Journal ol Analytical Toxicology, Vol. 16, November/December 1992

Modificationof Emit Assay Reagentsfor Improved Sensitivity and Cost Effectiveness in the Analysis of HemolyzedWhole Blood w . M i c h a e l A s s e l i n and d a n n a M. L e s l i e

R.C.M,P. Forensic Laboratory, 5201 Heather Street, Vancouver, B.C., Canada V5Z 3L7

~_Ab_stract~ This report describes an Improved method for the direct detection of a broad spectrum of drugs of abuse In hemolyzed whole blood by means of Syva Emlt| enzyme fmmunoassay. Improvements include a 1.5 to 10 fold increase In Emit assay sensitivity along with a 2 to 4 times Increase In the normal number of assays per kit. This was accomplished by enzyme substrata and cofactor supplementation with a commercially available product (Ralchem), assay reagent dlluUon, and extension of the absorbance measure time. The Emit drug abuse in urine (d.a.u.) assays used In this study Included amphetamine, barbiturate, methadone, methaqualone, opiate, benzodlazeplne metabolite, phencyclldlne, and propoxyphene. The Emit serum assays used were the benzodlazeplne and the trtcycllc antidepressant assays. The within-run coefficients of variation ranged from 0.25 to 0.66%, and the between-run coefficients of variation ranged from 0.45 to 1.00%. The proposed method allows for the analysis of hemolyzed whole blood using both Emit d.a.u, and serum assays. It Is sensltlve and can detect therapeutic or subtherapeutic concentrations of drugs In all assays tested. The method Is simple, rapid, and allows for the direct analysis of a methanollc extract of whole blood without lengthy sample concentration steps. The method allows for the detection of highly potent drugs and for long-term monitoring of drug metabolltes and conjugates. This could be beneficial for therapeutic drug monitoring, assessing patient compliance, and detection of previous drug use.

Introduction

Since the advent of homogeneous enzyme immunoassay techniques, such as Emit| for the detection of drags in urine, there has been considerable interest among analytical toxicologists in extending this technique to other biological fluids, such as whole blood, bile, and tissue homogenates. Analysis of these samples by Emit has not been previously possible because of high background absorbance levels (1). Attempts to analyze other, less proteinaceous samples such as saliva (2,3), vitreous humour (4), and tears (5,6) have been reported. These attempts suffered from a requirement for a preliminary dilution with a concurrent loss of sensitivity. In 1981, Peel and Perrigo (7) reported the first analysis of hemolyzed whole blood using Emit for the detection of cannabiholds. They used a methanolic precipitation protocol which required neither a prior dilution of the blood nor a tedious organic solvent extraction. Subsequently, we reported (8) the analysis of

hemolyzed whole blood using all 10 Emit drug abuse in urine (d.a.u.) assays. A recent report by Gjerde et al. (9) evaluated our method (8) using an automated procedure with 300 forensic blood samples and obtained good results. Several reports (10,11) have described a slight modification to our method where N, N-dimethylformamide was used instead of methanol. It was reported that this change in solvent resulted in a cleaner fraction, without the need to filter the resultant supernatant prior to analysis by Emit. Lewellen and McCurdy (12) used acetone in their procedure. They reported somewhat higher sensitivity for some of the d.a.u. assays relative to our method (8), but their protocol required more sample and a tedious evaporation step. Recently, our laboratory reported the use of the Emit serum tricyclic antidepressant (TCA) assay with hemolyzed whole blood using our initial method (13). This allowed for the detection of TCA's at therapeutic concentrations in blood or serum rather than only toxic concentrations in serum when used as directed by the manufacturer. In 1985, Sung and Neeley (14) reported an extremely cost effective method for the use of Emit reagents. These reagents were extensively diluted with buffer, followed by supplementation of Reagent A with substrate and cofactor. Using a centrifugal analyzer, they reported obtaining 2470 assays per kit without loss of precision or sensitivity, compared to the 334 assays per kit obtained using the manufacturer's protocol. In their report, the authors mentioned that the increase in substrate and cofactor concentrations had increased the enzyme reaction rate. Yu and Osterloh (15) reported a protocol using a commercially available substrate/cofactor supplement and a Syva Autocarousel. Although only interested in cost effectiveness, they did observe an increase in the sensitivity of the Emit methadone d.a.u, assay after reagent dilution and supplementation. Based on these observations (14,15), it appeared that supplementation of Emit reagents might increase assay sensitivity by altering the overall assay kinetics rather than simply increasing the number of assays obtainable from a kit. This potential increase in Emit sensitivity was noted by Fraser et al. (16), who in 1988 reported the detection of alprazolam in serum using several modified methods. One of these methods included supplementation of Reagent A with glucose-6-phosphate (G-6-P) and nicotinamide adenine dinucleotide (NAD+). This method resulted in a slight increase in assay sensitivity; however, few details of the method were described. The purpose of this report was to investigate the possibility of in-

