Elisabetta Bertol 1, Fabio Vaiano 1, Maurizio Borsotti 2, Massimo Quercioli 2 and Francesco Mari 1 *

Transcription

1 Journal of Analytical Toxicology 2013;37: doi: /jat/bkt063 Advance Access publication August 13, 2013 Article Comparison of Immunoassay Screening Tests and LC MS-MS for Urine Detection of Benzodiazepines and Their Metabolites: Results of a National Proficiency Test Elisabetta Bertol 1, Fabio Vaiano 1, Maurizio Borsotti 2, Massimo Quercioli 2 and Francesco Mari 1 * 1 Forensic Toxicology Division, Department of Health Sciences, University of Florence, Florence, Italy, and 2 Department of Security and Quality, Careggi Hospital of Florence, Florence, Italy *Author to whom correspondence should be addressed. francesco.mari@unifi.it For most diverse purposes, different immunoassay (IA) screening methods are usually used to detect benzodiazepines and their metabolites in urine. In this study, we compared the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIA w ; Enzyme- Multiplied Immunoassay Technique, EMIT w ; Fluorescent Polarization ImmunoAssay, FPIA w ; Kinetic Interaction of Microparticles in Solution, KIMS w and Immunochromatographic Techniques, IMC) with liquid chromatography tandem mass spectrometry (LC MS-MS). Twelve urine specimens were analyzed by 178 laboratories in Italy that participated in a National Proficiency Test, providing both qualitative and semi-quantitative results. Each IA was evaluated by the parameters: true positive, true negative, false positive (FP), false negative (FN), sensitivity (SENS), specificity (SPEC), positive predictive value, negative predictive value (NPV) and accuracy. SPEC was affected by a high FP rate for all IAs. The lowest SENS and NPV were provided by FPIA due to a high number of FN cases. Comparing IA semi-quantitative data with LC MS-MS results, an overestimation of benzodiazepine amount is noted. This paper draws attention to the problem of the careless use of IA tests for forensic purposes as they may provide FP and/or FN results that can lead to errors of great severity. Introduction Benzodiazepines are the drugs most frequently prescribed worldwide as tranquilizers, hypnotics, anesthetics, anticonvulsants or muscle relaxants to treat sleeplessness, depression, anxiety and epilepsy. Benzodiazepines are considered to be safer and more effective than barbiturates, but chronic use can produce dependence and abuse (1). Their intake, in combination with other central nervous system (CNS) depressants such as alcohol, may cause severe respiratory depression, which can lead to death (2 4). Benzodiazepine misuse is increasingly associated with suicidal poisoning (5), driving under the influence of drugs (DUID) (6) and drug-facilitated sexual assault (DFSA) (7 9). As such, benzodiazepines are among the most frequently encountered substances in clinical and forensic toxicological analyses, for which the simultaneous analysis of benzodiazepines and their metabolites in biological matrices is of great interest. The most representative difficulties in benzodiazepine analysis derive from the large number of these drugs (more than 50 different benzodiazepines are commercially available for clinical use) and from the possibility that many of them can be metabolized to multiple forms (many metabolites are drug substances in their own right) (10, 11). For these reasons, screening tests that can provide a rapid qualitative (or semi-quantitative) detection of benzodiazepines with high sensitivity (SENS) and specificity (SPEC) to include both abusive use and low-therapeutic doses are necessary, followed by instrumental techniques based on mass spectrometric detection. Immunoassays (IAs) are widely utilized for their rapidity, flexibility and semi-quantitative results. Urine is one of the preferred matrices for drug analysis, because the concentrations of benzodiazepines and their metabolites are higher in urine than in other biological specimens such as blood, oral fluid and hair. However, the capability of IAs to detect benzodiazepines is problematic due to the wide variation of the various representatives of this group of drugs in potency, structure, metabolism and elimination. The most critical limitation of IAs is related to the variable immunoreactivity of the antibodies to the diverse structural differences of the benzodiazepine class of drugs, leading to a large incidence of false positives (FPs) and false negatives (FNs) (12). In forensic laboratories, usually only positive results are investigated by instrumental techniques, while negative (and FN) ones are not. FNs can occur when benzodiazepine compounds with a low immunoreactivity rate are analyzed (i.e., Phase II metabolites, especially glucuronide forms) (13, 14). It is important to keep in mind these limitations, as in urine specimens benzodiazepine metabolites may be present and there is a risk of quantitative underestimation resulting in a false negativity (15). Analytical identification by IA is further complicated by the wide ranges of benzodiazepine therapeutic concentrations and metabolic pathways. Thus, these types of assays are not suitable to selectively identify the drug and to discriminate the parent drug from their metabolites (16). The present paper aims to evaluate the advantages and limitationsoftheuseofiasforforensicpurposesonthebasisofa National Proficiency Test (PT), for which we are the Reference Laboratory (RL). The study was conducted by comparing the liquid chromatography tandem mass spectrometry (LC MS-MS) method with the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIA w ; Enzyme-Multiplied Immunoassay Technique, EMIT w ; Fluorescent Polarization ImmunoAssay, FPIA w ; Kinetic Interaction of Microparticles in Solution, KIMS w and Immunochromatographic Techniques, IMC) to determine benzodiazepines and their metabolites, with particular attention to false result cases. Methods Chemicals and urine specimens Drug standards 3-hydroxyflunitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, a-hydroxyalprazolam, a-hydroxymidazolam, alprazolam, bromazepam, brotizolam, clonazepam, chlordiazepoxide, delorazepam, diazepam, flunitrazepam, halazepam (internal standard, IS), ketazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordiazepam, oxazepam, pinazepam, prazepam, temazepam and triazolam were purchased # The Author [2013]. 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2 Table I Number and typology of IA techniques used in the quality control program for benzodiazepines and their metabolites detection IA technique Laboratories CEDIA 13 EMIT-SYVA 27 EMIT-I.L. 10 EMIT-DIMENSION 26 EMIT-BECKMAN 11 EMIT-ARCHITECT 10 FPIA-ABBOTT 9 KIMS 39 Immunochromatography 33 from Lipomed, Inc. (Cambridge, MA, USA). Dichloromethane was acquired from Panreac Quimica S.L.U. (Castellar del Vallès, Spain). Glacial acetic acid was obtained from J.T. Baker (Deventen, Holland). Sodium hydroxide, ammonium acetate and isopropanol were supplied by Carlo Erba Reagenti (Milano, Italy). LC MS CHROMASOLV w methanol and b-glucuronidase enzyme (type HP-2 from Helix pomatia, 152,900 units/ml) were purchased from Sigma-Aldrich (St Louis, MO, USA). Water was obtained from B. Braun (Milano, Italy). Urine specimens were supplied by the Department of Security and Quality, Careggi Hospital of Florence, according to the scheme of the Regione Toscana External Quality Assessment (EQA). These specimens consisted of liquid urine from drug abusers ( pool of several patients): thus, the samples are not statistically representative of the real population containing not only benzodiazepines (but also other drugs of abuse such as opiates, cocaine, cannabinoids, barbiturates and amphetamines). Urines were filtered and sodium azide was added as preservative. Twelve samples were analyzed to detect benzodiazepines and their metabolites. Immunoassays IAs were performed by 178 different laboratories in Italy that participated in a PT based on an EQA program for the detection of benzodiazepines and their metabolites, performed by the Department of Security and Quality (Careggi Hospital of Florence) and promoted by the Tuscany Region. The IA techniques were CEDIA, EMIT (Syva, S; Instrumentation Laboratory, I.L.; Dimension, D; Beckman, B; Architect, A), FPIA, KIMS and IMC assays (Table I), and they were performed according to the manufacturers instructions, which indicate the method that each laboratory must apply for the instrument used, in order to provide the most reliable results and to compare with the others. Enzymatic hydrolysis and liquid liquid extraction The supplied urine samples were already mixed by the Department of Security and Quality, Careggi Hospital of Florence and 1.0 ml was added with 1.0 ml of ammonium acetate buffer ( ph 4.5), 10 ml of b-glucuronidase and 20 ml of 500 ng/ml solution of IS, and then incubated for 18 h at 508C. After cooling at room temperature, 0.5 ml of NaOH 2 N was added. A liquid liquid extraction (LLE) was performed by adding 5.0 ml of a 9 : 1 (v : v) dichloromethane and isopropanol mixture. After centrifugation at 4,000 rpm for 5 min, the lower Table II MRM transitions for each benzodiazepine, quantifier transition in bold Benzodiazepine Parent ion (m/z) Product ion (m/z) 3-Hydroxyflunitrazepam , Aminoclonazepam , Aminoflunitrazepam , Aminonitrazepam , 208 a-hydroxyalprazolam , 297 