Direct Semiquantitative Screening of Drugs of Abuse in Serum and

Journal of Analytical Toxicology, Vol. 23, July/August 1999

Direct Semiquantitative Screening of Drugs of Abuse in Serum and Whole Blood by Means of CEDIA | DAU Urine lmmunoassays Stefanie Iwersen-Bergmann* and Achim Schmoldt

Department of Legal Medicine, University of Hamburg, Butenfeld 34, D-22529 Hamburg, Germany

Abstract

The purpose of this study was to test the direct applicability of CEDIA DAU urine immunoassays to serum or whole blood. The performance of the urine assays for sensitive screening of amphetamines (AMP), benzoylecgonine (BZE), benzodiazepines (BENZ), methadone (MET), opiates (OPI), and tetrahydrocannabinol carboxylic acid (THCCOOH) was evaluated on the BM/Hitacbi 911 analyzer with unpretreated serum and whole blood. The limit of detection was 0 ng/mL for all tests. Cutoff values were set from 10 to 40 ng/mL for the different assays. The assays were found to be linear between the following concentrations: AMP 0-2500 ng/mL, BZE 0-1200 ng/mL, BENZ 0-1600 ng/mL, MET 0-600 ng/mL, OPI 0-720 ng/mL, and THCCOOH 24-60 ng/mL. Precision results (within run) for different concentrations were as follows: AMP 3.1-5.7%, BZE 2.4-6.6%, BENZ 4.3-8.0%, MET 2.0-5.5%, OPI 2.8-7.6%, and THCCOOH 1.4-2.4%. Between-run results were as follows: AMP 8.7-15.5%, BZE 6.4-7.5%, BENZ 8.2-15.8%, MET 2.7-5.1%, OPI 4.3-11.2%, and THCCOOH 2.6-7.4%. Sensitivity, specificity, and comparison of CEDIA semiquantitation with GC-MS quantitative results were performed on 500 original serum and whole blood samples. The data provided sufficient documentation to use the CEDIA urine-screening technique without any adaptation as a sensitive serum/whole blood screening for BZE, BENZ, MET, OPI, and THCCOOH. Se(um screening for amphetamines is not sensitive enough in the unchanged urine mode. It will require some adaptation to a serum mode (probably a higher sample volume [BM/Hitacbi 911] combined with protein precipitation of the sample).

Introduction

The widespread use of immunoassay technique for the screening of drugs of abuse in urine is based on its fast performance and good sensitivity. However, in many cases, immunological analysis of blood and serum is required, for example, when no urine sample is available. Depending on the technology of the manufacturer (FPIA, EIA, KIMS) and the measured wavelength of the analyzer, protein content or turbidity of serum or hemoglobin of whole blood samples may result in high background absorbance levels (1) or error messages (2). Therefore, numerous

*Author to whom correspondence should be addressed.

efforts have been made to adapt the urine immunoassays to serum or whole blood. Indirect application for EMIT or TDx/ADx was achieved by protein precipitation with methanol (3,4), acetone (5), trichloroacetic acid (6), or zinc sulfate (2). Other possi- bilities are the extraction of the blood sample (7,8) or simple dilution with saline (9). Disadvantages of these methods are that they are time consuming and probably result in a loss of sensitivity. This is of special interest when the result of the screening is required urgently, especially for clinical cases. Another problem specific to Germany is that suspected impaired car drivers or other road users can refuse to supply a urine sample but not a blood specimen. As a consequence, the number of cases in which

Table I. Spiked Low, Medium, and High Serum Concentrations

Serum concentrations (ng/mL) Assay Low Medium High

AMP (methamphetamine) 500 1000 2000 BZE (benzoylecgonine) 100 300 600 BENZ (nitrazepam) 100 200 400 MET (d,l-methadone) 150 300 600 OPI (morphine) 100 300 600 THCCOOH (THCCOOH) 15 30 60

Display CEDIA (ng/mL) 1.000

Serum -* Urine ~AP Serum

20O

0 0 200 400 600 800

Concentration (ng/mL)

Figure 1. Benzoylecgonine-spiked blank serum measured with urine, serum calibration, and spiked blank serum after protein precipitation.

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1.000 DjsPlaY--CEDIA (ng/mL) Serum * Urine o AP Serum

800 j . d

600 . . ~ f . - - J -

200 ~

0 200 400 600 800 Concentration (ng/rnL)

Figure 2. Morphine-spiked blank serum measured with urine, serum calibration, and spiked blank serum after protein precipitation.

Display CEDIA (ng/mL) 200 ~ Urine . Serum e hemoI.Serum I

150

100

50 ~ :

0 ~ ..... 50 100 200

Concentratio~ (ng/mL)

Figure 3. Benzoylecgonine-spiked blank serum, blank hemolytic serum, and blank urine measured with urine calibration.

only a blood sample (and no urine sample) is available for analysis is steadily increasing. The blood-sample Vacutainer tubes used by the police contain sodium or potassium fluoride. Because of these salts, blood samples for the analysis for drugs of abuse are more or less hemolyzed. Concerning blood samples, another important aspect is that in many cases only very limited sample material is available. Especially for forensic (impaired drivers, criminals) and clinical cases, it is necessary to detect very low concentrations of drugs of abuse. A procedure that can show sensitively which test parameters require a confirmation method saves time and assists in getting analytic data of high quality from a small quantity of sample, sometimes only 1 mL of blood.

