Franz Aberl and Robert Van Dine

Summary

Drugwipe® (Securetec Detektions-Systeme AG) is a pen-size detector for illegal drugs in saliva, in sweat, and on surfaces. It was first launched in 1995 to support drug law-enforcement police in their operations against smuggling and dealing of contraband. In 1996 the US Office for National Drug Control Policy (ONDCP) tested Drugwipe for its accuracy, sensitivity, and specificity in detecting invisible traces of narcotics on surfaces (1). Since then, Drugwipe has been included in the technology transfer program of ONDCP.

With the increasing interest in saliva and sweat testing on the part of traffic police, the Drugwipe device has been significantly improved over the years. Today, Drugwipe is available for the detection of cocaine, opiates, cannabinoids, benzodiazepines, and amphetamines/methamphetamines ("ecstasy"). Drugwipe can be used to test oral fluids or sweat samples, or to detect invisible traces of narcotics. Commercially available Drug-wipes include single, twin, and five-panel configurations. Drugwipe is especially designed for on-site applications and combines easy and rapid sampling with fast analysis. Drug-wipe is used widely in Germany as a routine sweat or saliva test for roadside screening for driving under the influence of drugs (DUID). In the current Roadside Testing Assessment (ROSITA) II project, Drugwipe is under evaluation as a saliva test. The basic technology for analysis is lateral-flow immunoassay (see Chapters 3-6).

This chapter will first describe the technological basis of Drugwipe, including its major technical features. The second part will cover the various evaluation studies that

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

have been performed using Drugwipe under controlled and general field conditions. Some of the data are not yet published.

Drugwipe can detect various benzodiazepines to as low as 5 ng/mL, and A9-tetrahydrocannabinol (THC) can be detected at 30 ng/mL. These sensitivities are currently unique for point-of-collection oral fluid/sweat test kits. The second part of this paper summarizes various published and unpublished data from trials and studies under controlled and general field conditions. Based on 1763 cases, a statistical evaluation by traffic police in Germany shows that more than 97% of all positive Drugwipe sweat tests are confirmed with positive blood results.

1. Technical Description of Drugwipe

The product concept of Drugwipe is guided by user and operational requirements of law-enforcement units around the world. The testing procedures are similar to a laboratory process and consist of sampling, sample transfer, sample preparation, analysis, and output of the result. All of these procedures are integrated into a single-unit device. The sampling step is based on wiping. Wiping is fast, easy, and requires very little cooperation of the person under evaluation. Optimal sample transfer is guaranteed by the geometric design of the device. The lateral-flow immunoassay is specifically optimized to analyze various sample materials for the native drug. The signal output is simply visual and unambiguous.

1.1. Design of the Device

Figure 1 shows the major components of Drugwipe. The wiping element is designed for the collection of various types of samples. The collection step itself consists of a sequence of wipes. This sequence differs from specimen to specimen and is standardized according to the type of specimen. Next, the sample is transferred to a lateral-flow immunoassay strip sitting inside the detection element. The design of the wiping and the detection element guarantees automatic and efficient sample transfer.

Analysis of the sample starts with dipping of the Drugwipe absorbent pad into a small container of tap water for 15 s. The water container is part of the Drugwipe device and holds the correct amount of water to properly develop the test result. A positive test result develops within 2 to 5 min in the readout window, in the form of one line on a single-parameter strip and two red lines on a double-parameter strip. The time depends on the concentration and the type of drug to be analyzed, with high concentrations showing results quicker than low concentrations. In addition, a single red line has to appear in the internal control region. The appearance of only a control line indicates a negative result, confirming the correct usage of the device and the absence of interfering substances. A positive test result is shown as a second red line in the read-

Wiping Element (Sample collector)

Wiping

Wiping Element (Sample collector)

Fleece

Absorbant

Fig. 1. Drugwipe® components.

Fleece

Absorbant

Detection Element

Water Container

Fig. 1. Drugwipe® components.

out window. This form of presenting test results is unique in the area of lateral-flow assays for small molecules (e.g., drugs of abuse). The underlying principle is explained later.

1.2. Test Principle

Lateral-flow immunoassays utilize the recognition and binding capabilities of antibodies to differentiate between the presence and absence of a particular analyte. The detection limit is mainly influenced by the affinity of the selected antibodies for the target analyte. The Drugwipe immunoassay follows the general principles of other lateral-flow immunoassays for small molecules, with the major exception that a positive test result is correlated with the appearance of a test line. The Drugwipe lateral-flow immunoassay strip is schematically shown in Fig. 2.

