CEDIA dau Benzodiazepine Screening Assay: A ... - Semantic Scholar

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Without hydrolysis, all urine samples tested negative. The cross-reactivities of Iorazepam in terms of nitrazepam calibration equivalents, varied from 108 to 178% ...
Journal of Analytical Toxicology, Vol. 22, July/August 1998

CEDIA dau BenzodiazepineScreeningAssay: A Reformulation* Robert C. Meatherall t

Biochemistry Laboratory,St. BonifaceGeneralHospital, 409 Tach6Avene, Winnipeg,Manitoba, CanadaR2H 2A6 Albert D. Fraser

Toxicology Laboratory, Queen ElizabethII Health SciencesCentre, 1278 Tower Road, Halifax, Nova Scotia, Canada B3H 2Y9

[Abstract The CEDIA dau Benzodiazepine assay has been reformulated to include online hydrolysisof urinary benzodiazepine glucuronide conjugates.The new antibody possessesenhanced cross-reactivities toward the low-closebenzodiazepines, which are excreted at low urinary drug-metabolite concentrations.The screeningmethod was evaluated using Iorazepam as the probe benzodiazepine. Four subjects each consumeda l-rag Iorazepam tablet. Sequential urine voids over the same time intervals were collected for the next 48 h. Twelve postdoseurine samples were collected from each subject. Positive resultswere obtained from 5-24 h to 2-35 h usinga 200-ng/mt nitrazepam calibration cutoff. There was no practical difference between hydrolyzing online with the supplied E. coil ~-glucuronidaseor offline with Helix pomatia 13-glucuronidase purchased separately.Without hydrolysis,all urine samples tested negative. The cross-reactivitiesof Iorazepam in terms of nitrazepam calibration equivalents, varied from 108 to 178% for Iorazepam concentrationsbetween 50 and 2500 ng/mL. Lorazepam glucuronide gave cross-reactivities(expressedas Iorazepam base) between 72 and 136% usingthe online hydrolysisprocedure with E. coli ~-glucuronidase.Offline hydrolysiswith Helix pomatia gave cross-reactivitiesbetween 84 and 134%. Without hydrolysis, Iorazepam glucuronide gave lessthan 4% cross-reactivityin the assay.

Introduction

Commercially available immunoassay kits are commonly used to screen for benzodiazepines in urine samples. The antibodies used in these kits are most often developed against one or several unconjugated benzodiazepines. Cross-reactivities toward the other benzodiazepines, which are similar in structure, facilitate their detection in the same screening assay. Most benzodiazepines, however, are eliminated renally as their glucuronide conjugates rather than as free drugs. The low * Presentedat the Societyof Forensic Toxicologists annual meeting, Salt LakeCity, Utah, October 1997. Author to whom correspondenceshould be addressed.

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cross-reactivities of the immunoassay antibodies toward the benzodiazepine conjugates result in compromised detection limits. The importance of first hydrolyzing the benzodiazepine glucuronide with a [3-glucuronidase preparation before screening by immunoassay has been well documented (1-6). Lorazepam has been one of the most difficult benzodiazepines to detect. Cross-reactivities of conjugated and free lorazepam are both low in most commercially available immunoassays. After a single dose, urinary lorazepam concentrations are usually less than 2000 ng/mL. When using EMIT It screening assay (Syva) with a 200-ng/mL cutoff, the drug was not detected in unhydrolyzed urine samples from subjects taking therapeutic doses of lorazepam, and it was detected in just 45% of the hydrolyzed urine samples from the same subjects (1). The cloned enzyme donor immunoassay (CEDIA)technology is based on the assembly of an active [3-galactosidase (nitrazepam in the case of the benzodiazepine assay) enzyme from two inactive component parts. Ahapten is attached to one of the enzyme parts in such a way as to not affect the enzymatic activity of the combined components. However, an antibody directed toward the hapten will prevent the joining of the enzyme components. When free hapten is added to the mixture via the serum sample, the antibody is consumed. Consequently, the enzyme activity is proportional to the amount of free hapten in the serum (7). Recently, the CEDIA dau Benzodiazepine assay kit was reformulated to include [3-glucuronidase in the reagent and an antibody with a higher cross-reactivity toward lorazepam. The kit performance was evaluated in four subjects by testing urine samples collected over a 48-h period following the ingestion of a single 1-mg lorazepam dose.

Methods Subjects

Four healthy volunteers, two males and two females, each ingested a 1-mg tablet of lorazepam (Novolorazepam |

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Journal of Analytical Toxicology, Vol. 22, July/August 1998

occurred between 12 and 24 h after the lorazepam dose. Using 200 ng/mL as the cutoff, subjects I to 4 screened positive at 5-24, 5-35, 2-35, and 2-32 h, respectively. For most of the urine samples, hydrolysis with 40,000 U of E. coli per milliliter of reaction mixture for 5 rain at 37~ gave a response that was 75 to 95% of the corresponding response obo tained using 5000 U Helix pomatia per milliliter of reaction

Novopharm, Toronto, ON, Canada) at 8:00 a.m. A predose and 12 timed urine collections over the following intervals were procured: 0-2, 2-5, 5-8, 8--11, 11-14, 14-24, 24-26, 26--29, 29-32, 32-35, 35-38, and 38-48 h. There were no medication restrictions other than the consumption of a benzodiazepine. The absence of benzodiazepine metabolites was confirmed by analysis of the four predose urine samples by gas chromatography-mass spectrometry (8). Aliquots of urine from each collection period were frozen at -70~ 1600 until analyzed.