As a first step, we tested whether the high protein content of serum samples interferes with the cloned enzyme donor immunoassay (CEDIA) and whether it is necessary to perform a special calibration with serum calibrators that are in lower concentration ranges than those supplied by the manufacturer. To do this, we selected the opiate (OPI) and benzoylecgonine (BZE) assays as test parameters. Serum and urine blanks were spiked with morphine or BZE and measured with the urine standard calibration mode (as recommended by the manufacturer) and with a self-spiked serum calibration with lower concentrations. Subsequently, hemolytic whole blood and acetone-precipitated serum samples were measured. This pretest showed the successful applicability of CEDIA DAU for the direct measurement of opiates and BZE in serum and whole blood. Therefore, a system- atic evaluation for all test parameters mentioned was begun.

Materials and Methods

250

200

Display CEDIA (ng/mL)

Urine * Serum o hemol. Serum

150

100

50

o

i

i

. J

50 1 O0 150 200

Standards Stock solutions for amphetamine (AMP), methamphetamine

(METH), methylenedioxymethamphetamine (MDMA), methyl- enedioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDEA), benzodioxolbutaneamine (MBDB), nitrazepam, diaze- pare, oxazepam, nordazepam, flunitrazepam, temazepam, clobazam, clonazepam, chlordiazepoxide, tetrazepam, bromazepam, midazolam, BZE, cocaine, methadone (MET), morphine, codeine, dihydrocodeine, tetrahydrocannabinol (THC), and tetrahydrocannabinol carboxylic acid (THCCOOH) and their deuterated internal standards were purchased from either Promochem GmbH

Concentration (ng/m L) Figure 4. Morphine-spiked blank serum, [)lank hemolytic serum, and blank urine measured wifll urine calibration.

Table II. Mean CEDIA Display

Within run (ng/mt) Assay Low Medium High tow

AMP 296 753 1449.4 315.8 BZE 106.7 257.5 495.3 100.2 BENZ 59.8 136 338.7 66.8 MET 142 258.8 507.7 146.1 OPI 99.9 281.7 549.1 103.2 THCCOOH 2.6 27.7 55.1 1.9

(Wesel, Germany) or Sigma Chemie (Deisenhofen, Germany). Stock solutions were diluted with ethanol to produce standard working solutions. The standard working solutions were used to

prepare serum and urine standards for the analysis.

Between day (ng/mt) Medium High

769 1527.1 262.8 494.9 164.4 365.1 264.2 513.1 294.5 564.4

28.3 54.6

Reagents All solvents were of reagent-grade quality.

CEDIA immunoassay reagents were supplied by Boehringer Mannheim (Mannheim, Germany). Reagents for the ADx were supplied by Abbott GmbH (Wiesbaden, Germany). Reagents and test tubes for RIA were supplied by Biermann (Bad Nauheim, Germany). Measurements were done on a y-counter (Berthold, Isernhagen, Germany). All procedures for the preparation of the

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immunoassay reagents were the same as those recommended by the manufacturer for the analysis of urine. The CEDIA test has two multiurine controls (for methamphetamine, BZE, morphine, nitrazepam, and methadone) and two controls (25 ng and 50 ng/mL) for THCCOOH which were used to check the accuracy

Table III. Within-Run and Between-Day Precision for CEDIA

and stability of the calibration. The 25-ng/mL THCCOOH control was routinely diluted to 20 ng/mL. The urine calibration curves proved to be stable for at least two weeks.

Within run (%CV) Between day (%CV) Assay Low Medium High Low Medium High

AMP 5.7 3.1 3.4 15.5 8.7 10.4 BZE 2.4 3.9 6.6 7.5 7.1 6.4 BENZ 8.0 7.0 4.3 15.8 10 8.2 MET 5.5 2.3 2.0 5.1 2.7 3.0 OPI 7.6 2,8 2.9 11.2 4.3 4.7 THCCOOH 2.4 1.4 7.4 2.6

Table IV. Results of 71 Blood Samples Analyzed by CEDIA and GC-MS for THC and THCCOOH

GC-MS GC-MS Case CEDIA THC THCCOOH Case CEDIA THC THCCOOH no. (ng/mL) (ng/mL) (ng/mt) no. (ng/mL) (ng/mL) (ng/mL)

37 41 - 12 38 77 3 60 39 66 0.8 10 40 67 1 10 41 35 9 42 71 19 233 43 60 2 11 44 58 2 10 45 39 1 22 46 22 - 15 47 65 2.2 19 48 65 1.5 22 49 72 11 120 50 48 9 30 51 34 1.4 17 52 66 1.8 25 53 65 4 40 54 67 3 27 55 70 10 54 56 62 2 20 57 17 1 15 58 65 3 25 59 65 2.2 23 60 65 5 30 61 22 - 15 62 25 - 20 63 15 1 16 64 17 1 20 65 20 1 21 66 18 - 20 67 72 5.8 50 68 32 2 10 69 34 2 18 70 39 5 24 71 65 3 25

1 18 - 6 2 46 - 5 3 19 - 34 4 69 2.6 57 5 72 6.1 111 6 74 11 367 7 75 4.3 41 8 77 1 27 9 63 1.4 13