Through the connection of the wiping element to the detection element, the sample medium (sweat, saliva) is automatically transferred into the sample application zone (area 2). This zone contains drug-specific antibodies coupled to colloidal gold particles. By means of the absorbent pad (area 1), water is drawn into the cassette and applied to the assay strip in a controlled process. The water supports the migration of the sample and the antibody conjugate along the strip. In the case of a sample containing drug molecules, the binding sites of the antibodies are saturated with the complementary hapten (drug) in the sample application zone. Downstream of the sample application zone, sample and antibody conjugate pass through a capture zone (area 3). This zone separates drug-saturated antibody-gold complexes from gold-antibody conjugates without the

Flow

4) Result Read Out Area

2) Sample Application Zone 3) Capture Zone

Fig. 2. Schematic drawing of the Drugwipe® lateral-flow immunoassay.

drug molecules. Only those gold conjugates that have been loaded with drug molecules are able to pass the capture zone and to migrate into the result readout area (area 4). In the result readout area, the gold conjugates are retained in a linear form on the strip in designated positions. Various design options for this area are possible. Figure 3 shows the test result for a five-panel Drugwipe test. The test is positive for all drugs.

1.3. Main Technical Features of the Drugwipe Device

Drugwipe provides detection capabilities for various drugs of abuse while maintaining maximum operational flexibility. Drugwipe is available in different test configurations:

• Single tests for cannabis, cocaine, opiates, amphetamines/methamphetamines, and benzodiazepines;

• Triple and twin test devices for amphetamines/methamphetamines/cannabis and cocaine/opiates;

• Five-panel device for cannabis, cocaine, opiates, amphetamines/methamphetamines (including "ecstasy").

Sweat or saliva samples can be analyzed according to the specimen available and operational needs. A sample volume of less than 10 ^L is sufficient for analysis. The small sample volume is a critical feature for point-of-collection (POC) testing. A significant percentage of drug consumers abusing designer drugs or cannabis suffer from a dry-mouth syndrome and are not able to provide sufficient saliva for testing in conventional devices.

The cut-off values are identical for each test configuration and are given in Table 1 .

All Drugwipe types are directed toward the native drugs that are the dominating compounds in sweat or saliva. The most sensitive assay is the Drugwipe

1) Absorbant Pad

Fig. 3. A five-panel Drugwipe® test—positive for cannabis, amphetamines, methamphetamines, cocaine, and opiates.

test for benzodiazepines. Aminoflunitrazepam can be detected in oral fluid down to a concentration of 5 ng/mL. Characteristic of the amphetamine assay is its broad cross-reactivity, covering a range of five important amphetamines and methamphetamines.

1.4. Collection of Saliva Samples

Salivary glands continuously secrete a mucous, colorless fluid into the mouth. The composition of saliva is determined by the composition of the blood plasma and the physico-chemical properties of the plasma compounds. Drugs of abuse are excreted in higher or lower concentrations than present in plasma.

When testing with Drugwipe, saliva is taken from inside the cheek or directly from the tongue. The wiping fleece is firmly wiped three times over the mucus membrane on each side. The saliva volume is defined by the void volume of the wiping fleece.

1.5. Collection of Sweat Samples

Sweating is described as a continuous excretion of water and small molecules through the skin. Sweat is produced as the body's response to exercise

Table 1 Drugwipe® Cut-Off Values

Cut-off in ng/mL

Cannabis

A-9-tetrahydrocannabinol

30

Cocaine

Cocaine

50

Opiates

Heroin

20

Morphine

20

Amphetamines

d-Amphetamine

200

Methylenedioxymethamphetamine

100

d-Methamphetamine

100

Methylenedioxyampthetamine

100

Methylenedioxyethamphetamine

500

Benzodiazepines

Aminofluni tazepam

5

Flunitrazepam

10

Nitrazepam

10

Temazepam

10

Diazepam

10

or thermal stress. The water evaporates and low-volatile compounds like drugs of abuse remain on the skin for a limited time. Sweat samples can be taken from various parts of the body surface. The forehead is a compromise among excretion characteristics, accessibility, and contamination risk. In earlier studies, Drugwipe was used to collect sweat from the armpit, but this sampling location has operational disadvantages.

1.6. The Drugwipe Reader

A laptop-/palmtop-controlled reader is available for recording of the Drugwipe result in an electronic format. Figure 4 is a picture of the reader, which is marketed under the brand name DrugRead® (Securetec). DrugRead is advantageous under poor light conditions or when the Drugwipe result must be obtained independent of a visual interpretation. A further benefit is that all test data are stored on a hard disk and can be further processed (e.g., mathematically interpreted) or printed. Calibration curves for the different target drugs can be implemented and used to correlate the Drugwipe signal with certain drug quantities. An example for a correlation curve is given in Fig. 5.

