The potential of oral fluid as a specimen in various drug testing programs

Transcription

1 The potential of oral fluid as a specimen in various drug testing programs *Christine Moore, Cynthia Coulter, Katherine Crompton, Warren Rodrigues, Michael Vincent, James Soares Immunalysis Corporation, 829 Towne Center Drive, Pomona, CA Tel: Fax: cmoore@immunalysis.com Abstract The purpose of this study was to determine the utility of oral fluid as a specimen for drug testing in various areas, such as traffic safety, workplace programs, pain management, and school drug testing. While saliva testing offers advantages over urinalysis, specifically ease of collection and difficulty of adulteration, its implementation on a routine basis has been hampered by insufficient or unknown volume, and lack of sensitivity of detection methods. In this report, neat oral fluid is collected (1 ml), using a Quantisal device, which dilutes the specimen with transportation buffer (3 ml). Adequate volume is then available to screen for multiple drug classes, at pharmacologically relevant concentrations using ELISA. Sufficient volume remains for the confirmation of many drug classes assuming multiple positive screens were obtained. Confirmatory assays were performed using GC/MS or LC/MS/MS.

2 1. Introduction Various laboratories offer drug test panels targeted at specific area of interest, including workplace, Medical or Healthcare Professional panels, traffic safety and school testing. Each area may require a specific drug or drug class to be added to the test. For example, a Med Pro panel is targeted at the detection of prescription and pain medications as well as the more common drugs of abuse. Workplace testing may only identify a small number of problem drugs, and traffic safety may require as wide a range of drug detection as possible. While blood and urine are more commonly used for these test profiles, oral fluid is increasing in popularity, due to its ease of collection, difficulty of adulteration and improving sensitivity of analytical techniques. There are many publications on the use of oral fluid in drug analysis, and most of these involve chromatographic screening if a wide range of drugs is monitored [1,2]. In this paper we describe the screening of oral fluid using immunoassay, which is faster and cheaper than chromatographic techniques, leaving adequate specimen volume for confirmatory techniques if necessary. One of the main issues with the quantitation of drugs in oral fluid is the difficulty of collection in terms of specimen volume. Some of the currently available devices do not indicate how much oral fluid is collected, thereby rendering any quantitative results meaningless without further manipulation in the laboratory [3]. Collection devices incorporating a pad or material for the saliva collection also need to be tested to determine how much drug is recovered from the pad before analysis, so that confidence in the quantitative result can be enhanced. This work employed the Quantisal oral fluid collection device, which collects a known amount of neat oral fluid. The efficiency of recovery of many drugs from the collection pad into the transportation buffer was determined, as well as the stability of the drugs in the buffer at room

3 temperature and at 4 o C. The procedures are currently in use in the National Roadside Survey for Experimental 2.1. Oral fluid collection devices Quantisal TM devices for the collection of oral fluid were used. The devices contain a collection pad with a volume adequacy indicator, which turns blue when one milliliter of oral fluid (+/- 10%) has been collected. The pad is then placed into transport buffer (3 ml), allowing a total specimen volume available for analysis of 4 ml (3 ml buffer + 1 ml oral fluid). This is specifically advantageous in cases where the specimen is positive for more than one drug and the volume of specimen available for analysis may be an issue. The oral fluid concentration is diluted 1:3 when using Quantisal TM collection devices, and drug concentrations detected were adjusted accordingly. 2.2 Screening specimens using immunoassay Oral fluid samples can be analyzed rapidly and inexpensively using immunoassay platforms, specifically enzyme linked immunosorbent assays (ELISA) [8,13]. ELISA technology is based upon the competitive binding to antibody of enzyme labeled antigen and unlabeled antigen in proportion to their concentration in the reaction well. The sensitivity of ELISA allows low amounts of sample to be used in order to provide adequate separation at relevant concentrations. Collection devices containing sodium azide in the extraction / transportation buffer, would be problematic in ELISA screening procedures using horse radish peroxidase as the enzyme label, and are not recommended.