10 38 0.5 10 11 39 - 7 12 66 2.1 30 13 67 O.9 5 14 66 11 27 15 66 0.5 27 16 93 4.1 98 17 84 7.5 109 18 47 4.4 14 19 30 0.8 6 20 28 - 11 21 65 2.6 17 22 72 3.5 34 23 74 2.4 64 24 79 1.5 19 25 70 3.7 84 26 66 0.8 11 27 39 0.7 17 28 71 12.7 177 29 66 0.8 27 30 33 4 7 31 73 0.8 36 32 68 1 39 33 47 1 14 34 73 3 73 35 16 1 37 36 10 0.5 5

Experimental

Pretest Serum and urine blank samples were spiked

with morphine (10, 50, 100, 200, 300, 500, 800, 1000, and 2000 ng/mL) or BZE (10, 50, 100, 200, 300, 500, 600, and 800 ng/mL) and measured with the urine standard calibration mode (as recommended by the manufacturer: BZE 300, 2000, and 5000 ng/mL; OP1300, 800, and 2000 ng/mL) and subsequently with a self-spiked serum calibration with lower concentrations (BZE 200, 500, 1000 ng/mL; OPI 150, 300, 500 ng/mL). Drug-free serum and urine specimens were obtained from laboratory personnel. After these measurements an acetone precipitation was performed for the spiked serum samples as follows: aliquots of 500 tJL were added dropwise to 1 mL acetone, then vortex mixed for I rain and centrifuged at 4000 rpm for 5 rain. The clear supernatant was evaporated under nitrogen and reconstituted in 500 IJL of ADx buffer solution. The reconstituted sample was again measured by BM/Hitachi 911 analyzer and compared with direct serum measurements. Hemolyzed blood samples were spiked and run like serum samples to see if red color interfered with the test. The instrument settings were the same as the settings for the urine specimens recommended by the manufacturer. The only exception was the THCCOOH assay. Routinely, a cutoff of 20 ng/mL instead of 25 ng/mL is used.

Limit of detection, cutoff, and precision The limit of detection was calculated by adding

three standard deviations to the mean of 21 replicates of a blank serum. Because of the necessary low limit of detection, cutoffs were set as follows: AMP, 20 ng/mL; BZE, 10 ng/mL; BENZ, 10 ng/mL; MET, 40 ng/mL; OPI, 10 ng/mL; THCCOOH, 10 ng/mL. For within-run precision, three controls of a low, medium, and high serum concentration were assayed in 21 replicates. Between-day precision was determined by assaying each of the low, medium, and high control in three replicates per day for 21 consecutive days. The low, medium, and high concentrations are shown in Table I. Linearity was analyzed by 11 dilution steps of a high concentration, starting 10% over the assay range.

Immunoassays (CEDIA, FPIA, RIA) Five hundred serum or whole blood hemolyzed

samples from clinical poisonings or impaired

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drivers were centrifuged and run on the BM/Hitachi 911 analyzer with CEDIA DAU reagent under the same conditions as urine samples. Following this initial testing CEDIA-negative samples (under the cutoff) were precipitated with acetone as described with the only difference being that 20 IJL of isopropanolic HCI was added before evaporation. The reconstituted samples were tested using the ADx analyzer and complete ADx reagent. Pretests with spiked acetone precipitated samples showed the ADx to have a nearly equal or partially better sensitivity (amphetamines) than CEDIA with the exception of the benzodiazepine test. Therefore, we used it as a method for cross checking CEDIA-negative samples. Additionally, CEDIA-negative samples were retested by RIA for morphine and by HPLC for benzodiazepines. Positive samples by either screening assay with a displayed drug concentration

higher than the CEDIA cutoffs were selected for confirmation by gas chromatography-mass spectrometry (GC-MS). Radio- immunoassay (RIA) was performed according to the manufac- turers instruction. A five-point standard curve from 2.5 to 250 ng/mL was generated. The cutoff positive calibrator was 5 ng/mL. The correlation coefficient for each standard curve was 0.998 or greater.

GC-MS Quantitative GC-MS was performed on 91, 102, and 21 sam-

ples positive for BZE, opiates, and methadone, respectively, as described elsewhere (10) and on 16 samples positive for amphetamines as described elsewhere (11). Quantitative GC-MS was performed on 71 samples positive for THCCOOH as follows:


4OOO

A

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7

0 0 450 900 1350 1800 2250 2700 3150 3600 4050 4500

Concentration (ng/m L)

1200 1080 96O 84O 720 600 48O 36O 240 120

0


D / / e . /

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120 240 360 480 600 720 840


/

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960 1080 1200

4000 Display CEDIA (ng/mL)

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2500

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1500

1000

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0 0 400 800

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1200 1600 2000 2400 2800 3200 3600 4000 Concentration (ng/m L)

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3500

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2500

2000

1500

1000

500

0 ~ 0 400 800

120

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15

0 1200 1600 2000 2400 2800 3200 3600 4000



F

0 12 24

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36 48 60 72 84


x - y

96 108 120

Figure 5. Results of the linearity test for AMP (A), BZE (B), BENZ (C), MET (D), OPI (E), and THCCOOH (F).

