Journal of Analytical Toxicology, Vol. 29, September2005
Diagnostic Performance of Triage for Benzodiazepines: Urine Analysisof the Dose of Therapeutic Cases TM
Emiko KurisakD*, Makiko Hayashida2, Makoto Nihira 2, Youkichi Ohno 2, Hirobumi Mashiko3, Takaaki Okano 3, Shin-ichi Niwa 3, and Kouichi Hiraiwa 1 ~Departrnentof Legal Medicine, FukushimaMedical UniversitySchool of Medicine, Hikarigaoka l, Fukushima,Japan; 2Departmentof Legal Medicine, Nippon Medical School, 1-I-5 Sendagi, Bunkyo-ku, Tokyo,Japan; and 3Departmentof Neuropsychiatry, FukushimaMedical University School of Medicine, Hikarigaoka 1, Fukushima,Japan
thienodiazepines.
only in the urine of patients with benzodiazepinepoisoning but also in the urine of patients given therapeutic doses (3). There are many reports on screening for benzodiazepines in urine using Triage (4,5). However,it is not clear whether the intake of a benzodiazepineis a therapeutic dose or toxic dose when results obtained by using Triage are positive for benzodiazepines. Although benzodiazepines are relatively nontoxic, poisonings with the multiple use of benzodiazepines are found frequently, and there are many benzodiazepine-related deaths in Japan (6). The results of a screening test are used to determine whether a subsequent test should be carried out for confirmation. The aim of the present study was to determine the usefulness of a screening test using Triage for detection of benzodiazepines in urine of patients given therapeutic doses of benzodiazepines. The results of screening using Triage were compared with results obtained by using EMIT d.a.u, benzodiazepine enzyme immunoassay kits. All of the assay results were checked by subjecting the urine samples to chromatographic confirmation.
Introduction
Experimental
Generally the first step in the detection of possible drug consumption in clinical and forensic analysis is immunologica] screening of a urine sample. Triage is one of the commercially available immunoassay kits for drug screening and is the most frequently used in Japan. The use of Triage plus TCA enables simultaneous testing for eight classes of drugs (i.e., amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine metabolites, opiates, phencyclidine, and tricyclic antidepressants) (1,2). Because benzodiazepines are widely prescribed as anxiolytic, hypnotic, and anticonvulsant drugs, they have been found not
Materials and reagents
[ Abstract ] We evaluated the diagnostic performance of Triage for benzodiazepines in 74 urine specimens from outpatients given
therapeutic doses of benzodiazepines and compared the results of EMIT assays. Resultsobtained in all urine samples were confirmed using liquid chromatography-mass spectrometry (LC-MS). Overall agreement between results of Triage and EMIT assayswas 73%. All of the Triage-positive samples were also positive by EMIT assays. Results of Triage and EMIT assayswere different for 20 samples obtained from patients given thienodiazepines (etizolam, brotizolam, and clotiazepam) and nitrobenzodiazepines (nitrazepam, flunitrazepam, and clonazepam). LC-MS confirmed parent drugs in urine specimens, consistent with the prescriptions of drugs. The low agreement between Triage and EMIT results in this study might be due to low sensitivity of Triage for thienodiazepines. Thienodiazines are frequently prescribed benzodiazepines, and Triage panel is the most frequently used screening kit in Japan. It should be noted that negative results obtained by a Triage test might not mean the absence of
* Author to whom correspondence should be addressed: Emiko Kurisaki, Department of Legal Medicine, Fukushima Medical Universily School of Medicine, Fukushima 960-1295, Japan. E-mail:
[email protected]
All reagents were of analytical or high-performance liquid chromatography (LC) grade. Etizolam and c]otiazepam were supplied from Welfide Co. (Osaka, Japan); ]ormetazepam was obtained from Shering (Osaka, Japan); brotizolam was obtained from Nippon Boehringer Ingelheim Co., Ltd. (Hyogo, Japan); cloxazolamwas obtained from Sankyo Co., Ltd. (Tokyo, Japan); triazolam and alprazolam were obtained from Pharmacia & Upjohn (Tokyo, Japan); and clobazam was obtained from Dainippon Pharmaceutical Co., Ltd. (Osaka,Japan). Other benzodiazepines were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Benzodiazepine metabolites, except for 1-hydroxytriazolam and 1-hydroxyetizolam, were not determined.
