Chemical testing of biological fluids is the most common objective means of diagnosis of use of drugs of abuse. The presence of a drug analyte in a biological specimen can be used as evidence of recent exposure. Saliva has many advantages over both blood and urine (1–4). Saliva collection is noninvasive. Saliva testing detects primarily the parent drug. Presence of the parent drug in blood and therefore in saliva may correlate with impairment. The feasibility of detecting drugs in saliva samples obtained from impaired drivers was first investigated by Peel et al. (1). They found that the presence of drugs in saliva correlated well with officer judgments of driving while intoxicated. We describe here a recently developed rapid saliva test that is based on the principle of competitive lateral flow immunoassay. The outcome of the test was expressed electronically through the use of digital photography.
Saliva was collected using the Cozart RapiScan collection pad and tube and tested using the Cozart RapiScan Saliva Drugs Test (Cozart Bioscience Ltd., Abingdon, UK). When placed in the mouth, the collection pad absorbed exactly 1 mL of saliva as indicated by a blue indicator in the handle. The saliva-soaked pad was then placed in the tube containing elution fluid and separated from the plastic handle. The 1 mL of saliva on the cellulose pad was diluted with 2 mL of run buffer fluid to a final volume of 3 mL. Four drops of the saliva/run fluid mixture was placed in the immunoassay cartridge using a transfer pipette (Fig. 1). Once fluid started to flow by capillary action, the cartridge was inserted into the hand-held Cozart RapiScan instrument for incubation. The saliva/run fluid rehydrated gold-labeled anti-drug antibodies contained within the cartridge. This mixture traveled by capillary action across an array of immobilized drug sites. Absence of or reduction in color development at an immobilized drug position indicated drug presence. The quality control position contained anti-IgG to ascertain that complete lateral transfer of specimen had been achieved. Incubation is timed for 4 to 12 min depending on the number of test bands on the cartridge. The test results are read electronically, processed by a computer chip and displayed as a written message. After 12 min for the 5 panel test (4 min for the single tests), if the quality control was satisfied, the screen on the Cozart RapiScan reader, displayed the results as “Positive” or “Negative” for each of the five drug classes. However, the instrument can be adapted to express the outcome as percent of drug line intensity. Presence of a given drug in the saliva results in a decrease in the corresponding standard drug line intensity. Detection limits were set based on testing a large number of negative saliva samples. These do not necessarily correspond to total absence of the drug line. The back-lit screen for reading results, timing, quality control, and error messages is similar to those used in mobile phones, onsite glucose analyzers, and hand-held computers. In addition to the message, if all results are negative, a green light appears above the power switch. If any of the results are positive a red light appears. The collector pad has a dead volume of approximately 1 mL and the Cozart RapiScan cartridge requires between 0.12 and 0.15 mL for completion. The same volume is required for single, dual, or multiple drug panels. The excess volume of saliva/run fluid mixture was designed to allow confirmation to be performed. For the purposes of this study, it allowed multiple testing from the same sample tube Crossreactivity for the Cozart RapiScan was determined by dropping Cozart RapiScan run buffer spiked with drugs at the concentrations indicated in the tables into the sample well and determining the response of the Cozart RapiScan test. In all the five tests performed simultaneously, the colloidal-gold labeled drug derivatives have been tested in the same manner at 10 000 ng/mL and found not to cause a positive for the other Cozart RapiScan drug tests.
The Cozart Microplate EIA assays were used to assay the mixed saliva and run buffer fluid which remained in the collection tubes. These are antibody-coated microtiter plates employing a drug derivative that is labeled with horseradish peroxidase. In the assay 25 L sample, calibrator, or control is added to each well of the coated microtiter plate followed by 100 L of working enzyme conjugate. After a 30 min incubation the plate is washed four times with 350 L wash buffer. Then 100 L of substrate solution containing a 3,35,5-tetramethyl benzidine is added to each well and incubated for a further 30 min. Finally, 100 L of stop solution (1 M sulfuric acid) is added to each well and the absorbance is read at 450 nm within 30 min (5). Concentrations were determined from the assay calibration curve run on the same plate as the salivabuffer specimens. Concentrations shown in Table 3 are of total opiates in the run buffer-saliva mixture.
Cannabis Study Dose-response relations for the different drugs were investigated using both buffer and saliva spiked with the drugs. The test was assessed using cannabis as an example. Figure 2a shows a typical dose-response plot for delta-9-THC and delta-9-carboxylic-THC with spiked buffer; Figure 2b shows the dose response curve for spiked saliva. Figure 2b shows that the test can detect the equivalent of 10 g of cannabis resin. The cutoff concentration was set at 50 ng/mL delta-9-THC and 10 ng/mL delta-9-THC carboxylic acid respectively. Figure 2c shows the Cozart RapiScan response with spiked and unspiked buffer-saliva mixture (2:1) which was equivalent to 150 ng/mL delta-9-THC in neat saliva. Table 1 shows the cross-reactivity of the Cozart RapiScan Saliva Test for the different cannabinoids. The measured saliva/plasma ratio for THC after smoking marijuana is 10 and is a function of the time since smoking (2). Cannabinoids in saliva are probably due largely to residual cannabinoids left in the mouth during ingestion or smoking of marijuana or marijuana products. Figure 3a shows the time-dose profile for the Cozart RapiScan Cannabis Saliva Test following smoking a “joint” and Fig. 3b shows the time course of the reported effect after smoking the joint. In the three volunteers, the Cozart RapiScan test was able to detect the presence of the drug in the saliva for at least 1 h and in some cases up to 2 h post smoking. This is not long enough to coincide with the period of driving impairment which may last from 6 to 8 h or longer after smoking. At the conclusion of these studies the cutoff of the Cozart RapiScan Cannabinoids test was lowered for further field trials. The Cozart RapiScan test also showed good correlation with reported “effect” following cannabis smoking. This is in agreement with Menkes et al. (6) who found that saliva levels of cannabis correlated with rapid heart rate and psychological feelings of “high.”