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Journa l o f A n a l y t i c a l Tox i co logy , Vo l . 23, Ju l y /Augus t 1999

10 ng and 20 ng of the deuterated standards (THC-d3, THC- COOH-d3) were added to 1 mL of centrifuged serum and transferred to an RP18 column (Baker) conditioned with 1 mL methanol and 1 mL distilled water. The column was then washed consecutively with 2 mL distilled water, 2 mL acetic acid (0.25 mol/L), and 2 mL distilled water; dried by passing air through it for 10 min; and subsquently centrifuged for 10 min (4000 rpm). Adsorbed drugs of abuse were eluted with 3 x 0.5 mL acetone. The eluent was evaporated to dryness under a stream of nitrogen at 50~ The residue was reconstituted with 150 IJL tetrabutyl- ammonia hydroxide/dimethyl sulfoxide (2:98, v/v). After 2-rain incubation at room temperature, 50 IJL iodomethane was added and the solution vortex mixed. The reaction was stopped after an additional 5 rain with 350 IJL HCI (0.1 tool/L). Extraction of the methylated products followed twice with 2 x I mL isooctane for 1 rain. After centrifugation the organic phases were decanted into a clean tube, dried under nitrogen, and reconstituted in 50 IJL isooctane.

A 1-1JL quantity of the sample was injected into the same GC-MS system as described for opiates. The temperature was pro- grammed from 100 (3-min hold) to 180~ at 30~ and to 290~ at 10~ (8-rain hold). The MS detected the following ions of the methylated products in selected ion monitoring mode: THC-d3 316/331,THC 313/328, THCCOOH-d3 316/360/375, and THCCOOH 313/357/372.

Standard curves were generated using a six-point standard curve from 10 to 500 ng/mL for amphetamine, BZE, cocaine, codeine, dihydrocodeine, MBDB, MDA, MDEA, MDMA, methamphetamine, morphine, and methadone. Six-point curves from 1 to 50 ng/mL were generated for THCCOOH and THC. The detection limits were 0.5 ng/mL for THCCOOH and THC; 5 ng/mL for benzoylecgonine, codeine, dihydrocodeine, and morphine; and 10

Table V. Results of 16 Blood Samples Analyzed by CEDIA and GC-MS for Amphetamines

GC-MS

ng/mL for amphetamine, cocaine, MBDB, MDA, MDEA, MDMA, methamphetamine, and methadone. The correlation coefficient for each standard curve generated was 0.998 or greater.

High-performance liquid chromatography (HPLC) Quantitative HPLC was performed on 47 samples positive for

benzodiazepines and for cross checking of the 453 benzodiazepine-negative samples. Five-hundred nanograms of the internal standard papaverine was added to 1 mL of centrifuged serum. The pH was adjusted to pH 8-9, 1 mL K2HPO 4 buffer (pH 8-9) was added, and the solution was vortex mixed for 5 min. After centrifugation, the supernatant was transferred to an RP18 (Baker) column conditioned with 2 mL methanol and 2 mL distilled water. The column was then washed consecutively with 2 x I mL distilled water and dried by passing air through for 10 min. Adsorbed drugs of abuse were eluted with 3 x 0.5 mL methanol. The eluent was evaporated to dryness under a stream of nitrogen at 50~ The residue was reconstituted in 50 pL methanol. HPLC analysis: a 20-1JL quantity of the sample was injected into the HPLC system. Instrumentation: LDC/Milton Roy Spectro- MonitorTMD detector, pump: TSP Constametric, wavelength 220 nm. The system was equipped with a RP18 LiChrospher| column, solvent system: (4.80 g H3PO 4 + 6.66 g K2HPO 4 in 1 L distilled water, adjusted to pH 2.3) + 458.8 g acetonitrile. Flow rate was 1 mL/min.

Standard curves were generated using a six-point standard curve from 40 to 500 ng/mL for nitrazepam, diazepam, oxazepam, nordazepam, temazepam, bromazepam, midazolam, and clobazam. Six-point curves from 10 to 50 ng/mL were generated for clonazepam, flunitrazepam, and tetrazepam. The detection limits were 30 ng/mL for nitrazepam, oxazepam, nordazepam, temazepam, bromazepam, and midazolam; 50 ng/mL for

diazepam and clobazam; and 10 ng/mL for clonazepam, flunitrazepam, and tetrazepam. The correlation coefficient for each standard curve generated was 0.998 or greater.

Sample CEDIA Amph. Methamph. MDMA MDEA MDA MBDB no. ( n g / m L ) ( n g / m L ) ( n g / m L ) ( n g / m L ) ( n g / m L ) ( n g / m L ) ( n g / m L )

1 21 134 - 33 4 7 50 -

2 0 - - 275 - -

3 0 - - 8 2 0 - -

4 9 8 215 - - - 100 -

5 0 - - - 4 0 - -

6 223 3 1 7 - 6 6 4 - - -

7 0 - - - 2 5 0 2 0 -

8 0 - - 242 - - -

9 0 - 180 - 30 - -

10 0 70 20 20 4 0 - -

11 0 - - 164 - - 100

12 0 . . . . . 170

13 0 20 - 80 - - 8 0

14 0 - - - 127 - 148

15 0 - - - 315 - -

16 0 - - - 212 - 180

* Abbreviat ions: A m p h = Amphetamine , Me thamph = Methamphetamine, M D M A = Methy lened ioxymeth - amphetamine, MDEA = Methy lenedioxyethamphetamine, M D A = Methy lened ioxyamphetamine , MBDB = Benzodioxolbuta neamine.

