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Automated on-line hydrolysis of benzodiazepines improves sensitivity of urine screening by a homogeneous enzyme immunoassay.

Benzodiazepines are used therapeutically as antidepressant, anxiolytic, anticonvulsant, hypnotic, muscle relaxant, and preanesthetic agents. Moreover, benzodiazepines are often abused for their euphoric effects. Consequently, benzodiazepines are commonly detected in toxicological analyses.

Immunoassays of benzodiazepines are plagued by several factors that make reliable detection difficult. Complicated metabolic pathways, including N-dealkylation, C-hydroxylation, and glucuronidation, effectively reduce the parent compound to more polar, and thus easily excreted, metabolites. Some of these metabolites are pharmacologically active; in some cases, they are present in higher concentrations and have a longer elimination half-life than the parent compound [1, 2]. Many, especially the glucuronide conjugates, which appear at high concentrations in the urine, do not readily cross-react with commercial immunoassays. In contrast, other benzodiazepine metabolites demonstrate high cross-reactivity, depending on the immunoassay utilized [3].

In the US, 13 benzodiazepines of various potencies are commonly prescribed. Therapeutic daily doses have substantially decreased with the newer generation of benzodiazepines [2]. Variations in dosage and in ultimate excretion patterns complicate the ability to accurately detect benzodiazepine use [4].

Accumulated research findings indicate pretreating urine specimens with [beta]-glucuronidase (EC increases the diagnostic sensitivity of benzodiazepine immunoassays. In addition, even untreated specimens with concentrations of benzodiazepine analytes below the cutoff concentration may screen positive because of the presence of highly cross-reactive benzodiazepine metabolites [3,5]. Also, oxaprozin (Daypro [R]; G.D. Searle), a structurally unrelated nonsteroidal antiinflammatory drug, produces false-positive results in several commercial immunoassays [6-8].

In this study we evaluated whether automated on-line hydrolysis of benzodiazepine analytes would improve the sensitivity of the Boehringer Mannheim Corp. (BMC) cloned enzyme donor benzodiazepine immunoassay (CEDIA [R]). We evaluated in parallel 1002 urine specimens that were screened by two CEDIA benzodiazepine assays: the current assay with no enzymatic pretreatment, and a modified assay in which [beta]-glucuronidase (200 U/mL; BMC) was added to the R1 reagent system (5 [micro]L of [beta]-glucuronidase solution per milliliter of R1) for automated on-line hydrolysis of conjugated benzodiazepine analytes. (The modified CEDIA has also been evaluated recently by Bellet et al. [9].) All specimens were analyzed with a Hitachi 717 (BMC) automated chemistry analyzer, with a positive cutoff concentration of 200 [micro]g/L (nitrazepam). All instrument settings were according to published manufacturer's guidelines for the CEDIA benzodiazepine assay.

All CEDIA presumptive positive specimens (n = 50) were assayed by GC/MS. Before GC/MS analysis, the urine specimens were hydrolyzed with [beta]-glucuronidase (Sigma Chemical Co.), followed by solid-phase extraction (Clean Screen [R] SPE columns, ZSDAU020; United Chemical Technologies, Horsham, PA). The extracts were derivatized with methyl-(tert-butyldimethylsilyl)-trifluoroacetamide (Pierce Chemical Co.) for quantification of nordiazepam, oxazepam, temazepam, lorazepam, hydroxyethylflurazepam, [alpha]-hydroxyalprazolam, and [alpha]-hydroxytriazolam. All specimens that were negative by GC/MS were subsequently reextracted and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (Pierce Chemical Co.) for quantification of 7-aminoclonazepam, 7-aminoflunitrazepam, and 7-aminonitrazepam.

A Hewlett-Packard (HP) 5890 Series II gas chromatograph and HP 7673 automated liquid sampler interfaced to an HP 5972A mass-selective detector (MSD) were utilized. The gas chromatograph was equipped with a cross-linked 95% dimethyl/5% diphenyl polysiloxane capillary column [HP-5MS; 30 m x 0.25 mm (i.d.) x 0.10-[micro]m-thick film]. Automated injections were made in the splitless mode. The MSD was operated in the selected-ion monitoring mode with mass ions monitored at a dwell time of 20 ms. A multipoint calibration curve (25-500 [micro]g/L) and pentadeuterated internal standards (Radian Corp., Austin, TX) were used for quantification; i.e., quantification was based on the ion peak area ratio of the analyte to its corresponding pentadeuterated internal standard analog. The limit of quantification (LOQ) was established as 25 [micro]g/L. Analytes were identified on the basis of retention time ([+ or -]1%) and ion ratios ([+ or -]20%) by comparison with the corresponding values for the internal standards.

