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

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