Subject I (female) ,

Analysis Urine samples were analyzed with the Microgenics CEDIAdau Benzodiazepines Assayaccording to the manufacturer's protocol on a Hitachi 717 analyzer (Boehringer Mannheim, Montreal, PQ, Canada). The assaywas run in the semiquantitative mode. Supplied calibrators containing nitrazepam at 0, 200, 800, and 5000 ng/mL were used to prepare a log-logit calibration curve within the instrument. The E. coli [3-glucuronidase enzyme was added to the R1 reagent, which contained sheep polyclonalbenzodiazepine antibody and half of a recombinant [3-galactosidase enzyme. When the urine sample was added to the R1 reagent, the lorazepam glucuronide was hydrolyzed. The free lorazepam then binds with the benzodiazepine antibody. After a 5-min incubation at 37~ the second reagent, R2, which contained the other half of the [3-galactosidaseenzyme attached to a benzodiazepine (presumably nitrazepam), was added to the reaction well. The two [3-galactosidaseenzyme halves can combine to form an active unit only when the benzodiazepine antibody is bound to the lorazepam in the sample. A second Hitachi channel was set up with the omission of the E. coli ~-glucuronidase from R1 reagent. Each urine sample was assayed with and without E. coli hydrolysis.The E. coli enzyme activity in the sample plus R1 reagent mixture was calculated to be about 40,000 Fishmann units per milliliter. A second 1-mL urine aliquot was hydrolyzed with 5000 units of Helix pomatia ~-glucuronidase for 2 h at 56~ (1) and analyzed on the Hitachi channel without E. coli [3-glucuronidase. Creatinine concentration was measured on all urine samples using the Jaffe method (9).

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Resultsand Discussion The response in the CEDIAdau Benzodiazepine assay as apparent nitrazepam equivalents (ng/mL) is displayed in Figure 1 for each of the four subjects. Without hydrolysis, all urine samples screened as negatives using 200 ng/mL as the cutoff concentration. With hydrolysis, the peak response

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Figure 1. CEDIA Benzodiazepine response in subjects' timed-interval urine collection. - [ ] -, prehydrolysis, 5000 U H e l i x p o m a t i a per milliliter of reaction mixture;- A -simultaneous hydrolysis, 40,000 U E. c o i l per milliliter of reaction mixture; - 0 - unhydrolyzed.

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mixture for 2 h at 56~ The hydrolysis conditions using Helix pornatia have been previously shown to be optimal for benzodiazepine conjugates (10). In another study using urine spiked with lorazepam glucuronide, hydrolysis was shown to be nearly complete under these conditions (11,12). The E. coli used in the CEDIA test kit appears to have hydrolytic activity toward lorazepam glucuronide similar to the E. coli Type 1X-A

obtained from Sigma Chemical (St. Louis, MO), which was used in the hydrolysis optimization studies (10). The convenience of having the l~-glucuronidase incorporated into the reaction mixture outweighs the disadvantage of suboptimal hydrolysis yield. The time to the first negative result was consistently reduced by 3 h (e.g., 38 to 35 h) when E. coli was used in place of Helix pomatia. The E. Coli [3-glucuronidase was purchased separately from Boehringer Mannheim. A 5-mL Subject I (female) bottle containing approximately 20 million lOOO Fishmann units costs $100 (U.S.). The shelf-life is approximately 1 year when stored at 4~ Each ~ " soo / \ --~-...~ 110 test kit requires 90 pL of the enzyme added directly to the R1 reagent. The additional cost for the l~-glucuronidase is less than 2r per test. In Figure 2, the lorazepam response is corrected for urinary creatinine for the same four z 0 6 12 18 24 30 36 42 subjects. The response is expressed as apparent Collection t i m e (h) nitrazepam equivalents per milligram of creatinine (ng/mg). In each case, the curves are Subject 2 (male) smoothed. If a 100-ng/mg creatinine cutoff is i lOOO used, subjects 1 to 4 screened positive at 5-38, soo 5-35, 2-35, and 2-32 h, respectively. Creatinine correction extends the positive interval for subject I from 24 h out to 38 h. There is no difference in the positive time interval for the other three subjects. More trials are needed to assess possible the benefits of creatinine corrections. The cross-reactivity of lorazepam in the CEDIA 6 12 18 24 30 36 42 48 dau Benzodiazepine assay was determined by supCollection tlme (h) plementing drug-free urine with lorazepam free base. The cross-reactivities ranged from 108 to Subject 3 (female) ,~ looo 178% for lorazepam concentrations between 50 and 2500 ng/mL. At each lorazepam concentra800 tion there was little difference between the results from unhydrolyzed urine and urine which E~ has been hydrolyzed with E. coli or Helix pomatia l~-glucuronidase. The findings are summarized in Figure 3. Therefore, neither the hydrolysis procedure nor the [3-glucuronidase 0 6 12 18 24 30 36 42 48 enzymes affected the CEDIAassay. Collection time (h) Lorazepam glucuronide was purchased from Alltech Applied Science (State College, PA) and used to supplement drug-free urine. The urine Subject 4 (male) looo was spiked such that the lorazepam base concen800 trations were the same as those used in Figure 3. These urine standards were subjected to the CEDIA Benzodiazepine assay without hydrolysis and with E. coli and Helixpomatia hydrolysis as described. The results are summarized in Figure 4. The unhydrolyzed urines all gave cross-reactivities below 4%. The E. coli-treated urines gave 6 12 18 24 30 36 42 48 cross-reactivities between 72 and 136%. The Collectlon tlme (h) Helix pornatia-treated urines gave cross-reactivFigure2. CEDIA Benzodiazepine response corrected for creatinine in subjects' timed-interval ities between 84 and 134%. Most of the spiked urine collection. - [] -, prehydrolysis, 5000 U Helix pomatia per milliliter of reaction mixture; urines gave a higher response using Helix - A -simultaneous hydrolysis, 40,000 U E. coil per milliliter of reaction mixture; - 9 - unhypomatia than the corresponding urine treated drolyzed. with E. coil This reflects the slightly suboptimal

