Qualitative determinations of other compounds (ambroxol, bro- mazepam-M, carbamazepine-M, clotiapine, cyproheptadine, lev- orphanol, lorazepam, naloxone ...
Journalof AnalyticalToxicology,Vol. 29, October 2005
[ TechnicalNote
Performance Characteristics of 7-Aminoflunitrazepam Specific Enzyme-Linked Immunosorbent Assays Dong-Liang Lin1, Rea-Ming Yin 1, Cheng-Hsing Chen 2, Ya-Lei Chen 3,*, and Ray H. tiu 4 1Instituteof Forensic Medicine, Ministry of Justice, Taipei, Taiwan;2ping-An Hospital, Pingtung Hsien, Taiwan;3Departmentof Biotechnology, National KaohsiungNormal University, KaohsiungHsien, Taiwan;and 4Departmentof Medical Technology, Fooyin University, KaohsiungHsien, Taiwan have demonstrated that conventional immunoassays (IA) for benzodiazepineswere not effectivefor the two-step IA/gaschromatography-mass spectrometry (GC-MS) testing protocols adapted for the high-volume testing environment. One of these studies (5) also demonstrated that a 7-aminoflunitrazepam (7amino-FM2)-specific enzyme-linked immunosorbent assay (ELISA)by Cozart Bioscience (Oxfordshire, U.K.)can be effective under this testing environment. More recently, 7-amino-FM2-specific ELISAs from other sources have become available. This study has been conducted to (1) provide a broader understanding on the cross-reacting characteristics of these reagents toward a comprehensive list of benzodiazepines; (2) compare the performance characteristics of these ELISAs,especially in relating the ELISA'sapparent 7amino-FM2 concentrations to the definite concentrations of 7-amino-FM2 as determined by GC-MS; and (3) understand the characteristics of data derived from urine specimens collected from patients under the treatment of FM2 (with or without diazepam) or other benzodiazepines.
Abstract[ With 7-aminoflunitrazepam (7-amino-FM2)-speciflc ELISAsnow readily available from several commercial sources(e.g., Cozart Bioscience, Immunalysis, this study was conducted to evaluate the performance characteristicsof these productswhen applied to the two-step testing protocol as commonly practiced in today's workplace drug-testing programs. Cross-reactingcharacteristicsof these two assaystoward a list of 25 benzodiazepineswere evaluated. These assayswere then applied to the analysis of urine specimens collected from patients treated with flunitrazepam (FM2) and/or other benzodiazepines. Resulting data were evaluated against gas chromatography-mass spectrometry (GC-MS) test data to ascertain corresponding cutoffs suitable for the two-step immunoassay/GC-MS testing strategy. Both Cozart and Immunalysis ELISAsare highly specific to 7-amino-FM2, with the latter reagent generating slightly higher responses. Diazepam and FM2 (parent compound) are the only compounds with significant cross-reacting characteristics. With the ELISA reagents' optimal dynamic ranges set between 0 and 25 ng/mL, urine specimens should be diluted by a factor of 5 prior to ELISA testing. If 30 ng/mL 7-amino-FM2 is adapted as the GC-MS cutoff, the corresponding ELISA cutoffs range is approximately 100-200 (or 20-40 when diluted by a factor of 5) ng/mL. Reagent lot and specimen characteristics (with or without the presence of cross-reacting compounds) affect the correlation of data derived from ELISA and GC-MS tests.
Introduction Flunitrazepam (FM2) is extensivelyused for treating insomnia in many parts of the world. It has been associatedwith drug-assisted sexual assault cases (1,2) and is also one of the benzodiazepines that may affect human performance and behavior (3). Earlier studies (4,5)
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0 * Author to whom correspondence should be addressed: Dr, Ya-Lei Chen, Department of Biotechnology, National Kaohsiung Normal University, 62, Shen-Chung Road, Yen-Chao, Kaohsiung Hsien, Taiwan. E-mail: dan1001@ms31 .hinet,net.