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381

Journal of Analytical Toxicology, '4ol. 16, NovembedDecembor 1992

creasing Emit assay sensitivity when used to analyze methanolic supematants from whole blood, without the need to concentrate the sample or use tedious extraction procedures. The proposed method was compared directly to our initial approach (8) with three Emit assays: serum benzodiazepine, d.a.u, benzodiazepine, and serum TeA. Indirect comparison of the two methods was perfomaed with the lbllowing assays: amphetamine, barbiturate, methadone, opiate, methaqualone, propoxyphene, and phencyclidine (PEP). The proposed method showed between a 1.5- and 10-fold increase in assay sensitivity and resulted in twice as many tests per kit for d.a.u. assays and four times as many for serum assays,

mixed for 5 min (Vortex Genie) and then centrifuged at 3000 rpm for 5 rain at -10~ The clear supematant (1.0-1.5 mL) was filtered with a 0.45-pro filter attached to a 3-mL tuberculin syringe. As much sample as possible was removed fi'om the filter assembly by purging with air. After equilibration to room temperature, the filtered supematant was transferred into the sample cups of the Aulocarousel (70 I.tLper assay). It was important to prevent preferential evaporation of methanol over time, so each sample cup was then covered with a small piece of plastic wrap (Stretch 'n Seal). Once covered, the same cup was used repeatedly for all assays.

Assay preparation Materials A Syva Emit system consisted of a Stasar-IlI spectrophotometer (Gilford Instrument Labs), an Emit Clinical Processor Model CP-5000, a Syva Autocarousel sampler, a Gillord 3021 Vacuum Receiver, and DeVilbiss pump. The spectrophotometer was operated in the absorption mode at 340 nm and the microflow cell was set at 30~ The pipettor--diluters were set to aspirate 50 p_L,of sample and to deliver the sample plus 250 ~ of buffer. The CP-5000 was timed to measure absorbances at 15 and 215 s and to calculate the difference over a 200-s reaction time. The methanolic supernatant was filtered with a 0.45-~,rn disposable filter assembly (Gelman Acrodisc, Product No. 4184) and a 3-mL tuberculin disposable syringe (Becton Dickinson & Co. Ltd.). Autocarousel sample cups were covered with a small piece of plastic wrap (Stretch 'n Seal, DOW Chemical Co.). The following eight Emit d.a.u, assays were purchased from the Syva Company: amphetamine, barbiturate, methadone, methaqualone, opiate, benzodiazepine metabolite, phencyctidine, and propoxyphene, Emit serum benzodiazepine and tricyclic antidepressant (TEA) were also from Syva. Reagent A of each assay contained antibody, substrate (glucose-6-phosphate [G-6-P]), and coenzyme (nicotinamide adenine dinucleotide [NAD+]) in 0.055M Tris-HC 1. Reagent B contained a drug derivative labeled with glucose-6-phosphate dehydrogenase and 0.05% sodium azide in 0.055M Tris-HC 1. Buffer concentrate contained 0.825M Tris-HC1 buffer, pH 8.0 and 0.05% sodium azide. Glueose-6phosphate/NAD+ supplement (RAICHEM, Product No. 84111) was purchased from Reagents Applications, Inc. HPLC grade methanol was purchased from Caledon. The following drugs or metabolites (obtained from the Department of Health and Welfare, Ottawa) were prepared in ethanol at a concentration of approximately 1 mg/mL calculated as the free base or acid: dextroamphetamine, secobarbital, oxazepam, methadone, methaqualone, morphine, phencyclidine, propoxyphene, and nortriptyline. Each ethanolic stock solution was diluted 1:10 with water as working stock solutions. These working solutions were quantitatively added to 1.0 mL drug-free bovine blood over the desired concentration range of each assay. The blood, previously frozen, also contained 1% sodium fluoride and 0.25% potassium oxalate. All reagents and standards were stored in a refrigerator and allowed to equilibrate lbr at least 2 hours at room temperature prior to use.

Procedure Blood samples To a 13-mL plastic centrifuge tube was added 1 mL of whole blood followed by 2 mL of methanol. The sample was vigorously

382

All Emit d.a.u, assays were prepared similarly. Reagents A and B were each reconstituted with 6.0 mL distilled water as directed by Syva. Each reagent was then further diluted by adding 6.0 mL of the appropriate buflbr to give a total volume of 12.0 mL. Buffers for this purpose were prepared from the supplied Syva buffer concentrate (which had been diluted as directed) by adjusting the pH from pH 8.0 to the pH listed in the package insert. The Raichem G-6-P/NAD+ supplement, received as a lyophilized powder, was reconstituted with 2.5 mL distilled water as directed just prior to use, The reconstituted supplement (0.2 mL) was then added to each milliliter of prepared Reagent A. Thus, for each Emit assay, a total of 2.4 mL Raichem supplement was added to 12.0 mL Reagent A to yield a final total volume of 14.4 mL. The final volume of Reagent B remained at 12.0 mL. All Emit serum assays were prepared similarly. Reagents A and B were each reconstituted with 3.0 mL distilled water as directed. To this was added 9.0 mL of buffer at the appropriate pH, yielding a total of 12.0 mL. To 12.0 mL Reagent A was added 2.4 mL Raichem supplement, as for the Emit d.a.u, assays.