Results and Discussion

Pretest Figures 1 and 2 show the results of spiked blank

serum measured with urine or serum calibration and after protein precipitation. Acetone-precipitated samples gave the same results as unpretreated serum for benzoylecgonine and 10-30% lower results for morphine. The use of the serum calibration curve or the standard urine calibration curve gave approximately the same results. For that reason and ease of use, the standard urine calibration curve was selected for further determi- nations. Figures 3 and 4 show that red color due to hemoglobin does not interfere with the test. However, it has to be noted that high viscosity of blood samples (e.g., postmortem blood) may cause problems for the pipette station of the

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Ioumal of Analytical Toxicology, Vol. 23, July/August 1999

BM/Hitachi 911 analyzer. A precipitation step is thus recommended for very viscous samples to be on the safe side. Spiked blank urine samples delivered the same results as spiked serum samples. Thus, serum proteins do not interfere with the test, and direct measurement without any preliminary treatment of serum samples is therefore possible, in principle. After these successful pretests, the main study was begun.

Table Vl. Results of 91 Blood Samples Analyzed by CEDIA and GC-MS for Benzoylecgonine and Cocaine

GC-MS Case CEDIA BZE Cocaine Case CEDIA BZE Cocaine no, (ng /mt ) (ng/mt) (ng/mL) no. (ng/mt) (ng/mL) (ng/mL)

Limit of detection and precision In all assays tested the limit of detection was 0 ng/mL. This

result was corroborated by all the following measurements. Whereas urine blank measurements (i.e., dependent on turbidity, bacterial contamination, etc.) often give threshold displays of 10-30 ng/mL and even as high as 100 ng/mL, serum measurements show distinctively lower thresholds, most often a zero dis-

play. Of course, this is dependent on the quality of the serum sample too. Table I lists the spiked low, medium, and high serum concentrations, and Table II shows the mean concentrations with

GC-MS CEDIA. Table III lists the average within-run and between-day precision for CEDIA. For the low- THCCOOH concentration of 15 ng/mL, no coefficient of variation (CV%) is given. This is because the display for about half of the samples showed 0 ng/mL as a result. However, in real samples, a combination of different cannabinoids that cross- reacts with the antibody is present. As can be seen in Table IV, CEDIA gave a signal above the cutoff for very low concentrations (cases 1, 2, 11, 13, 19, 33, and 36). Therefore, the test is sufficiently sensitive for the direct detection of low cannabinoid concentrations in blood.

1 730 1397 27

2 540 612 -

3 85 103 -

4 172 239 -

5 253 403 -

6 405 380 -

7 842 1516 - 8 40 65 - 9 629 599 -

10 163 170 26

11 152 122

12 962 1090

13 1500 2160 124

14 917 1540

15 333 470

16 510 704

17 208 335

18 169 221

19 848 1320 65

20 168 184

21 I0 /0

22 ~99 392

23 353 593

24 941 1720 33

25 379 481

26 222 283

27 310 142

28 622 1160

29 737 1340

30 1000 878 46

31 354 410

32 430 493

33 400 848 10

34 490 554

35 0 155

36 927 931

37 169 216

38 445 296

39 1380 944 40 1290 944 41 708 597

42 1610 1130

43 1300 1030 10 44 732 553

45 268 333

46 82 97

47 455 531 27

48 1200 1780 51

49 681 653 -

50 477 354 -

51 1300 2900 -

52 1100 -

53 737 1340 -

54 622 1160 -

55 222 283 -

56 1140 1460 60

57 38 53 -

58 611 638 -

59 235 295 -

60 467 511 113

61 922 1030 -

62 1020 1500 80

63 1150 683 16

64 143 80 -

65 447 328 -

66 1020 897 68

67 512 305 -

68 601 349 -

69 488 486 -

70 113 96 -

71 169 217 -

72 90 225 -

73 112 244 10

74 242 320 20

75 732 663 -

76 1290 910 34

77 1022 965 -

78 737 1340 -

79 1002 1480 100

80 1200 1700 80

81 1380 2420 -

82 2300 2800 100

83 2000 1980 - 84 1200 1300 - 85 86 92 - 86 73 70 -

86 88 100 - 88 46 44 - 89 199 246 - 90 212 298 - 91 388 417 -

Linearity Figures 5A-F show the results of the linearity

test for the different groups. The assays were found to be linear between the following concentrations: AMP 0-2500 ng/mL, BZE 0-1200 ng/mL, BENZ 0-1600 ng/mL, MET 0-600 ng/mL, OPI 0-720 ng/mL, and THCCOOH 24-60 ng/mL. The pretests have already shown that linearity begins at 10 ng/mL for opiates and benzoylecgonine, respectively. Because of the very low limits of detection, very low cutoffs from 10 to 40 ng/mL were selected as follows: AMP 20 ng/mL, BENZ 10 ng/mL, MET 40 ng/mL, OPI 10 ng/mL, and THCCOOH 10ng/mL.

Method comparison Comparison of GC-MS quantitative and CEDIA

semiquantitative results are shown in Tables IV-IX and Figures 6A-D.

