug Testing Index

Drug Category

Positivity prevalence rate: oral fluid drug testing Jan.-Oct. 2001 (n = 77,218)

Drug testing index: federally mandated urine drug testing* Jan .-Dec. 2001 (n = 1,000,000)

Drug testing index: general workforce urine drug testing* Jan .-Dec. 2001 (n = 5,200,000)

THC

3.22

1.72

3.17

Cocaine

1.12

0.60

0.69

Opiates

0.23

0.26

0.29

Phencyclidine

0.03

0.05

0.02

Amphetamines

0.47

0.29

0.29

TOTAL

5.06

2.92

4.46

*Urine test date according to Quest Diagnostics' Drug Testing Index for workplace drug tests performed January to December, 2001 by Quest Diag. (data source can be found at http://www. questdiagnostics.com/business/b_bus_lab_emp_drugtesting_index.html)

*Urine test date according to Quest Diagnostics' Drug Testing Index for workplace drug tests performed January to December, 2001 by Quest Diag. (data source can be found at http://www. questdiagnostics.com/business/b_bus_lab_emp_drugtesting_index.html)

for screening of presumptive positive samples. These presumptive positive samples are then confirmed using a combination of GC and MS. In the early days of drug testing, urinalysis went through a series of changes and modifications until reliable and legally defensible procedures were established. In many ways, oral-fluid testing has followed a similar path, with some exceptions. In oral-fluid testing, significant advances in screening and confirmation technologies have allowed the use of comparatively minute amounts of sample with similarly reliable results. In addition, positive oral-fluid testing can be indicative of a more recent time frame of drug use. It is expected that future technological development will make it possible to correlate oral-fluid testing with drug impairment and not just drug presence, as is currently possible with blood samples (12-16).

References

1. Substance Abuse and Mental Health Administration. Mandatory Guidelines for Federal Workplace Programs. Fed Regist 1988;53:11,970-11,989.

2. Verstraete A. Roadside Testing Assessment (www.ROSITA.org), 2000.

3. Road Safety (Drug Driving) Act 2003. Parliament of Victoria, Australia, 2003.

4. Malamud D and Tabak L. Saliva as a diagnostic fluid. Ann NY Acad Sci 1993; 694:276-279.

5. Samyn N, Verstraete A, van Haeren C, and Kintz P Analysis of drugs of abuse in saliva. For Sci Rev 1999;11(1):1-19.

6. Mucklow JC., Bending MR, Kahn GC, and Dollery CT. Drug concentration in saliva. Clin Pharmacol Ther 1978;24:563-570.

7. DiGregorio GJ, Piraino A, Nagle B, and Knaiz E. Basic biological sciences— secretion of drugs by the parotid glands of rats and human beings. J Dental Res 1977;56(5):502-50.

8. Niedbala RS, Kardos KW, Fritch, D. F., et al. Detection of marijuana use by oral fluid and urine analysis following single-dose administration of smoked and oral marijuana. J Anal Toxicol 2001;25:289-303.

9. Niedbala RS, Kardos KW, Fries T, and Davis A. Immunoassay for detection of cocaine/metabolites in oral fluids. J Anal Toxicol 2001;25:62-68.

10. Niedbala RS, Kardos K, Waga J, et al. Laboratory analysis of remotely collected oral fluid specimens for opiates by immunoassay. J. Anal. Toxicol. 2001;25:310-315.

11. Cone E, Presley L, Lehrer M, et al. Oral fluid testing for drugs of abuse: positive prevalence rates by Intercept™ immunoassay screening and GC-MS-MS confirmation and suggested cutoff concentrations. J Anal Toxicol 2003;27:169-172/

12. Cone EJ, Kumor K, Thompson LK, and Sherer M. Correlation of saliva cocaine levels with plasma levels and with pharmacologic effects after intravenous cocaine administration in human subjects. J Anal Toxicol 1988; 12, 200-206.

13. Menkes DB, Howard RC, Spears GFS, and Cairns ER. Salivary THC following cannabis smoking correlates with subjective intoxication and heart rate. Psycho-pharmacology 1991;103:277-279.

14. Chait LD and Zacny JP. Reinforcing and subjective effects of oral A9-THC and smoked marijuana in humans. Psychopharmacology 1992;107:255-262.

15. O'Neal CL, Crouch DJ, Rollins DE, Fatah A, and Cheever ML. Correlation of saliva codeine concentrations with plasma concentrations after oral codeine administration. J Anal Toxicol 1999;23:452-459.

16. Pitts FN, Allen RE, Aniline O, and Yago LS. Occupational intoxication and long-term persistence of phencyclidine (PCP) in law enforcement personnel. Clin Toxicol 1981;18(9): 1015-1020.

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