Instrumentparameters The Syva instrumentation was set up with the following parameters: temperature, wavelength, delay time, measure time, mode, sample volume, reagent volume, buffer volume, 30~ 340 nm; I5 s; 200 s; absorption; 50 laL; 50 ~L; and 250 IlL.

Results and Discussion Sung and Neeley (14) have shown that the limiting factor in the dilution of Syva's Emit reagents is the final concentration of substrate (G-6-P) and cofactor (NAD+). They also observed that the optimum concentrations of G-6-P and NAD+ for enzyme activity should be 5.3raM and 5. l mM, respectively, in the final sample container. When Emit reagents are used as directed by Syva (either manually or with an Autocarousel), the final concentration of G-6-P is 3.67mM and that of NAD+ is 2.22mM. These are significantly lower than the optimum concentrations suggested by Sung and Neeley. When Emit reagents are used with the COBAS-BIO analyzer as directed by Syva, the final concentrations of G-6-P and NAD+ are 3.86mM and 2.34mM, respectively (17), virtually the same as the Syva manual protocol. Sung and Neeley (14) used final concentrations of G-6-P and NAD+ of 5.30raM and 5.06raM, respectively. Yu et al. (15) used the commercial G-6-P/NAD+ supplement (Raichem) supplied by Reagents Applications at final concentrations of G-6-P and NAD+ of 2.96mM and 2.52mM respectively, following a Raichem protocol. These concentrations are clearly not optimal. Both Sung and Neeley (14) and Yu et al. (15) observed that the overall rate of the enzyme assays increased after addition of the G-6-P/NAD+ supplement. Yu et al.,

Journal of Analytical Toxicology, Vol. 16, November/December 1992

using the Emit d.a.u, methadone assay, tound it to be twice as sensitive after modification. Our object was to modify the Yu protocol in order to significantly increase the sensitivity of the Emit assays. We also wanted to use the general concepts put forward by Sung and Neeley in order to increase the number of assays per kit. Briefly, the Raichem procedure involves the 1:3 dilution of prepared Syva reagents using the Emit buffer (pH 8.0) and the addition of 0.1 mL Raichem to each milliliter of diluted Reagent A, resulting in 19.8 mL Reagent A and 18.0 mL Reagent B. Our

Table I. Comparison of Final MIIlimolar (mM) Concentrations of Glucose-6-phosphate (G-6-P) and Nicotinamide Adenine Dinucleotide (NAD+), and Number of Assays Obtainable Per Kit Using Five Different Methods Method Syva (manual) Syva (COBAS) Sung et al. (COBAS) Raichem (manual) Proposed(manual)

Final [G-6-P] (mM)

Final [NAD+] (mM)

Tests pet kit

3.67 3.86 5.30 2.96 4.92

2.22 2.34 5.06 2.52 4.31

120 334 2470 360 240

proposed method is similar to the Raichem procedure, except that the Syva Reagents are diluted 1:2 rather than 1:3, and the Emit buffer used is corrected for pH for each separate reagent rather than using pH 8.0 for all assays. In addition, twice as much Raichem supplement is added in order to approach the optimum concentrations of G-6-P and NAD+. The proposed method increases the number of assays per kit to 240 from the 120 assays obtained with an Emit d.a.u, kit or from the 60 assays obtained from an Emit serum kit when either is used as directed by the manufacturer. The Raichem procedure allows for 360 assays per kit. A summary of final G-6-P and NAD+ concentrations and assays per kit using the different procedures is found in Table I. In order to offset the effect of reagent dilution, Sung and Neeley increased the assay temperature from 30 to 37~ and increased the reaction time from 30 to 180 s (COBAS-BIO, Syva protocol). The Raichem procedure 1br manual or Autocarousel operation calls for a similar increase in temperature to 37~ and an increase in reaction time to 60 s. On raising the temperature to 37~ we noticed the expected increase in reaction rate, but also a concurrent decrease in precision. Sung and Neeley did not observe significant rate increases at low theophylline concentrations when increasing the temperature to 37~ Because our goal was to optimize sensitivity at low concentrations, we chose to operate at a temperature of 30~ with a longer measure time of 200 s. In order to directly compare our previous method (8) to the

Table II. Comparison of the Within-Run Precision Data Obtained by Use of the Proposed Method and Our Previous Method (8)*

Assay Emit serum benzodiazepine

Emit d.a.u. benzodiazepine

Emit serum TCA

Day

n

Proposed method x (mA) SD (mA)

1 2 3 4 Mean

9 9 9 8

244.6 249.1 249.0 249.6

1 2 3 Mean

8 8 8

1 2 3 4 Mean

9 9 10 9

405.1 403.9 401.1

383.9 383.2 384.3 386.2

1.51 1.27 1.41 1.41

1.17 1.54 2.54

1.62 0.97 1.64 1.20

CV (%)

n

Previous method x (mA) SD (mA)

0.62 0.51 0.57 0.56 0.57

9 10 9

153.6 154.4 153.8

0.29 0.41 0.88 0.45

9 5

0.42 0.25 0.43 0.31 0.35

9 10 10 9

0.88 0.84 1.48

CV (%) 0.57 0.55 0.96 0.69

339.0 347.8

3.00 2.30

0.89 0.66 0.78

243.3 241.9 243.4 246.4

0.71 0.74 1.08 1.67

0.29 0.31 0.44 0.68 0.43

'The methanolic supernatant obtained from drug-free whole bovine blood was analyzed using the Emit serum and d.a.u, benzodiazepine assays and the serum tricyclie anfidepressant assay.