Amphetamines Results for 16 blood samples analyzed for

amphetamines by CEDIA and GC-MS are shown in Table V. Referring to the cutoffof20 ng/mL, 13 samples were false negatives. These results show that the amphetamine urine test is not suitable for serum testing without adaptation. One reason is the lower cross-reactivity of commonly used amphetamine derivatives such as MDMA (69%), MDEA (21%), and MBDB (60%) compared with methamphetamine. Another reason is that serum proteins may cause interferences in this test, which is not the case with other assays. This

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became evident when the sample volume was increased on the BM/Hitachi 911 analyzer from 3 IJL (as recommended for the urine tests) to 18 IJL to increase sensitivity for amphetamine detection. With the so-adapted calibration, spiked urine concentrations of 150 ng methamphetamine/mL were shown as

Table VII. Results of 47 Blood Samples Analyzed by CEDIA and HPLC for Benzodiazepines (ng/mL)

HPLC CEDIA Benzo 1 Benzo 2 Benzo 3 Benzo 4

61 Midaz* 70 720 Diaz 288 Nordaz 89 3100 Diaz 317 Nordaz 170 41600 Diaz 3650 Nordaz 1360 Oxaz. 108 1700 Diaz 1320 Nordaz 260 100 Diaz 190 Fluni 24 Clonaz 20 113 Clonaz 60 2600 Diaz 125 Nordaz 306 Fluni 10 22500 Diaz 1058 Nordaz 588 535 Oxaz 446 153 Bromaz 125 - 428 Diaz 184 2600 Tetraz 600 - 1270 Diaz 90 Nordaz 160 Clonaz 80 16800 Diaz 2830 Nordaz 1300 Oxaz 190 3200 Diaz 103 Nordaz 320 Clonaz 57 14 Fluni 13 Clonaz 13 10 Clonaz 16 - 3800 Chlordiaz 1500 Oxaz 250 746 Tetraz 240 1200 Diaz 190 Nordaz 780 959 Midaz 508 2761 Tetraz 650 1300 N itraz 900 1300 Diaz 240 Nordaz 780 Nitraz 130 165 Fluni 23 17600 Diaz 2800 Nordaz 1500 Fluni 40 5900 Diaz 1500 Nordaz 1500 Fluni 16 5700 Diaz 600 Nordaz 5000 Fluni 60 Limit High Diaz 98 Nordaz 194 3100 Temaz 2300 444 Diaz 440 Nordaz 170 107 Diaz 14 Nordaz 60 Fluni 5 9320 Diaz 4240 Nordaz 4760 Oxaz 128 Limit High Diaz 200 Nordaz 180 5800 Diaz 300 Nordaz 154 Oxaz 80 900 Nitraz 800 180 Bromaz 155 5130 Diaz 258 Nordaz 166 Oxaz 90 250 Clobaz 230 30 Midaz 40 10 Clonaz 18 4700 Diaz 400 Nordaz 370 Oxaz 100 15 Fluni 20 235 Diaz 64 Nordaz 107 Oxaz 154

Clonaz 170

Clonaz 40

* Abbreviations: Diaz = Diazepam, Nordaz = Nordazepam, Oxaz = Oxazepam, Nitraz = Nitrazepam, Fluni = Flunitrazepam, Temaz = Temazepam, Clonaz = Clonazeparn, Chlordiaz = Chlordiazepoxide, Tetraz = Tetrazeparn, Bromaz = Bromazepam, Midaz = Midazolam, Clobaz = Clobazam.

A 3.500

3.000

2.500

2.000

1.500

1.000

5OO

0


e �9 �9 e.e �9

�9 �9149

500 1.000 1.500 2.000 2.500

GCMS (ng/mL)


B 3 ,~ 3.000 ~-y

2.000

t .500

1.000 t % � 9 $ �9 ee e *

5OO

0 500 1.000 1.500 2.000 2.500 3.000

GCMS (ng/mL)

Display CEDIA (ng/rnL) C 500 x-y

400 ,

2OO

117(I

0 11~ 200 300 401) 5011 600 700

GCMS lng/mL)

Display CEOIA (ng/mL)

120 x-v

8O

60 ~ ' t , ~

40 �9

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0 0 50 1110 150

GCMS (ng/mL)

Figure 6. Comparison of GC-MS quantitative and CEDIA semiquantitative results for BZE (A), OPI (8), MET (C), and THCCOOH (D).

3.500

3.000

8O0 900

Table VIII. Results of 21 Blood Samples Analyzed by CEDIA and GC-MS for Methadone (ng/ml)

CEDIA GC-MS CEDIA GC-MS

600 320 460 270 870 210 250 315 275 177 394 100 487 80 534 200 303 390 246 288 290

310 222 496 170 276 314 246 385 387 461 360

360 300 360 370 266 160 90

120 310 300

200

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Table IX. Results of 102 Blood Samples Analyzed by CEDIA and GC-MS for Opiates

GC-MS Morphine

Case Morphine glucuronide Codeine Dihydrocodeine no. CEDJA (ng/mL) (ng/mL) (ng/mL) (ng/mt)