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Journal of Analytical Toxicology, Vol. 29, September 2005
Urine specimens Urine samples were collected from 74 outpatients of the Department of Neuropsychiatry, Fukushima Medical University Hospital. Written agreement was obtained from each patient before sample collection, and drug prescriptions were also obtained. Forty-nine of the 74 patients were given benzodiazepines with or without other antipsychotic drugs (BZ group). Multiple benzodiazepines were prescribed for 20 of the patients in this group. The prescribed benzodiazepines are listed in Table I. The other 25 patients were not given any benzodiazepines (noBZ group). Twenty-twoof those 25 patients were given other antipsychotic drugs. The urine specimens were stored in glass test tubes at -20~ until analysis. Screening assays Triage Panels for Drugs of Abuse plus TCA were purchased from Biosite Diagnostics (San Diego, CA).The cutoff for Triage screening is 300 ng/mL oxazepam glucuronide. EMIT d.a.u. benzodiazepine enzyme immunoassay kits were purchased from Syva Co. (Palo Alto, CA) along with EMIT Calibrator A Level 1 (a cutoff calibrator containing 200 ng/mL oxazepam) and EMIT Calibrator A Level 2 (a high calibrator containing 1000 ng/mL oxazepam).
Methods Screening assays Triage and EMIT d.a.u, immunoassays were performed according to the manufacturers' instruction. EMIT d.a.u, immunoassay was performed using an ETS| instrument. Each turbid urine specimen was centrifuged at 3,000 rpm for 5 min.
LC-mass spectrometry (MS) analysis Each aliquot (0.5 mL) of urine specimens was hydrolyzed at pH 5 with 10,000 units of 13-glucuronidase in IM potassium acetate buffer. The mixture was incubated at 55~ for 4 h. After cooling, 3 mL of 0.1M borate buffer (pH 9.0)was added, and then the entire mixture was extracted three times with 4 mL of 1-chlorbutane. The combined extract was evaporated to dryness at 50~ under N2 gas. The residue was dissolved in 200 ]JL of the mobile phase and subjected to LC-MS. An LC-MS analysis was performed using an 1100 MSD (Agilent Technologies, Inc.) equipped with a Zorbax XDA C18 column (2.1 x 150 mm, Agilent Technologies, Inc.). Column temperature was 30~ and flow rate was 0.25 mLlmin. The analysis was done using electrospray ionization in the positive
Table I. Names and Numbers of Prescribed Benzodiazepines Benzodizazepines
Number of Prescriptions
Diazepam Etizolam Tdazolam Brotizolam Nitrazepam Alprazolam Estazolam Flunitrazepam Bromazepam Lormetazepam Flurazepam Clonazepam C]otiazepam Clobazam
16 15 9 8 5 5 5 5 3 2 2 2 1 I
Table II. Precision for Benzodiazepines* Compound Name
(min)
tR
SIM ion (m/z)
LOD (ng/mL)
Bromazepam Flurazepam Clonazepam 1-OH Triazolam 1-OH Etizolam Estazolam Nitrazepam Alprazolam Triazolam Brotizolam Etizolam Flunitazepam Clobazam Lormetazepam Diazepam Clotiazepam
7.33 7.66 7.70 8.52 8.52 8.84 8,91 9,09 9.20 9.47 9.51 9.63 9,98 10.00 10.45 10,57
316.0 388.1 316,1 359.0 359.0 295.0 282,1 309,0 343.0 395.0 343.1 314,1 301,1 335.0 285.0 319.0
0,5 0,5 0.5 1.0 0.25 0.5 2.0 1.0 0.5 0,5 0,25 0.5 0.5 0.5 0.5 0,5
* t~ = retention time, LOD = limit of detection, and LOQ = limit of quantitation.