Table III. Between-Run Precision Data bsing the Proposed Method vs. Our Previous Method (8)

Assay Serum benzodiazepine D.A.U. benzodiazepine Serum TCA Mean

n

Proposedmethod x (mA) SD (mA)

CV (%)

n

Previous method x (mA) SD (mA)

35

248.0

2.48

1.00

28

153.9

1.12

0.73

25

403.4

2.48

0.62

14

342.1

5.05

1.48

37

384.4

1.74

0.45 0.69

38

243.7

1.97

0.81 1.00

CV (%)

383

Journal of Analytical Toxicology, Vol, 16, November/December 1992

proposed method, three different Emit assays were used with each on the same day. The assays chosen for this comparison were the Emit d.a.u, and serum benzodiazepine and the Emit serum TCA assays. Following our previous method protocol, the d.a.u, benzodiazepine assay was prepared to 6.0 mL (as directed by SYVA) and the serum assays were prepared by adding 3.0 mL of water followed by a further addition of 3.0 mL of appropriate pH buffer (as described previously) to yield a total volume of 6.0 mL. No G-6-P/NAD+ supplement was added to these three assays and the measure time was set for 90 s instead of 200 s (8). The same three assays were prepared using the proposed method as described in the Assay Preparation section to allow for a direct comparison. Our experience has shown that optimum assay performance is achieved after the reconstituted reagents have been allowed to stand for at least 12 h. Therefore, all assays were prepared 24 h prior to use. Replicate negative samples prepared from bovine blood were analyzed by both methods on consecutive days. These data are shown in Table II. The within-run precision of the three assays using both methods is excellent, with the percent coefficient of

variation (%CV) being consistently less than 1%. The betweenrun precision for each of the three assays using both methods is shown in Table III. Previously, a mean %CV of 1.00% was observed (8), compared to 0.69% using the proposed method. The overall precision of the proposed method was very good and generally better than that observed with our previous method. Standard curves for the benzodiazepine d.a.u, assay using hemolyzed whole blood supplemented with oxazepam are shown Figures 1 (previous method) and 2 (proposed method). Replicate analyses of negative whole blood samples yielded a mean %CV of 0.45% for the proposed method and 0.78% for the previous method (Table II). From this data, the minimum detection limit of the d.a.u, benzodiazepine assay was calculated to be 60 ng/mL oxazepam for the previous method (3 SD) and 20 ng/mL for the proposed method (3 SD). This direct comparison of the two methods indicated an approximate three-fold increase in assay sensitivity by using the proposed method. Similar standard curves for the serum benzodiazepine assay using whole blood supplemented with oxazepam are shown in Figure 3 (previous) and Figure 4 (proposed). Analysis of repli-

d.a.u. BENZOOL(ZEPINE ASSAY (pmMous)

serum BENZODIAZEPINE

mA 340 nm MIMMUM CUTOFF: (repicate datlJ:

J

P

188

MINIMUM CUTOFF(replicate data):

180

366

ASSAY (previous)

mA 3 4 0 nm

1TO lS8

345 ~

"

THERAPEUTICR~QE : 60 - 600 r~hrL (o~

1./

34~ 0

I

I

l

I

I

I

I

I

I

20

dO

8D

80

100

120

140

100

IdO

~ r

THERAPEUTIC RANGE =

188

60 - 500 nolmL (oxmnlllam)

16GI 146 200

0

I

I

I

I

I

I

I

I

I

20

40

60

80

100

120

140

160

180

nO/r4. OXAZEPAM 8ado! 1

8erlol I

Figure 1. Standard curve of the Emit d.a.u, benzodiazepine metabolite assay using a methanolic extract of whole blood with our previous method (8). Concentration of oxazepam (ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Table I1.

Figure 3. Standard curve of the Emit serum benzodiazepineassay using a methanolic extract of whole blood with our previous method (8). Concentration of oxazepam (ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Table II.

serum Bi~IZODIAZEPIME ASSAY Ipropoeed)

rla.u. BENZODIAZEPINE ASSAY Ipropoeedl

m& 3,~ am

m~ 340 nm 63O

MIMMUM CUTOFF (replicate data):,

620

200

ng/mL OXAZEPAM

2~

j

J

MINIMUM CUTOFF:

,,+-

/

=

j

-

285 mpucNs =~11

20 ng/mL

510 27~ 500 490 2~

48O

470

i

i

i

l

10

20

80

40

r 60

i

60

l

~

i

70

80

gO

100

ng/mL O;(AZEP~M $erlt= 1

Figure 2. Standard curve of the Emit d,a.u, benzodiazepine metabolite assay using a methanolic extract of whole blood with the proposed method (8). Concentration of oxazepam (ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Table II.