1 2400 100 614 41 -

2 182 6 57 - -

3 345 23 232 26 -

4 72 14 90 - -

5 641 14 50 - 396

6 226 10 90 - 40

7 1118 176 824 32 -

8 135 13 96 - -

9 428 40 200 -

10 371 10 154 10 10

11 319 16 34 -

12 1500 27 580 - 697

13 263 18 120 - 83

14 201 21 118 -

15 30 - 40 -

16 716 52 412 16

17 328 27 145 17

18 9~6 30 199 - 3360

19 0 - 25 -

20 112 78 - -

21 2430 16 128 - 2890

22 38800 164 2320 2430

23 51 - 40 -

24 84 20 80 -

25 0 20 24 -

26 181 - 100 -

27 299 10 171 -

28 523 46 184 -

29 183 10 80 -

30 860 54 286 -

31 116 10 80 -

32 235 - - 311

33 101 - 39 -

34 90 - 28 -

35 146 26 131 -

36 116 15 39 -

37 234 31 128 - 41

38 68 10 47 -

39 300 28 170 -

40 298 94 63

41 189 20 155 -

42 662 28 141 - 103

43 512 15 169 - 78

44 84 6 25 -

45 374 71 257 11

46 368 33 168 -

47 1300 115 506 -

48 747 30 278 -

49 817 30 367 -

50 92 - 60 - 51 720 11 276 -

52 1100 48 504 19

53 112 17 33 - -

54 253 8 24 - 250

146 ng/mL, whereas the same concentration in spiked serum was shown as only 20 ng/mL. Lower concentrations than 150 ng methamphetamine/mL could not be detected in serum at all, whereas detected urine concentrations went down to 10 ng/mL (results not shown). It will therefore be necessary to adapt the urine test for a sensitive detection of amphetamines in serum. A combination of enhancement of sample volume on the BM/Hitachi 911 analyzer and preliminary treatment of the sample (protein precipitation) is very promising. Amphetamine precipitation with organic solvents is problematic because of the loss of the very volatile amphetamines during the dry- down step of the organic supernatant. Addition of methanolic HC1 prevents this amphetamine loss, but it also causes a loss of about 30% of cannabinoids (12). However cannabinoids can be directly measured (as will be shown later in this paper). A good alternative for organic solvents is the precipitation by zinc sulfate. This method was shown to work for sensitive determination (cutoff 25 ng/mL) of methamphetamine with TDx (2), but we did not test it here.

BZE In contrast to the amphetamines, BZE could be

detected at very low concentrations. All immunological positive samples (at a cutoff of 10 ng/mL) could be confirmed by GC-MS. Two false negatives were detected by GC-MS with results of 155 ng BZE and 1100 ng BZE/mL, respectively. These two samples were extremely hemolytic and viscous. As we showed in the pretest, red color does not interfere with the assay. It was the viscosity of the samples that made it difficult for the pipette to get enough sample volume. Probably, in each case, no sample got into the assay at all. Therefore, blood samples have to be controlled for their viscosity before running the assays. Highly viscous samples should be centrifuged or treated with acetone or both. The linearity test (Figure 5B) showed the CEDIA BZE assay to be linear from 0 ng/mL to 1200 ng/mL. All assays showed that at low concentrations the linearity is best (with exception for THCCOOH). The good correlation of GC-MS and CEDIA results (Table VI, Figure 6A) show that a sensitive semiquantitative BZE detection is possible in serum and whole blood without any adaptation.

Benzodiazepines Direct and sensitive benzodiazepine measure-

ment is also possible in serum. No false positives and no false negatives could be detected. This is probably because the CEDIA benzodiazepine assay does not use a monoclonal antibody like the other CEDIA tests (13), but polyclonal. Because of the

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very good cross-reactivities, benzodiazepines with very low therapeutic blood levels can nevertheless be detected in serum at normal therapeutic range (flunitrazepam [109%], clonazepam [71%], alprazolam [220%]). The good cross-reactivity for metabo-

Table IX. Results of 102 Blood Samples Analyzed by CEDIA and GC-MS for Opiates (Continued)

GC-MS

Morphine Case Morphine glucuronide Codeine Dihydrocodeine no. CEDIA (ng/mC) (ng/mC) (ng/mL) (ng/mC)

55 561 46 174 -

56 235 -

57 1200 65 735 -

58 860 54 470 -

59 183 6 92

60 523 46 188

61 653 43 185

62 310 31 158

63 507 -

64 1111 - -

65 116 17 80 -

66 103 17 70

67 1700 50 170 5400

68 1100 40 450 -

69 503 50 280 50

70 348 15 60 99

71 175 - 78

72 169 13 86 -

73 71 14 37 - -

74 860 40 214 -

75 1500 83 817 -

76 757 12 224 -

77 118 7 53 -

78 1970 120 838

79 444 40 120 -

80 220 19 64 -

81 517 20 162 -

82 0 - 28 -

83 388 19 171

84 1060 30 320 20 -

85 219 13 97

86 189 24 82

87 340 10 100 -

88 83 7 30 -

89 457 16 157 -

90 279 10 150 -

91 406 25 100 -

92 1126 50 349 -

93 9O 10 9O -

94 219 15 95

95 300 20 180 - -

96 1160 60 350 40 -

97 174 20 184 - -

98 523 34 2OO - -

99 235 40 271

100 662 - - 316

101 503 60 240 -

102 869 60 266 20 -

lites like 7-amino-clonazepam (96%) and 7-amino-flunitrazepam (81%) further enhances sensitivity. Another point is that the CEDIA urine Benzodiazepine Hs assay offers the opportunity of automatic glucuronidase cleavage by the analyzer. Although glu-

curonidated products do not play such a big role in serum as in urine, this step might bring a slight but important increase in sensitivity for direct serum measurements. It was possible to confirm all positive (cutoff 10 ng/mL) immunological results with another method (Table VII). All CEDIA-negative samples were tested by another immunoassay (FPIA) and by HPLC. Although the determination of drugs of abuse works well with

- FPIA after acetone precipitation (14,15), exact cut- 311 offs for all benzodiazepines are not available.