540
LOQ (ng/mL)
Correlation Coefficient
Recovery (%)
CV (%) n= 5
1.0 1,0 1,0 1.0 1.0 1.0 5.0 1,0 1,0 1.0 1.0 1.0 1,0 1.0 1.0 1.0
0.9961 0.9961 0.9978 0.9971 1.0000 0.9995 0.9998 0.9973 0.9999 0,9937 0.9999 0.9943 0.9909 0.9909 0,9999 0,9997
116 89 58 84 84 90 107 114 108 99 110 86 114 106 98 95
3,49 0,58 4.34 1.11 1.11 1.16 8.18 9,97 1.50 0.29 0.75 0.91 2.00 1,37 0.84 0.69
Journalof AnalyticalToxicology,Vol. 29, September2005
ion mode. Quantitation was carried out by selected ion monitoring (SIM). The drying gas (nitrogen) temperature was set at 320~ drying gas flowat 10 L/rain, nebulizer pressure at 40 psi, and Vcap at 3500 V. The fragmentor voltage was set at 100 V. Mobile phase A was programmed as follows: 0.5% acetic acid: 0.5% acetic acid in acetonitrile (80:20, v/v) at 0 rain, 30:70 at 6 min, and 20:80 at 8 min. Mobile phase B was programmed as follows: 10mM ammonium acetate (pH 6.8): acetonitrile (80:20, v/v) at 0 min, 30:70 at 6 min, and 20:80 at 8 min. The atmospheric pressure ionization-electrospray ionization method was used for ionization. Retention times and SIM ions of the compounds are listed in Table II. Under these conditions, all of the benzodiazepines, except for 1-hydroxytriazolam and 1-hydroxyetizolam, were separated well (Figures I and 2). _
patients included thienodiazepines (etizolam, clotiazepam, and brotizolam) and 7-nitro benzodiazepines (nitrazepam, flunitrazepam, and clonazepam). LC-MS results
The degree of precision of LC-MS analysis is summarized in Table II. The limit of detection calculated for a signal-to-noise ratio of 3 is shown. Linearity was then tested between the limit 7.540
k
j 1o 9
Medazepamm/z 270 Diazepamm/z 285 Oxazeman m/z 287
.~8,831
Estazolam m/z 295
.642
Chlordiozepoxidem/z 300
10.341
Fludiazepamm/z 304
7.028
Flumazenil m/z 303
, •9.059
._
Alprazolam m/z 309
Flunitrazepamm/z 314
7.6~o~.
Results
Bromazepam,clonazepam m/z 316 Clotiazepamm/z 319
1 0 . 5 2 8
9"1901~30
Screening results
The results for Triage and EMIT assay are summarized in Table III. Overallagreement was 73% (n = 74). In the BZ group, only 22 (45%) of the 49 urine specimens were positive using Triage and 42 (86%) of the specimens were positive using EMIT d.a.u. All of the EMIT-positivespecimens were also positive by Triage. In the noBZ group, only 2 (8%) of the 25 urine specimens were positive using both assays. No benzodiapzeines were detected using LC-MS in these two positive samples, and it was not clear why they were positive by Triage and EMIT. One of the patients was not given any drugs, and the other patient was given the antipsychotics haloperidol and thioridazine and the antiparkisonian drug biperiden. There has been no report of these drugs giving positive results by cross-reactivity. For comparison of the performances of the two screening tests, Table IV shows a summary of Triage and EMIT results of urine specimens obtained from patients, each given a single benzodiazepine. These urine specimens were divided into three groups by the results of the immunoassay tests: in groups 1 and 2, the results by Triage were consistent with the results by EMIT except for few specimens, and the group 3 included 17 specimens that showed disparate results (negative by Triage and positiveby EMIT). In the group 3, the benzodiazepines prescribed to
Triazolam, etizolam m/z 343
8.529
1-OH TRZ, 1-OH ETZ m/z 359
~.-
Flurazepamm/z 388
~9.460
Brotizolam m/z 395
,8.914
Nitrazepamm/z 282
. . . . . 2. . . . . . .4. . . . . . .6 .
'o . . . . 12 " ' ,
8
1
1
Timo (rain) Figure 1. Chromatogramof a standardmixtureof benzodiazepines(1 ng).