384

2415 0

l

i

l

i

l

i

i

I

i

10

20

SO

40

60

(10

70

80

gO

100

no/mL OXAZEPAM 8atlas 1

Ftgure 4. Standard curve of the Emit serum benzodiazepine assay using a methanolic extract of whole blood with our proposed method, Concentration of oxazepam (ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Tables II and IV.

Journal of Analytical Toxicology, Vo[. 16, November/December 1992

cate negative blood samples (Table II) yielded mean %CV values of 0.69% (previous) and 0.57% (proposed). The minimum detection limit for the serum benzodiazepine assay was calculated to be 45 ng/mL oxazepam for the previous method (3 SD) and 12 ng/mL for the proposed method (3 SD), reflecting an overall four-fold increase in assay sensitivity. The minimum detection limit of the serum benzodiazepine assay using the proposed method with actual forensic samples known to be negative for benzodiazepines is shown in Table IV. These samples were both ante and post mortem and were analyzed over a one-year period. The mean %CV of 134 such samples was determined to be 1.63%. Using this data and that presented in Figure 4, the minimum detection limit based on actual forensic cases was calculated to be 26 ng/mL oxazepam (3 SD) compared to 12 ng/mL using replicate negative bovine blood. The minimum detection limit (3 SD) of the d.a.u, benzodiazepine assay with actual forensic samples had previously been estimated to be approximately 150 ng/mL oxazepam (8). Based on a direct comparison of the minimum detection limits of the d.a.u, and serum benzodiazepine assays, the serum assay has been chosen over the d.a.u, assay for the routine analysis of blood.

The serum TCA assay was compared directly on consecutive days using the previous and proposed methods. The within-run precision based on replicate analysis of negative bovine blood samples is shown in Table II. The mean %CV for the previous method was 0.43% versus 0.35% for the proposed method. The between-run precision is shown in Table III, with a %CV of 0.81% (previous) versus 0.45% (proposed). A standard curve of nortriptyline using the serum TCA assay and the proposed method is shown in Figure 5. Based on replicate analyses of negative bovine blood, the minimum detection limit was calculated to be 8 ng/mL (3 SD). Our laboratory has recently reported the use of the Emit serum TCA assay with whole blood using our previous method (13). In that report, the minimum detection limit of nortdptyline, based on the analysis of negative bovine blood, was determined to be 30 ng/mL. In this report, the minimum detection limit was determined by the analysis of 173 actual forensic whole blood samples over a one-year period. Using a %CV of 0.76% (Table IV) and data presented in Figure 5, the minimum detection limit for the proposed method was calculated to be 14 ng/mL, an overall two-fold AMPHETAMINE ABSAu

Table IV. Data from Analysis of Ante and Post Mortem Forensic Blood Samples Using Nine Different Emit Assays with the Proposed Method

n

Mean % CV

Mln. det. limit (3 $D) (ng/mL)

129 174 184 169 179 181 173 134

0.73 0.52 0.79 0.86 0.75 0.63 0.76 1.63

12 10 32 22 23 16 t4 26

169

1.41

10

Assay Amphetamine Barbiturate Methadone Opiate Methaqualone Propoxyphene Tricyclic Benzodiazepine (serum) Phencyclidine

"These samples were determined to be negative for the assayed drugs and were analyzed In batches of 10-20 over a one-year pedod.

625

ml~ 140 nm

515 "MINIMUM CUTOFF: 4gG

476

448 4361

20 - ~

425

l

I

I

10

lO

lid

I

I

iA

t

iSu

gO

100

Figure 6. Standard curveof the Emit d.a.u, amphetamineassay using a methanolic extract of whole blood with the proposed method. Concentration of amphetamine (ng/mL) versus the change in milliabsorbance (mA) at 340 rim. See Table IV.

BARBITURATE ASSAY

~

600

MINIMUM CUTOFF:

14.g/mL

I

Ildel 1

serum TRICYCUC AI#TIDEPRESBAN'TASSAY mE 040 nm

I

40 50 80 70 ng/n'L AIMFHETP~iNE

nglmL

mA 340 am

480 , MINIMUM CUTOFF :

/

425 I

7

418 I

:t

..,

J

386 L 0

.oy,

60 - 150 ~llmL

i

i

10

20

i

i

i

i

i

BO 40 60 aO 70 ng/mL IIDRTRIPTYLINE

i

I

i0

gO

I''~176 'T^L

430

100

8erle| 1

Figure 5. Standard curve of the Emit serum tricyclic antidepressantassay using a methanolic extract of whole blood with the proposed method. Concentrationof nortriptyline(ng/mL) versusthe changein milliabsorbance (mA) at 340 nm. See Table IV.

10

20

I 30

I 4O

I 50

60

70

80

go

100

nOlmL 8EOOBARBITAL Carlos 1

Figure 7. Standard curve of the Emit d.a.u, barbiturate assay using a methanollc extract of whole blood with the proposed method. Concentration of secobarbital (ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Table IV.