- Huang (8) showed that the FPIA urine assay was - not able to detect 70 ng of several benzodiazepines - after serum extraction with butyl chloride. Table X - shows that the CEDIA test is able to detect 10

ng/mL for some benzodiazepines. Therefore, it 32o appeared necessary to check the immuno-nega- 803 tives by the more sensitive HPLC method, but no

_ false negatives were discovered. The use of polyclonal antibodies is very good for the specific and sensitive detection of benzodiazepines, and this is the main point when doing an immunological screening. Because of the use of polyclonal antibodies the correlation of HPLC to CEDIA semiquantitative results must be very noisy when more than one benzodiazepine and metabolites are present in the sample (Tables VII and X).

Methadone All samples positive for methadone could be

confirmed by GC-MS. Correlation of CEDIA semiquantitation with GC-MS was low (Table VIII and Figure 6C). We did not distinguish between d- and /-methadone in the study. The addition of a fifth, very low calibrator to the standard curve may improve the correlation (communication of Boehringer Mannheim). Patients who receive methadone for substitution show usually blood levels of 50-400 ng/mL. A cutoff of 40 ng/mL will therefore be low enough to detect this.

Opiates Opiate detection with CEDIA was very specific

and sensitive in serum and blood. All positives were confirmed by GC-MS. Three false negatives were found at very low (see Cases ]9, 25, and 82, Table IX) concentrations. Two of these "false negatives" contained no morphine at all but did contain morphine glucuronide (cross-reactivity of the opiate test for morphine-3-glucuronide is 81%). As already shown in the pretest (Figure 2), CEDIA has good linearity at low concentrations. Semiquantitative CEDIA results were usually higher than GC-MS results even when the

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Journal o f Analytical Toxicology, Vol. 23, July/August 1999

Table X. CEDIA Display with Different Concentrations of Benzodiazepines in Spiked Serum Samples

Spiked concentrations of benzodiazepines

0 10 25 50 100 250 500 1000 2000

Midazolam 0 6 22 42 88 468 800 Limithigh* Limithigh CIonazepam 0 8 14 40 115 356 767 1852 3989 Alprazolam 0 0 12 48 ] 27 638 3556 Limit high Limit high Diazepam 0 0 6 29 83 387 Limit high Limit high Limithigh Flunitrazepam 0 0 5 19 63 267 1372 Limit high Limit high 7-amino- 0 0 5 24 57 248 908 1854 4476 Flunitrazepam

* Limit high = requires dilution, out of calibration range.

different cross-reactivities of codeine, morphine, morphine glucuronide, and dihydrocodeine were taken into consideration (Table IX, Figure 6B).

THCCOOH For THCCOOH, all CEDIA results higher than 10 ng/mL could

be confirmed by GC-MS. Linearity could be detected from 24 ng/mL up to 60 ng/mL, but GC-MS results did not correlate with CEDIA semiquantitation (Table IV, Figure 6D). Similar results for THCCOOH CEDIA assay were found by Cagle et al. (12) after acetone pretreatment of blood. With a cutoff of 25 ng/mL, they found FPIA semiquantitation to show a much better correlation with results of GC-MS than CEDIA. Sensitivity differences of both tests were not tested. Other authors chose a 25-ng cutoff for FPIA after acetone precipitation to get presumptive positives (< 2 ng THC/mL) by GC-MS (16). Although there is almost no correlation between CEDIA semiquantitation and GC-MS results, all CEDIA-positives could be confirmed by GC-MS. The low correlation is due to the CEDIA assay's cross-reactivity to other cannabinoids and metabolites. Therefore, very sensitive detection of cannabis misuse in unpretreated serum/blood is possible for concentrations far below 2 ng THC/rnL.

Conclusions

The data provided sufficient documentation to use the CEDIA screening technique without any adaptation as a very reliable method to detect analytically low but toxic concentrations of BZE, BENZ, MET, OPI, and THCCOOH in serum or whole blood without any pretreatment of the samples and without any changes at the BM/Hitachi 911 analyzer for routine urine measurements. Serum screening for amphetamines is not sensitive enough in the unchanged urine mode. It will require some adaptation to a serum mode (probably a higher sample volume [BM/Hitachi 911] combined with protein precipitation of the sample). This procedure saves time and assists in economic use of very small sample specimens.

Acknowledgments

The authors wish to thank Boehringer Mann- heim for support and their kind assistance.

References

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13. A.H.t3. Wu, E. Forte, G. Casella, K. Sun, G. Hemphill, R. Foery, and H. Schanzenbach. CEDIA for Screening drugs of abuse in urine and the effect of adulterants. J. Forensic Sci. 40' 614-618 (1995).

14. M. Bogusz, R. Aderjahn, G. Schmitt, E. Nadler, and B. Neureither. The determination of drugs of abuse in whole blood by means of FPIA and Emit-dau immunoassays--a comparative study. Forensic Sci. Int. 48:27-37 (1990).

15. R.D. Maier, M. Erkens, H. Hoenen, and M. Bogusz. The screening for common drugs of abuse in whole blood by means of EMIT-ETS and FPIA-ADx urine immunoassays. Int. J. Leg. Med. 105:115-119 (1992).

16. C.R. Goodall and B.J. Basteyns. A reliable method for the detection, confirmation, and quantitation of cannbinoids in blood. J. Anal. Toxicol. 19:419-426 (1995).

Manuscript received June 22, 1998; revision received October 23, 1998.

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