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Urine Ioruepam (ng/mL) Figure 3. CEDIA Benzodiazepine response toward urine spiked with Iorazepam free base. - [] -, prehydrolysis, 5000 U Helix pomatia per milliliter of reaction mixture; - A -simultaneous hydrolysis, 40,000 U E. coli per milliliter of reaction mixture;- 9 - unhydrolyzed.

hydrolysis conditions also observed in the urine samples of subjects ingesting lorazepam.

Acknowledgment We are grateful for the financial support and supply of CEDIA Benzodiazepine kits from Boehringer Mannheim Corporation.

Fig, re 4. CEDIA Benzodiazepine responsetoward urine spiked with lorazepam glucuronide. - [] -, prehydrolysis,5000 U Helix pomatia per milliliter of reaction mixture; - A -simultaneous hydrolysis, 40,000 U E. coli per milliliter of reaction mixture;- 9 - unhydrolyzed.

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R. /vleatherall. Benzodiazepine screening using EMIT II| and TDx| urine hydrolysis pretreatment required. J. Anal. ToxicoL 18: 385-390 (1994). A.D. Fraserand R. Meatherall. Comparative evaluation of five immunoassaysfor the analysis of alprazolam and triazolam metabolites in urine: effect of lowering the screening and GC-MS cut-off values. J. Anal. Toxicol. 20:217-223 (1996). A.B. Boussairi, J.P. Dupeyron, B. Hernandez, D. Delaitre, L. Beaugnet, R Espinoza, and O. Diamant-Berger. Urine benzodiazepines screening of involuntarily drugged and robbed or raped patients. Clin. Toxicol. 34" 721-724 (1996). R Simonsson,A. Linden, and S. Lindberg. Effect of ~glucuronidase on urinary benzodiazepine concentrations determined by fluor-

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escence polarization immunoassay. Clin. Chem. 41" 920-923 (I 995). P.T. Mearrick and J.S. Chahl. Screening for benzodiazepines in urine after hydrolysis of glucuronide conjugates. Cfin. Chem. 31" 152 (1985). O. Beck, P. Lafolie, P. Hjemdahl, S. Borg, G. Odelius, and P. Wirbing. Detection of benzodiazepine intake in therapeutic doses by immunoanalysis of urine: two techniques evaluated and modified for improved performance. Clin. Chem. 38:271-275 (1992). D.R. Henderson, S.B. Friedman, J.D. Harris, W.B. Manning, and M.A. Zoccoli. CEDIA, a new homogeneous immunoassay system. Clin. Chem. 32:1637-1641 (1986). R. Meatherall. GC-MS confirmation of urinary benzodiazepine metabolites. J. Anal. Toxicol. 18:369-391 (1994). Beckman Synchron CX| Delta Systems creatinine assay, Kit #443340, Beckman Instruments Inc., Mississauga, ON, Canada. R. Meatherall. Optimal enzymatic hydrolysis of urinary benzodiazepine conjugates. J. Anal. Toxicol. 18:382-384 (1994). C.L. O'Neal and A. Poklis. Evaluation of (R,S) oxazepam and Iorazepam [3-glucuronide primary reference materials. Presented at the Society of Forensic Toxicologists annual meeting, Baltimore, MD, October 1995. C.L. O'Neal and A. Poklis. Validation of benzodiazepine beta glucuronide primary reference materials for hydrolysis and quality assurance controls. Forensic $ci. Int. 79:69-81 (I 996). Manuscript received September 8, 1997; revision received December 16, 1997.

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