385

Journal of Analytical Toxicology, Vol. 16, November/December 1992

increase in assay sensitivity. When analyzed with our previous method, 12 different TCA's were detectable in the low or subtherapeutic concentration range. Using the proposed method, these same tricyclics were all detectable in the subtherapeutic range. Thus, the Emit serum TCA assay could be useful for therapeutic drug monitoring (TDM). Cross-reactivity with phenothiazines is known to occur with this assay (13). In addition, a number of antihistamines, particularly diphenhydramine, also cross react with this assay (13). This cross-reactivity may well be useful from a forensic point of view where "shot-gun" type screening is required. Of course, in such cases, whenever an Emit assay result is positive, confirmatory methods such as GC/MS must be employed. A positive Emit TCA assay response could signify the presence of a TCA, a phenothiazine, an antihistamine, or any combination of these. The following Emit assays were indirectly compared to each of the two methods: amphetamine, barbiturates, methadone, opiates, methaqualone, propoxyphene, TCA's, and phencyclidine (PCP). The minimum detection limit for each was determined by calculating the SD and %CV of over 100 actual forensic blood

METHAQUALONE ASSAY

ASSAY

PROPOXYPHENE

mA 340 nm 350~

mA 340 nm 295t

samples known to be negative for the targeted drugs. These forensic samples were analyzed using the proposed method in batches of 10 to 20 samples over a one-year period. The number of actual forensic samples analyzed ranged from 129 to 184 with a mean of 166. The overall mean %CV and minimum detection limit to 3 SD for each assay (calculated from each respective standard curve, Figures 5 to 12) are shown in Table IV. A comparison of our previous method (8) and the proposed method is shown in Table V. The increase in sensitivity for each assay based on forensic cases was determined to range from 1.5 to 10 fold, with a mean of 4.4. Excluding the PCP and serum benzodiazepine assays, the overall %CV was less than 1.0% (Table IV) with minimum detection limits between 10 and 32 ng/mL, which are all within the therapeutic range for these drugs. The serum benzodiazepine and PCP assays had overall mean %CV values of 1.63 and 1.41%, respectively, with corresponding minimum detection limits (3 SD) of 26 and 10 ng/mL The minimum detection limits to 3 SD shown in Table IV represent the absolute minimum detection limits and may be somewhat low for routine toxicological drug screening. As such, the authors rec-

MINIMUM CUTOFF:

34G~

/

MINIMUMCU'TOFF:

16n~mL

2901 2851 260] 2751

~6~

2701

TI'~APEUnr

320 ;" ~

2851

~'~

~ E

=

1-10 ug/I'zl,.

315~

2601 t 10

255'

i

l

|

20

30

i

i

i

50 60 70 ng/mL PROPOXYPHENE 40

i

i

60

go

310

100

;

t

I

I

O

10

20

BO

I

8erie= 1

X

f

I

;

80

gO

11)0

8r162

Figure 8. Standard curve of the Emit d.a.u, propoxyphene assay using a methanolic extract of whole blood with the proposed method. Concentration of propoxyphene (ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Table IV.

Figure 10. Standard curve of the Emit d.a,u, methaqualone assay using a methanolic extract of whole blood using the proposed method. Concentration of methaqualone (ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Table IV.

OPIATE ASSAY

METHADONE ASSAY InK 340 nm

mA 34Ohm ~*Or

~I

I

40 60 eO 70 ~l/n~L MEI'HAOUN.,O~E

4,5O

.,N,U~ . :

/

/

44O

MNMUM (~JllOFF :

~

r

22 rG/mL

430 420 410

2

4

0

0 I 10

~ i 20

t 8D

t t t t 40 60 eO 7a no/mL METHADONE

t lid

I 90

100

8oflls 1

Figure 9. Standard curve of the Emit d.a.u, methadone assay using a methanolic extract of whole blood with the proposed method. Concentration of methadone (ng/mL) versus the change in milliabsorbance (mA) at 340 rim, See Table IV.

386

, ~

~

30 - 800 lll/nL

0

20 - 200 .WmL

i

i

i

10

20

BD

~

i

L

i

40 60 80 70 ng/mL MORPHINE $1rl|s

i

i

80

gO

100

1

Figure 11. Standard curve of the Emit d.a.u, opiate assay using a methanolic extract of whole blood with the proposed method. Concentration of morphine (ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Table IV.

Journal of Analytical Toxicology, Vol. 16, N o v e m b e r / D e c e m b e r 1992

ommend using a more conservative and practical cutoff corresponding to 6 SD. For most assays (excluding PCP and the serum benzodiazepine assays), this conservative cutoff would correspond to approximately 20 milliabsorbance (mA) units above the negative calibrator (30 mA units for the PCP and serum benzodiazepine assays). Ante and post mortem forensic cases that were positive using the proposed Emit method with the serum benzodiazepine or serum TCA assays were quantified by GC and confirmed by GC/MS. These results are shown in Tables VI and VII, respectively. Both the benzodiazepine data (Table VI) and the TCA data (Table VII) show that the proposed method can detect low or subtherapeutic concentrations with mA changes greater than 20 mA above the negative calibrator. Therapeutic concentrations of TCA's were easily detected with a minimum detection limit of 14 ng/mL nortriptyline as described above. In addition, it is apparent that this assay cross reacts with diphenhydramine and thioridazine at higher concentrations. The actual case data showed the proposed method to be very sensitive. A forthcoming report will describe positive case data results for other Emit assays using the proposed method. A number of reports have already