Materials and Methods
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Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
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Journal of Analytical Toxicology, Vol. 29, October 2005
ELISAKit (216-0480,Ver: 08/2000) were purchasedfrom Cozart Bioscience(Abingdon,Oxfordshire,U.K.)and Immunalysis (San Dimas, CA). Standards (in 1 or 0.1 pg/mL methanolic solu-
tions), including the analyte (7-amino-FM2),internal standard (7-amino-FM2-dT), and other benzodiazepines for cross-reactivity studies were purchased from Cerilliant (Austin, TX). All clinical urine specimens were from patients admitted to Pin-Annhospital (Pin-Tong, Table I. Cross-Reactivities ( % ) of 7-Aminoflunitrazepam-Specific ELISAs Taiwan) and were collected by a staff nurse t o w a r d Benzodiazepines and Their Metabolites followinga procedure established by the hospital. Fooyin University IRB procedure exCozart Immunalysis Substance and empts urine specimen collection through Manufacturer* ExperimentaP Manufadurer* ExperimentaP Conc. (ng/mt) non-invasive means. Specimens in Groups A (n = 13), B (n = 13), and C (n = 9) were from Diazepam 12 (85) 13 patients treated with other benzodiazepines, -10 --w 11 9.9 such as lorazepam, alprazolam, and clon100 7 9.0 -2.2 azepam (without FM2 or diazepam), FM2 4.3 -I000 > 2.5 0.55 (without diazepam), and FM2 plus diazepam, > 0.25 0.63 -I0,000 respectively. All specimens were kept at 4~ Flunitrazepam 83 (12) until analysis. 5 < 20 18 -34 10 25 100 250 500
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---
- 0.998) within the 10-200 ng/mL range studied. Using the criteria commonly adapted for defining LOD and LOQ, the LOQ and LOD were 2.0 and 0.50 ng/mL. Specifically, LOD is defined as the lowest concentration at which the monitored ion ratios are within ___20% of that observed for the 50 ng/mL standard, while LOQ is defined as the lowest concentration at which the observed concentration is within • 20% of the targeted value.
Correlation of ELISAapparentand GC-MS 7-amino-FM2 concentrations Cross-reactivitydata shown in Table I indicate diazepam as the only compound (other than FM2 parent compound) that crossreacts with the ELISAsto a significant extent. To further understand the effect of diazepam on the correlation of the ELISA and GC-MS test data, specimens in Groups B and C were analyzed (second and third section of Table II). Resulting correlation data for these two groups are shown in Figures 2A-C, and the correlation of the apparent 7-amino-FM2data derivedfrom these two ELISAsare shown in Figure 3. The associatedcorrelation parameters derived from Figures 2A--Care listed in Table IV. Data shown in Table IV indicate that the apparent 7-aminoFM2 concentration (that was equivalent to 30 ng/mL as determined by GC-MS) derived from the Immunalysis reagent is higher than that derived from the Cozart reagent. Furthermore, these apparent concentrations derived from Group C specimens (patients with both flunitrazepam and diazepam exposure) are higher than the corresponding data derived from Group B specimens (patients with flunitrazepam but without diazepam exposure). Thus, data derived from two sets of clinical specimens further confirm that Immunalysis reagent indeed has slightly higher cross-reactivities than the reagent from Cozart. It is interesting to note that data
derived from Group C specimens result in a better correlation with the GC-MS data for the Immunalysis than the Cozart reagent. One important factor in a two-step testing protocol is the selection of a cutoff concentration for the GC-MS test and an appropriate corresponding apparent analyte concentration for the immunoassay protocol. With this in mind and based on the excretion profile (5-10) of 7-amino-FM2 and the GC-MS assay's LOD and LOQ established in this study, the cutoff for the GC-MS protocol is set at 30 ng/mL. Based on the correlation equations derived from data generated by the ELISAs using various sets of specimens, the equivalent ELISA apparent 7amino-FM2 concentrations were found to range approximately 100-200 ng/mL (or 20-40 when diluted by a factor of 5). Both ELISA kits can be effectively applied to the two-step testing protocol with the recommendedcutoffs and fivefolddilution of test specimens.