7.746
Medazepamm/z 270 Diazepam m/z 285
12,249
8312 6.399
Oxozeman m/z 287
.659
Estazolam m/z 295
7.321 1
0
.
Chlordiazepoxidem/z 300
~
Fludiazepam m/z 304 .. A ~
6339. ~0946'678
Flumazenil m/z 303
8.826
Alprazolam m/z 309
8.~-6~.198
Flunitrazepamm/z 314
~17'75.2
Bromazepam, clonazepamm/z 316 Clotiazepam m/z 319
9.395
Triazolam, etizolam m/z 343
.~2
1-OH TRZ, 1-OH ETZ rn/z359 2,404
4.855
7.092
Flurazepamm/z 388 6.635
Brotizolam m/z 395
~j%8.991
9",'","',,,
. . . . . . . . .~
,'.
. . . . .,,. .
,'~'
Nitrazepam m/z 282
Time (rain) Figure2. Chromatogramof a urinespecimenfrom a patientgiventherapeuticdosesof benzodiazepines.
541
Journal of Analytical Toxicology, Vol. 29, September 2005
of quantificationand 1000 ng/mL for each compound. Recovery Table III. Correlation of EMIT and Triage Results was calculated as absolute area of an extracted drug (50 ng/mL) from urine versus a nonextracted ethanolic standard EMIT injected in LC-MS. Within-run precision was performed from repeated analysis of spiked runs at 50 ng/mL for each com+ Total pound. + 24 0 24 Benzodiazepine metabolites, except for 1-hydroxytriazolam 20 30 50 and 1-hydroxyetizolam, were not determined by LC-MS because standards of benzodiazepine metabolites could not be 44 30 74 Total obtained. However, parent benzodiazepines were detected in the urine samples from the BZ group, and these were consistent with the drugs prescribed, except in Table IV. Summary of Triage and EMIT Results for Urine Specimens From two cases, even though both Triage and Patients Each Given a Single Benzodiazepine EMIT results were negative. No benzodiazepines were detected using LC-MS in ScreeningResults any of the specimens in the noBZ group. Prescribed Subgroups of Daily Dose (Positive/Total) Table V shows the results of LC-MS Benzodiazepines Benzodiazepines (mg) EMIT Triage analysis in 10 urine specimens after etizolam and triazolam intake at therapeutic N-Desmethyldiazepines 2-6 4/4 3/4 doses. The LC-MSassay used in this study Group 1 Diazepam Triazolo-benzodiazepines 4 1/1 1/1 Estazolam was able to separate triazolam and eti1,5-Benzodiazepines 20 1/1 1/1 Clobazam zolam but not the triazolam metabolite 6/6 5/6 1-OH triazolam or the etizolam metabolite Tfiazolo-benzodiazepines 0.125-0.75 0/2 0/2 1-OH etizolam because their retention Group 2 Triazolam N-Desmethyldiazepines 6 0/1 0/t Bromazepam times were very close and their molecular 3-Hydroxy-benzodiazepines 2 0/1 0/1 Lormetazepam weights are almost the same. Therefore, Triazolo-benzodiazepines 0.4-0.8 1/2 1/2 Alprazolam total concentration of 1-OH triazolam and 1/6 1/6 1-OH etizolam was expressed as the conThienodiazepines 1.5 7/7 0/7 centration of 1-OHtriazolam in the case of Group 3 Etizolam Thienodiazepines 10 1/1 0/1 Clotiazepam multiple doses of triazolam and etizolam. Thienodiazepines 0.25 2/4 0/4 Blotizolam As shown in Table V, etizolam and its 7-Nitro-benzodiazepines 5 1/1 0/1 Nitrazepam metabolites were detected in the urine 7-Nitro-benzodiazepines 1-2 3/3 0/3 FLunitrazepam specimens by LC-MS after etizolam in7-Nitro-benzodiazepines 3 1/1 0/1 CIonazepam take, although the results of Triage test 15/17 0/17 were negativeand those of EMITwere positive. In the case of triazolam intake, the concentrations of triazolam and its Table V. LC-MS Results for Urine Specimes Incuding Etizolam, Triazolam, and metabolite in urine specimens were very Their Metabolites low or undetectable, and the results of ScreeningResults both screening tests were negative. Case Benzodiazepines LC-MS Results No. (Daily Doses,mg) EMIT Triage (ng/mL) ~