The individual points in the calibration curve are mean values of 10 single measurements. Drugwipe starts to show positive signals between 20 and 30 ng of A-9-THC per mL of saliva.

Fig. 4. DrugRead®—the Drugwipe® reader.

2. Drugwipe as a Saliva Test

2.1. Roadside Drug Testing Assessment (ROSITA II): Confirmation of the Drugwipe Cut-Off Values (2,3)

In 2003, the Office of National Drug Control Policy (ONDCP) and the National Highway Traffic Safety Administration (NHTSA) sponsored an initial comparative laboratory evaluation of eight commercially available oral-fluid testing devices. Under supervision of the Walsh Group (TWG), the Center for Human Toxicology (CHT) of the University of Utah conducted the analytical evaluation at their laboratories in Salt Lake City, UT.

Human oral fluid was collected from drug-free individuals, pooled, and purified by freezing, thawing, and centrifugation. Standard solutions were prepared through the addition of known amounts of drugs to the purified saliva. The fortified solutions were assayed by gas chromatography (GC)-mass spec-trometry (MS) or liquid chromatography (LC)-MS for the quantitative levels of spiked drugs. Drug-free saliva was used as negative control.

In the case of Drugwipe, 10 ^L of each drug-saliva solution was added to the wiping fleece and the test was performed as described in the instructions for use (4). The negative control experiments were repeated five times, and spiked

l/S

225

<D

H

200

14-

175

o

£

150

isl c 0)

0) c

125

c

100

0)

75

>

50

ra 01

25

et

0

10 100 1000 10000 Concentration delta 9-THC in ng/mi

Fig. 5. DrugRead® calibration curve for Drugwipe® Cannabis. The relative intensity of the test line shown is dependent on the concentration of A9-tetrahydrocannabinol.

(positive) saliva samples were tested 10 times. The applied concentrations of drugs were selected in such a way that the cut-off values of Drugwipe were challenged. Three concentration levels of each drug were applied.

The Drugwipe evaluation results are summarized in Table 2.

For each Drugwipe type, the official Drugwipe cut-off level is also given in Table 2. The percentage of correct results represents the proportion of Drug-wipe data that correctly identifies control vs drug-spiked samples. Drug-free and fortified saliva samples with drug concentrations below the official cut-off level of the device were scored as negatives. Saliva samples with drug concentrations above the cut-off level were expected positives. At the time of evaluation, the Drugwipe amphetamines testing device did detected the100-ng/mL concentration with low signal intensity. After this evaluation campaign, the manufacturer of Drugwipe lowered the official cut-off level for amphetamines to 200 |g/mL.

Overall, Drugwipe was 90% accurate in identifying positive saliva samples with drug concentrations above the cut-off levels and 100% accurate in identifying negative saliva samples without or spiked with drug concentrations below the cut-off levels. In this study, Drugwipe was the only instrument-free device that performed according to the specifications and was able to detect THC concentrations at the 50 ng/mL level. The 30 ng/mL level (THC cut-off) was not challenged. THC is the most prevalent drug in the driving population, and a low cut-off for THC is critical for roadside applications.

Table 2

Independent Evaluation of the Drugwipe® Cut-Off Values With Spiked Saliva Samples

Drugwipe type Cannabis Amphetamines Methamphetamines Cocaine Opiates

Cut-off value in ng/mL

30

200

100

50

20

Cone, in

Correct

Cone, in

Correct

Cone, in

Correct

Cone, in

Correct

Cone, in

Correct

ng/mL

results in %

ng/mL

results in %

ng/mL

results in %

ng/mL

results in %

ng/mL

results in %

Negative saliva

0

100

0

10

0

100

0

100

0

10

Positive saliva

Level I

20

100

25

100

25

100

10

100

20

40

Level II

50

100

100

100*

100

100

40

100

100

90

Level III

100

100

500

100

500

100

200

100

500

100

*See text for explanation.

*See text for explanation.

2.2. Testing Saliva With Drugwipe/Drugread: A Controlled Study With Methylenedioxymethamphetamine (5)

In 1999, the Instituto Municipal d'Investigació Médica (IMIM) in Barcelona, Spain, performed a controlled, double-blinded study with methylenedioxymethamphetamine (MDMA; "ecstasy") and eight volunteers. Each subject was orally administered a single dose of 100 mg MDMA or placebo. At time zero (0, predose) and at 1.5, 4, 6, 10, and 24 h after MDMA administration, a sample of saliva (1-2 mL) was collected by spitting into a plastic tube and immediately stored at -20°C. Samples were analyzed for MDMA and metabolites by GC-MS. In this study, 2 ^L of each saliva sample were applied to the wiping fleece of Drugwipe Amphetamines (test kit for amphetamines), and the test was performed according to the instructions for use. Drugwipe results were measured with the DrugRead device and compared with the MDMA concentrations determined by GC-MS.