Manual pretreatment with enzyme before immunoassay analysis can be very time consuming. By using [beta]-glucuronidase added to the R1 reagent of the CEDIA benzodiazepine assay and subsequent on-line incubation with the urine specimen, conjugated benzodiazepines were cleaved to yield more immunoreactive analytes. Compared with the current CEDIA, the modified CEDIA demonstrated a 26% increase in the number of presumptive positive results, from 37 to 50 specimens. Furthermore, all benzodiazepine-positive specimens demonstrated an increase in 0 absorbance rates (mA/min) with the modified assay, the increase ranging from 10 to 269 (mean, 72.56; SD, 70.19).

Results of the GC/MS analyses for the specimens that were negative by the current CEDIA, but positive by the modified CEDIA, are listed in Table 1. The data demonstrate that [beta]-glucuronidase pretreatment improves immunoassay detection of several benzodiazepine analytes, even at concentrations less than the cutoff calibrator. Benzodiazepine analytes for which detection was improved include lorazepam, nordiazepam, oxazepam, and temazepam. Of the 50 total specimens, 3 could not be confirmed by the chosen GC/MS assays and were subsequently submitted to BMC for analysis by alternative GC/MS methods. Two of these specimens were found to be positive for oxaprozin, and one was positive for alprazolam. Combining these GC/MS results with the CEDIA screening results gives a specificity of 99.8% for the current and modified CEDIA methods.

Our study shows that addition of [beta]-glucuronidase to the R1 reagent of the CEDIA DAU benzodiazepine assay increases the detection of benzodiazepine analytes in urine specimens. To confirm the presence of benzodiazepine analytes in urine, a technique should be able to detect concentrations less than the immunoassay cutoff value. This sensitive on-line hydrolysis method provides a rapid and efficient urine screen for the presence of benzodiazepines.

This study was funded in part by Boehringer Mannheim Corp.


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[9.] Bellet NF, Scholz HA, Opel ML, Rytter H, Galloway FR, Khanna P. High sensitivity CEDIA [R] DAU benzodiazepine assay utilizing [beta]-glucuronidase. J Anal Toxicol 1997;21:90-1.

Jeri D. Ropero-Miller, Diana Garside, and Bruce A. Goldberger * Univ. of Florida College of Med., Dept. of Pathol., Immunol., and Lab. Med., P.O. Box 100275, Gainesville, FL 32610-0275; * author for correspondence: fax 352-846-1586, e-mail
Table 1. GC/MS results for benzodiazepine analytes. (a)
[DELTA] absorbance rate,
mA/min (a)

Specimen Current Modified
no. CEDIA CEDIA GC/MS result, [micro]g/L

25 -39 10 Nordiazepam <25; oxazepam 51;
 temazepam <25
28 -51 3 Nordiazepam <25; oxazepam 35;
 temazepam <25
236 -81 53 Lorazepam 334; [alpha]-
 <25; [alpha]-hydroxytriazolam <25
628 -10 24 Nordiazepam <25
674 -80 19 Lorazepam 334
685 -20 7 Lorazepam 48; 7-aminoclonazepam 202
786 -7 16 [alpha]-Hydroxyalprazolam 84
799 -85 49 Nordiazepam <25; lorazepam 199
831 -52 217 Lorazepam >500
850 -47 192 Nordiazepam <25; oxazepam 134;
 temazepam >500
882 -2 8 Nordiazepam <25; oxazepam 27;
 hydroxyethylflurazepam <25
910 -53 4 Nordiazepam <25; oxazepam 42;
 temazepam 26
984 -34 32 Nordiazepam <25; oxazepam 103

(a) rate [greater than or equal to] mA/min is considered positive.
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Title Annotation:Technical Briefs
Author:Ropero-Miller, Jeri D.; Garside, Diana; Goldberger, Bruce A.
Publication:Clinical Chemistry
Date:Sep 1, 1997
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