PHENCYCUDINE ASSAY ~r

~Om MINIMUM CUTOFF :

3gO~ 380~ 375 F 370 I388~

demonstrated the confirmation of drugs and/or metabolites using our previous Emit methodology or variations thereof (7-12,18). The proposed method for the direct Emit analysis of forensic hemolyzed whole blood samples was capable of increased sensitivity relative to our previous method (8). By altering the assay kinetics and operating conditions, assay sensitivity has been increased substantially (1.5 to 10 fold). This increase was achieved without resorting to tedious drying, concentration, or extraction procedures prior to analysis by Emit. It should be stressed that this methodology is directed at the detection of probable drug, drug metabolite, and drug conjugate at low concentrations in whole blood. This goal is of prime concem in forensic toxicology. Often little or no drug usage information is available, such that rapid, sensitive, and cost-effective drug screening of small volumes of forensic samples can assist the forensic toxicologist in further analysis or confirmation. The direct analysis of methanolic extracts of whole blood often results in negative findings for a particular assay. With low detection limits, as reported herein, negative results provide convincing immunological data for the absence of a given drug group, thereby allowing the toxicologist to focus on the possible presence of other drugs or drug classes. Positive Emit results are strictly presumptive until confirmed by other standard techniques. It is well known that the Emit assays are capable of detecting drug metabolites and drug conjugates. As such, it is possible that confirmation by GC/MS or other techniques may not be possible. Drug conjugates are generally extremely polar in nature and are usually not extracted by traditional organic solvents. In addition, many nonconjugated polar drug metabolites show poor extraction efficiency as well as poor chromatographic behavior. Most laboratories do not possess stan-

3eO~ 354SP

1o - 250 =tlhtL

r I

0

]~

5

I

I

I

l0 16 ZO nO/rid. PHENCYCLIBINIE

I

15

30

8erlse 1

Case

Figure12. Standard curve of the Emit d.a.u, phencyclidineassay using a methanolic extract of whole blood using the proposed method. Concentration of phencyclidine(ng/mL) versus the change in milliabsorbance (mA) at 340 nm. See Table IV.

Amphetamine Barbiturate Methadone Opiate Methaqualone Propoxyphene Tricyclic Benzodiazepine Phencyclidine

Min. det. limit (3 SD) (ng/mL) Previous Proposed method method 25 100 75 75 75 140 30 150 15

12 10 32 22 23 16 14 26 10 Mean

ChangeIn mA

1 2 3 4 5

21 22 24 31 50

6

68

7

72

8

80

IncreaseIn sensitivity

9 10

88 91

2 x 10 x 2.3 x 3.4 x 3.4 x 8.7 x 2 x 6 x 1.5 x 4.4 x

11

94

12 13

94 97

14 15

99 126

Table V. Minimum Detection Limits Using the Previous Method (8,13) and the Proposed Method for 9 Emit Assays

Assay

Table Vl. Positive Ante and Post Mortem Forensic Blood Samples Using the Emit Serum Benzodlazeplne Assay with the Proposed Method* Drug(s) Lorazepam Diazepam Lorazepam Nordiazepam Diazepam Nordiazepam Diazepam Nordiazepam Diazepam Nordiazepam Diazepam Nordiazepam Temazepam Diazepam Nordiazepam Diazepam Nordiazepam Flurazepam Desalkylflurazepam Diazepam Diazepam Nordiazepam Nordiazepam Temazepam

Conc. (ng/mL) 130 < 5 42 < 40 3 12 20 68 17 25 29 45 223 38 65 20 11 3 12 113 80 225 433 340

"Drugs were quantified using GC and confirmed by GC/MS.

387

Journal of Analytical Toxicology, Vol. 16, November/December 1992

Table VII. Positive Ante and Post Modem Forensic Blood Samples Using the Emit Serum Tricyciic Antidepressant Assay with the Proposed Method* Case

Changein mA

1 2 3 4

21 39 74 79

5

79

6 7 8

82 84 85

Drug(s) Oiphenhydramine Thiorirlazine Amitdptyline Amitriptyline N0rtriptyline Amitriptyline Nortriptyline Doxepin Amitriptyline Amitriptyline Nortripty[ine

The authors wish to thank their colleagues in the Toxicology section for their assistance and support. Sincere thanks are extended to Ms. A. Lyth for her photographic skills.

Conc. (ng/mL) 830 330 55 140 70 126 50 32(] 49 112 66

"Drugs were quantified using GC and confirmed by GC/MS,

dard samples or data for many drug metabolites. The presence of cross-reacting drugs may also pose a problem. Unsuccessful initial attempts to confirm Emit positive results may require hydrolysis and subsequent derivatization prior to GC or GCJMS analysis. Our laboratory currently uses the proposed method with whole blood and the following 16 Emit assays: amphetamines, barbiturates, cannabinoids, cocaine metabolite, methadone, methaqualone, opiates, propoxyphene, PCP, serum benzodiazepine, acetaminophen, valproic acid, phenytoin, carbamazepine, theophylline, and lidocaine. In addition to the increased assay sensitivity discussed above, the proposed method allows for 240 assays per kit compared to 120 assays for d.a.u, kits and 60 assays for serum kits as described by Syva when using an Autocarousel. This increase in assay usage is substantial and can be further increased by the use of clinical analyzers. Thus the proposed method allows for substantial increases in assay sensitivity, as well as a marked decrease in cost, without tedious protocols.