Conclusions
Both Cozart and Immunalysis ELISAs are highly specific for 7-amino-FM2, with diazepam and FM2 (parent compound) as the only compounds with significant cross-reacting characteristics. Immunalysis reagent appears to exhibit slightly higher cross-reactivity than the Cozart reagent; however, this characteristic does not make it less effective in predicting the concentration of 7-amino-FM2 when the specimens are collected from donors with both flunitrazepam and diazepam exposure. With the ELtSAreagents optimal dynamic ranges set between 0 and 25 ng/mL, specimens should be diluted by a factor of 5 prior to ELISA testing (dilution is generally not required for GC-MS analysis). Using 30 ng/mL 7-amino-FM2 as the GC-MS cutoff, the corresponding ELISA cutoffs are approximately 100-200 (or 20-40 when diluted by a factor of 5) ng/mL depending on the reagent and Table IV. Correlation of ELISA and GC-MS Data Derived from Specimens whether the specimens also contain significant Collected from Patients Treated with FM2 and Other Benzodiazepines levels of cross-reacting compounds, such as diazepam (and its metabolites). ELISA 7-Amino-FM2 Conc. (y) Corresponding ELISA and Resulting
Specimen Group [3
Group C
Group B and C
Correlation Equation
Correlation Equation
to 6 (or 30 in Original Specimen)ng/ml GC-MSConc.(x)*
Cozart
y= "14.57+ 1.227x
0~5412
Immunalysis
y= 11.13 § 1.921x
0.4000
Cozart
y = 18.82 + 0.07111x
lmmunalysis
y= 30.50 + 0.7897x
0.003113 32.2 (~ 160 in undiluted specimen) 0.1660 35.2 (~ 180 in undiluted specimen)
Cozart Immunalysis
y = 21.85 + 0.7117x y= 18.78 + 1.456x
0.2250 0.3088
21.9 l- I10 in undiluted specimen) 22.6 (~ 115 in undiluted specimen)
26.1 27.5
* With the dynamicrangesof the ELISAsset between0 and 25 ng/mL specimensweredilutedby a factorof 5 priorto ELISAtesting(undilutedfor GC-MSanalysis).
722
Acknowledgments The authors are thankful to Miss Ya-Ching Lee for collecting the urine samples used in this study and the financial support provided by the (Taiwanese) National Science Council (NSC 93-2745-M-242-002, NSC 93-2745-M242-003, and NSC-93-2745-P-521-002).
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Journal of Analytical Toxicology,Vol. 29, October 2005 2. M.A. EISohly and S.J.Salamone. Prevalence of drugs used in cases of alleged sexual assault. ]. Anal. Toxicol. 23:141-146 (1999). 3. O.H. Drummer. Benzodiazepines---effects on human behavior and performance. Forensic Sci. Rev. 14:1-14 (2002). 4. A.M. Barrett, K. Walshe, P.V. Kavanagh, S.M. McNamara, C. Moran, J. Burdett, and A.G. Shattock. A comparison of five commercial immunoassaysfor the detection of flunitrazepam and other benzodiazepines in urine. Addict. Biol. 4:81-87 (1999). 5. P.H. Wang, C. Liu, W.I. Tsay, J.H. Li, R.H. Liu, T.G. Wu, W.J. Cheng, D.L. Lin, T.Y. Huang, and C.H. Chen. Improved screen and confirmation test of 7-aminoflunitrazepam in urine specimens for monitoring flunitrazepam (Rohypnol) exposure. J. AnaL Toxicol. 26:411-418 (2002). 6. S.J.Salamone, S. Honasoge, C. Brenner, A.J. McNally, J. Passarelli, K. Goc-Szkutnicka, R. Brenneisen, M.J. EISohly, and S. Feng. Flunitrazepam excretion patterns using the Abuscreen OnTrak and OnLine immunoassays: comparison with GC-MS. ]. Anal.
Toxicol. 21: 341-345 (1997). 7. H. Snyder, K.S. Schwenzer, R. Pearlman, A.J. McNally, M. Tsilimidos, S.J. Salamone, R. Brenneisen, M.A. EISohly, and S. Feng. Serum and urine concentrations of flunitrazepam and metabolites, after a single oral dose, by immunoassay and GC-MS. J. Anal. Toxicol. 25:699-704 (2001). 8. Z. Lin and O. Beck. Procedure for verification of flunitrazepam and nitrazepam intake by gas chromatographic-mass spectrometric analysis of urine. J. Pharm. Biomed. Anal. 13:719-722 (1995). 9. M.A. EISohly, S. Feng, S.J. Salamone, and R. Wu. A sensitive GC-MS procedure for the analysis of flunitrazepam and its metabolites in urine. ]. Anal. Toxicol. 21:335-340 (1997). 10. A. Negrusz, C.M. Moore, T.L. Stockham, K.R. Poiser, J.L. Kern, R. Palaparthy, N.L. Le, P.G.Janicak, and N.A. Levy. Elimination of 7-aminoflunitrazepam and flunitrazepam in urine after a single dose of Rohypnol. ]. Forensic Sci. 45:1031-1040 (2000).
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