4 All ELISA kits were obtained from Immunalysis Corporation (Pomona, CA). Calibrators and controls were diluted 1:3 with Quantisal buffer prior to the assay. The initial dilution ensures that the concentrations reported are already converted to neat saliva. Calibrators, controls, or oral fluid was aliquoted into the corresponding ELISA plate. The corresponding enzyme conjugate (100 μl) was added to each well and the plates were incubated at room temperature for 60 minutes. The wells were washed six times with deionized water (6 x 350 μl). The wells were inverted and vigorously slapped dry on absorbent paper to ensure all residual moisture was removed. Substrate reagent (100 μl) was added and the plates were further incubated for 30 minutes. Finally, dilute acid (100 μl) was added to stop the reaction and the plates were read at a dual wavelength of 450nm and 650 nm. Cannabinoids An exception to the protocol was for the detection of cannabinoids in oral fluid, which required an additional pre-incubation step in order to achieve the sensitivity required for a screening cut-off concentration of 4 ng/ml. Calibrators and controls were diluted 1:3 before the assay. An aliquot (25 μl) of calibrator, control or specimen was added to each well of the plate. Pre-incubation buffer (25 μl) was added and the plates were incubated at room temperature for one hour. Enzyme conjugate (50 μl) was then added and the plates were incubated for 30 minutes. The wells were washed six times with deionized water (6 x 350 μl). Substrate reagent (100 μl) was added and the plates were further incubated for 30 minutes. Finally, dilute acid (100 μl) was added to stop the reaction and the plates were read at a dual wavelength of 450nm and 650 nm.

5 The specimen volumes were determined using the proposed Federal cut-off concentrations for five of the drug classes [14]; and using pharmacologically relevant concentrations for the other drugs based on literature review. The manufacturer recommended the volume of oral fluid to be placed into the microplate (Table 1). A standard curve consisting of a drug free negative oral fluid specimen, and drug free oral fluid specimens spiked at 50% and 200% of the cut-off concentrations were analyzed with every batch. The sample volume was pipetted directly from the collection device into the microplate. Specimens screening positively using ELISA, were carried forward to confirmation using chromatographic procedures. 2.3 Extraction efficiency from the pad The recovery of many of the drug classes from the collection pad has been determined and published in various journals [4 12]. Briefly, oral fluid was fortified with drugs at the screening cut-off concentration. A collection pad was placed into the fluid until the volume adequacy indicator turned blue showing that 1 ml (+-10%) of oral fluid had been absorbed. The pads were then placed into the Quantisal buffer (3 ml), capped, and allowed to remain at room temperature overnight, to simulate transportation to the laboratory. The following day, the pads were removed using serum separators, and an aliquot (1 ml) of the specimens was analyzed according to the described procedures. In general, the procedure was repeated 6 times for each drug.

6 3. Results and Discussion 3.1 Screening specimens using immunoassay The optimal sample sizes for ELISA screening assays for 20 drug classes were determined using the point at which the greatest discrimination between positive and negative samples was achieved, and was drug dependent. Once the sample volume has been determined and the degree of separation achieved at the cut-off concentration, the procedures required only 475 μl of total volume for the analysis of 20 drug classes. Between 3 and 3.5 ml of total specimen remained for confirmatory testing assuming multiple screen positives were obtained. In a normal working day (8 hours) using one instrument (depending upon the degree of automation), up to 400 oral fluid specimens were screened over the 20 drug classes. 3.2 Extraction efficiency from the pad The determination of drug recovery from the collection pad is an essential part of oral fluid method validation, since any quantitative result is affected. The recovery of most of the drugs in the study was determined as described, either in-house or by other independent research groups. All the drugs were recovered at a concentration of over 80% with the exception of secobarbital (Table 2). Since the transportation buffer is controlled at ph 6.8, acidic drugs tend not to be as efficiently removed from the collection pad. The ability to screen for a wide range of drug classes rapidly and inexpensively is an advantage for laboratories involved in high volume analysis. While some authors have suggested the replacement of immunoassay with liquid chromatography with tandem mass spectrometric detection (LC-MS-MS) [15], high volume screening will continue to

7 be significantly cheaper and easier than chromatographic techniques, particularly in test areas where positivity rates are expected to be low (e.g. workplace, school screening). 4. Summary Oral fluid is a useful matrix for the detection of drugs, and can be applied in various testing situations. The observed collection, lack of adulteration possibilities as well as improving collection and analytical techniques are continuing to enhance its utility. Using the Quantisal collection device, twenty drug classes were screened using immunoassay, consuming less than 0.5 ml of specimen. Over three milliliters of sample were then remaining for the confirmation of presumptive positives using chromatographic mass spectral techniques. 5. References 1. Wylie FM, Torrance H, Anderson RA, Oliver JS. Drugs in oral fluid Part I. Validation of an analytical procedure for licit and illicit drugs in oral fluid. Forens Sci Int 2005; 150 (2-3): Concheiro M, de Castro A, Quintela O, Cruz A. Lopez-Rivadulla M. Confirmation by LC-MS of drugs in oral fluid obtained from roadside testing. Forens Sci Int 2007; 170 (2-3): Kauert GF, Ramaekers JG, Schneider E, Moeller MR, Toennes SW. Pharmacokinetic properties of delta-9-tetrahydrocannabinol in serum and oral fluid. J Anal Toxicol 2007; 31(5): Moore C, Rana S, Coulter C. Simultaneous identification of 2-carboxytetrahydrocannabinol, tetrahydrocannabinol, cannabinol and cannabidiol in oral fluid. J Chromatogr Biomed Applns 2007; 852:

8 5. Moore C, Coulter C, Rana S, Vincent M, Soares J. Analytical procedure for the determination of the marijuana metabolite, 11-nor-Δ9-tetra-hydrocannabinol-9- carboxylic acid (THCA), in oral fluid specimens. J Anal Toxicol 2006; 30(7): Moore C, Vincent M, Rana S, Coulter C, Agrawal A, Soares J. Stability of Δ9- tetrahydrocannabinol (THC) in oral fluid using the Quantisal collection device. Forens Sci Int 2006; 164 (2-3): Quintela O, Crouch D, Andrenyak D. Recovery of drugs of abuse from the Immunalysis Quantisal oral fluid collection device. J Anal Toxicol 2006; 30: Moore C, Rana S, Coulter C. Determination of meperidine, tramadol and oxycodone in human oral fluid using solid phase extraction and gas chromatography-mass spectrometry. J Chromatogr B. Biomed Applns 2007; 850: Moore C, Coulter C, Crompton K, Zumwalt M. Determination of benzodiazepines in oral fluid using LC/MS/MS. J Anal Toxicol 2007; in press 10. Rana S, Moore C, Agrawal A, Coulter C, Vincent M, Soares J. Determination of propoxyphene in oral fluid. J Anal Toxicol 2006; 30(8): Rodrigues W, Wang G, Moore C, Agrawal A, Vincent M, Soares J. Development and validation of ELISA and GC-MS procedures for the quantification of dextromethorphan and its main metabolite dextrorphan in urine and oral fluid. J Anal Toxicol 2007; in press 12. Internal data, Immunalysis Corporation, Pomona, CA 13. Smink BE, Mathijssen MP, Lusthof KJ, de Gier JJ, Egberts AC, Uges DR. Comparison of urine and oral fluid as matrices for screening of thirty-three benzodiazepines and benzodiazepine-like substances using immunoassay and LC- MS(-MS). J Anal Toxicol 2006; 30(6):

9 14. Bush DM. The U.S. Mandatory Guidelines for Federal Workplace Drug Testing Programs: Current status and future considerations. Forens Sci Int 2007; in press 15. Allen KR, Azad R, Field HP, Blake DK. Replacement of immunoassay by LC tandem mass spectrometry for the routine measurement of drugs of abuse in oral fluid. Ann Clin Biochem 2005; 42:

10 Table 1. Volume of oral fluid required for immunoassay screening at the stated cut-off concentration Drug Cut-off concentration (ng/ml) Quantisal volume (μl) Amphetamine / Methamphetamine 50 / / 10 Barbiturates Benzodiazepines 10 1 Cannabinoids 4 25 Carisoprodol Cocaine Dextromethorphan Fluoxetine Ketamine Meperidine Methadone Methylphenidate Opiates Oxycodone Phencyclidine Propoxyphene Sertraline Tramadol Tricyclic antidepressants Zolpidem Total volume: 475 μl

11 Table 2. Drug recovery from the Quantisal collection device Drug class Mid-range concentration tested (ng/ml) Percentage recovery (%) THC; CBD; CBN; 2-c-THC ; 71.9; 79.7; THC THCA THC Amphetamine; Methamphetamine 50; ; Cocaine; Benzoylecgonine 20; ; Codeine; Morphine; 6-acetylmorphine 40; 40; ; 92.6; Methadone Oxazepam Tramadol Oxycodone Meperidine Benzodiazepines (14) 10 Range Propoxyphene Dextromethorphan Phencyclidine Secobarbital THC = tetrahydrocannabinol; CBD = cannabidiol; CBN = cannabinol; 2-c-THC = 2-carboxy- THC; THCA =11-nor-Δ 9 -tetra-hydrocannabinol-9-carboxylic acid