Discussion Although overall agreement between results of the two screening tests was only 73% in the present study, good correlations between results of Triage and EMIT were reported by Edinboro (7) (n = 106, agreement 85%) and by Buechler et al. (8) (n = 193, agreement 93%). There are several possible reasons for the discrepancy between our results and the results of previous studies. Schuts et al. (9) reported that false-negative results obtained by immunoassays might be caused by conjugate formation, poor cross542
1 2 3 4 5 6 7 8 9 10
Etizolam (3) Etizolam(0.7) Etizolam(1.5) Etizolam(2) Etizolam(3) Etizolam (3) Etizolam(3) Triazolam (0.125) Triazolam (0.25) Etizolam(1.5), Triazolam (0.125)
Pos Pos Pos Pos Pos Pos Pos Neg Neg Pos
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
*nd, not detected. t Total concentration of metabolites is expressedas 1-OH triazolam.
Etizolam (2.35), 1-OH Etizolam (179.34) Etizolam (0.87), 1-OH Etizolam (55.65) Etizolam (7.27), 1-OH Etizolam (519.42) Etizolam (nd), 1-OH Etizolam (6.45) Etizolam (2.44), 1-OH Etizolam (256.68) Etizolam (2.71), 1-OH Etizolam (342.33) Etizolam (9.01), 1-OH Etizolam (447.83) Tiazolam (nd*), 1-OH Triazolam (nd) Triazolam (nd), 1-OH Triazolam (10.25) Etizolam (14.69), Triazolam (1.31), 1-OH Triazolam~ (1844.59)
Journal of Analytical Toxicology, Vol. 29, September 2005
reactivity caused by structure alteration, and/or low concentration. The low agreement in the present study compared with those in previous studies might have been due to poor reactivity of Triage with thienodiazepines and 7-nitrobenzodiazepines (Table IV). It has been reported that flunitrazepam, one of the 7-nitrobenzodiazepines, might not be detected by Triage at dose in the therapeutic range (10). Thienodiazepine derivatives, in which a benzene ring of the benzodiazepine structure is replaced with a thiophene ring, are one of the most frequently prescribed benzodiazepines in Japan. Although these drugs are not available in the U.S. or U.K. (11), they are widely used in Korea, Italy, and Japan. Thienodiazepines were not examined in previous studies (7,8). As shown in TableV, the results of EMIT assays were positive, and etizolam and its metabolite 1-OH etizolam were detected in urine using LC-MS, but the results of Triage were negative. This suggests low sensitivity of Triage for thienodiazepines and its metabolites. On the other hand, the concentrations of triazolam and its metabolite 1-OH triazolam in urine were low or undetectable using LC-MS, and both Triage and EMITresults were negative. These results might be due to low levels of triazolam and its metabolite in urine. The findings revealed that Triage assays show false-negative results for specimens obtained from patients who have taken therapeutic doses of thienodiazepines and suggest that detection of thienodiazepines and their metabolites in urine might not be possible by Triage, even in cases of toxic level intake.
Conclusions In this study, we performed screening tests using Triage and EMIT assays for benzodiazepines in urine samples from patients who had been taking therapeutic doses of the drugs. All of the assay results were checked by subjecting the urine samples to chromatographic confirmation. There was a low agreement between Triage and EMIT results in the present study. This discrepancy might be due to low sensitivity of Triage for thienodiazepines. Thienodiazepines such as etizolam are frequently prescribed benzodiazepines in Japan. A Triage panel is a convenient screening kit for urine and is most frequently used in Japan. However,it should be noted that negative results obtained by Triage might not mean the absence of thienodiazepines, even at toxic levels.
Acknowledgment We are grateful to Dr. Masaru Tereda for the gift of 1-OH etizolam.
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