Figure 6 shows the concentration curve of MDMA in the saliva of one of the volunteers after oral administration of 100 mg of MDMA. The maximum in the concentration curve is reached after approx 2 h. The concentration of MDMA in saliva at this point is more than 3000 ng/mL. The window of detection with GC-MS is at least 24 h. The window of detection with Drugwipe/ DrugRead is approx 10 to 12 h and can be adjusted by changing the cut-off value of the Drugwipe/DrugRead system. All subjects showed a similar concentration curve in saliva, and Drugwipe gave a positive result for all volunteers at 1.5 and 4 h after drug administration. After 6 h, only one out of eight subjects gave a negative result. This subject showed the lowest MDMA concentrations among all the volunteers at that time. At 10 h after MDMA administration, it was still possible to detect consumption in five of the eight subjects. For the three subjects who had negative test results, the concentrations of MDMA in their saliva as determined by GC-MS were below the cut-off levels of the Drugwipe device. At 24 h, no positive results were reported.

The concentration curve of the Drugwipe/DrugRead system correlated well with the concentration curve measured with GC-MS. Provided that the sample volume is thoroughly controlled and the Drugwipe/DrugRead system is calibrated to a specific drug, quantitative measurements can be performed. The apparent slower disappearance rate in the DrugRead signal was probably due more to a saturation effect in the Drugwipe test than to the contribution of MDMA metabolites. In fact, MDMA was reported as the principal analyte that could be detected in saliva, while its principal metabolites were found only in minute amounts. Overall, the Drugwipe/DrugRead system not only detects consumption but also recent use of MDMA.

0 2 4 6 8 10 12 14 16 18 20 22 24 Time after Administration in h

Fig. 6. Concentration curve of methylenedioxymethamphetamine (MDMA) in saliva measured with gas chromatography-mass spectrometry and with Drugwipe®/ DrugRead® after a controlled oral administration of 100 mg MDMA.

2.3. Drugwipe Amphetamines: Sweat vs Saliva in a Driver Population

In the winter of 2003, Drugwipe Amphetamine was evaluated by the Institute for Legal Medicine at the University of Munich, Germany, for sensitivity, specificity, and accuracy. Traffic police in Munich selected drivers from the street who were suspected of DUID. Drivers were brought to the Institute for Legal Medicine for blood samples. At the same time, sweat from the forehead and from inside the palm, as well as saliva, were collected with the Drugwipe device. All Drugwipe tests were administered on a voluntary basis. All volunteers were tested three times (one saliva test and two sweat tests). In the final analysis, Drugwipe test results were correlated to those of the blood samples.

Table 3 summarizes the results of this comparative study with Drugwipe Amphetamines. Accuracy in the context of this study describes how reliable Drugwipe Amphetamines is in predicting the blood results of a suspected driver. Sensitivity refers to the percentage of positive blood samples, and specificity the percentage of negative blood samples corrected identified by Drug-wipe. Between 76 and 80 persons were tested with Drugwipe and approx 30% were tested positive in the blood for amphetamines or ecstasy. Seventy percent were amphetamine free. The accuracy of Drugwipe Amphetamines was in the range of 92 to 97% depending on the specimen and the sampling location on the body. Specificity ranged between 89 and 96%, whereas sensitivity was always 100%. It was also noted that the best accuracy and specificity were achieved with sweat samples from inside the palm of the hand. Saliva samples and sweat

0 2 4 6 8 10 12 14 16 18 20 22 24 Time after Administration in h

Fig. 6. Concentration curve of methylenedioxymethamphetamine (MDMA) in saliva measured with gas chromatography-mass spectrometry and with Drugwipe®/ DrugRead® after a controlled oral administration of 100 mg MDMA.

Accuracy, Sensitivity, and Specificity of the Drugwipe® Results in Relation to the Blood Status of Drivers Suspected for Driving Under the Influence of Drugs

Specimen

Sampling location

Accuracy

Sensitivity

Specificity

Number of cases

Sweat/skin

Forehead

92%

100%

89%

80

Inside the

97%

100%

96%

76

palm

Saliva

Tongue

94%

10%

91%

78

taken from the forehead were comparable with each other in terms of accuracy and specificity but were obviously not as good as palm sweat. It appears that contaminants on the hand have no major influence on assay accuracy.

The findings of this study are in contrast with the work of other researchers, who claim that sweat is of less value for the detection of recent drug use. In this study, testing of sweat has been shown to be comparable to saliva testing in terms of assay accuracy, specificity, and sensitivity.

3. Drugwipe as a Sweat Test

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