Conclusions The data presented demonstrate the applicability of supplementing the Emit assay reagents with enzyme substrate (G-6-P) and enzyme cofactor (NAD+). Advantages of this procedure include the following: (1) an observed 1.5- to 10-fold increase in Emit assay sensitivity as compared to our previous method (8,13); (2) no lengthy concentration or extraction procedures are required prior to the analysis of the methanolic extract by Emit; (3) assay reagent supplementation is simple and rapid to perform; (4) reagent supplementation allows for twice as many assays per kit for d.a.u, assays and four times as many for serum assays; (5) the proposed method allows for the detection of therapeutic drug concentrations in hemolyzed whole forensic blood samples lor all assays; and (6) the p,'oposed method can be applied to at least 16 different Emit assays.

388

Acknowledgment

References 1. E.L. Slightom and H.H. McCurdy. Enzyme immunoassay: Novel approaches to tissue and fluid analysis. In Advances in Analytical Toxicology, Vol. 1, R.C. Baselt, Ed., Biomedical Publications, Foster City, California, 1984, pp. 19-40. 2. C.W. Gorodetzky and M.P. Kullberg. Validity of screening methods of drugs of abuse in biological fluids, I1. Heroin in plasma and saliva. Clin. Pharmacof. Ther. 15:579-87 (1974). 3. M.G. Homing, L. Brown, J. Nowlin, K. Lertratanangkoon, P. Kellaway, and T.E. Zion. Use of saliva in therapeutic drug monitoring. Clin. Chem. 23:157-64 (1977). 4. J. Vogel and C.N. Hodnett. Detection of drugs in vitreous humor with an enzyme immunoassay technique. J. Anal Toxicol. 5" 307-309 (1981). 5. E Monaco, R. Mutani, C. Mastropaolo, and M. Tondi. Tears as the best practical indicator of the unbound fraction of an anticonvulsant. Epilepsia 20:705-10 (1979). 6. F. Monaco, S. Piredda, R. Mutani, C. Mastropaolo, and M. Tondi. The free fraction of valprolc acid in tears, saliva and cerebrospinal fluids. Epilepsia 23:23-26 (1982). 7. H.W. Pee~ and B.J. Perrigo. Detection of cannabinoids in blood using Emit. J. Anal ToxicoL 5:165-67 (1981). 8. W.M. Asselin, J.M. Leslie, and B. McKinley. Direct detection of drugs of abuse in whole hemolyzed blood using the Emit d.a.u, unne assays. J. Anal. Toxicol. 12:207-15 (1988). 9. H. Gjerde, A.S. Christophersen, B. Skuterud, K. Klemetsen, and J. Modand. Screening for drugs in forensic blood samples using Emit urine assays. Forens. Sci. InL 44:179--85 (1990). 10. L.M Blum, R.A. Klinger, and F. Rieders. Direct automated Emit d.a.u, analysis of N,N-dimethylformamide-modified serum, plasma, and postmortem blood for benzodiazepine, benzoyleogonine, cannabinoids, and opiates. J. Anal ToxicoL 13:285-88 (1989). 11. R.A. Klinger, L.M. Blum, and F. Rieders. Direct automated Emit d.a.u, analysis of N,N-dimethylformamide-modified serum, plasma, and postmortem blood for amphetamines, barbiturates, methadone, methaqualone, phencyclidine, and propoxyphene. J. Anal Toxicol. 14:288-91 (1990). 12. L.J. Lewellen and H.H. McCurdy. A novel procedure for the analysis of drugs in whole blood by homogeneous enzyme immunoassay (Emit). J. Anal ToxicoL 12:260-64 (1988). 13. W.M. Asselin and J.M. Leslie. Direct detection of therapeutic corn centrations of tricyclic antidepressants in whole hemolyzed blood using the Emit tox serum tdcyclic antidepressant assay. J. Anal Toxicol. 15:167-73 (1991). 14. E. Sung and W.E. Neeley. A cost-effective system for performing therapeutic drug assays 1. Optimization of the theophylline assay. Clin. Chem. 31:1210-15 (1985). 15. S.S. Yu and J. Osterloh. A cost effective Emit d.a.u, assay with improved sensitivity. Clin. Chem. 33" 976 (1987). 16. A.D. Fraser, W. Bryan, and A.F. Isner. Modification of the Emit tox benzodiazepine assay for screening of aiprazolam in serum. J. Anal ToxicoL 12" 197-99 (1988). 17. Emit therapeutic drug assays on the COBAS BIO centrifugal analyzer, Syva Co., Palo Alto, CA, publ. no. 8A944-3, October, 1983, 18, M. Bogusz, R. Aderjan, 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. Forens. ScL Int. 48:27-37 (1990). Manuscript received November 31, 1990; revision received November 8, 1991.