a-hydroxymidazolam , 203 Alprazolam , 281 Bromazepam , 209 Brotizolam , 214 Clonazepam , 270 Chlordiazepoxide , 283 Delorazepam , 165 Diazepam , 193 Flunitrazepam , 268 Halazepam (IS) , 241 Ketazolam , 193 Lorazepam , 303 Lormetazepam , 317 Midazolam , 291 Nitrazepam , 207 Nordiazepam , 165 Oxazepam , 269 Pinazepam , 269 Prazepam , 271 Temazepam , 283 Triazolam , 315 organic layer was transferred into a tube, dried under a gentle stream of nitrogen at 408C and the residue dissolved in 100 ml of LC MS CHROMASOLV w methanol. An aliquot of 3 ml was injected into the LC MS-MS system. LC MS-MS Analysis was conducted using an HPLC Agilent 1290 Infinity system (Agilent Technologies, Palo Alto, CA, USA) interfaced with an Agilent 6460 Triple Quad LC/MS (Agilent Technologies), coupled at an electrospray ion source (ESI). The column used was a Zorbax SB-C18 Rapid Resolution HT ( mm, 1.8 mm, Agilent Technologies), heated at 308C. The mobile phase initially consisted of 5 mm aqueous acid formic (A) and acetonitrile (B) 90 : 10. Gradient elution was carried out by increasing within 3 min, then running isocratically for 1 min; post-time was 1.5 min. The flow rate was 0.4 ml/min and the injection volume was 3 ml. The ESI configuration was: gas temperature 3258C; gas flow rate 10 L/min; nebulizer 20 psi; capillary 4,000 V. Multiple reaction monitoring (MRM) analysis was performed in the positive mode, with a dwell of 70 ms. Two transitions for each substance were chosen for identification; the most intense was used for quantification purposes (Table II). Data acquisition and elaboration were performed by the Agilent MassHunter Workstation Software. Calibration levels were prepared by spiking blank urine with the benzodiazepine standards at 0.5, 1.0, 10.0, 50.0, 100.0, and 1,000.0 ng/ml. Quantitative results were obtained by evaluating five replicates of each sample. Results Immunoassays Each IA was evaluated by the calculation of different statistical parameters: true positive (TP), true negative (TN), FP, FN (according to the concentration of the benzodiazepines detected in the 660 Bertol et al.

3 LC MS-MS controls), SENS, SPEC, positive predictive value (PPV), negative predictive value (NPV) and accuracy (ACC). The equations used in these calculations are presented in Table III. Table IV presents the qualitative results for each IA, using LC MS-MS analysis as a reference point for determining positive and negative samples, considering the manufacturers stated cutoff used for the IAs. From a quantitative point of view, the study was affected by the low number of responses provided by the participating laboratories, as shown in Table V. Concentration values correspond to the combined concentration of each substance, which is not a straightforward sum of each relevant molecule amount, instead it is necessary to take the individual cross-reactivity (and therefore the cutoff) for each analyte into account. Thus, concrete knowledge of the IA used appears crucial to avoid a misleading interpretation of the results, together with a complete case evaluation, especially in forensic toxicology. The concentration Table III Equations used for the device performance evaluations Parameter SENS SPEC PPV NPV ACC Calculation TP=ðTP þ FNÞ TN=ðTN þ FPÞ TP=ðTP þ FPÞ TN=ðTN þ FNÞ ðtp þ TNÞ=ðTP þ TN þ FN þ FPÞ Table IV Qualitative results of the 12 samples analyzed using LC MS-MS analysis as reference for determining positive and negative cases IA TP FP TN FN SENS SPEC ACC PPV NPV Cutoff (ng/ml) CEDIA EMIT-S EMIT-I.L EMIT-D EMIT-B EMIT-A FPIA KIMS IMC Not all participating laboratories provided a qualitative result. Table V Quantitative results of the various IA expressed in ng/ml, with percent coefficient of variation (%CV) in brackets range was ng/ml; it is possible to note that FPIA and KIMS tests yield lower values in the range, while CEDIA and EMITs values tend to be higher. In addition, KIMS and FPIA show a better percent coefficient of variation. Since the 12 urine specimens were prepared for a PT, they presented an imbalance between the positive and negative cases in favor of the positive ones; therefore, our evaluations concern this kind of population examined. LC MS-MS In our laboratory, validated LC MS-MS analyses of benzodiazepines and their metabolites are routinely used to confirm screening test results. The LLE employed in this study yielded recoveries above 90% for all benzodiazepines. Correlation coefficient values for the concentration range of 0.5 1,000.0 ng/ml were Limits of detection (LODs) and quantification (LOQs) were measured by evaluating the signal/noise ratio (S/N) and were fixed at the concentration with a S/N ratio of.3 and.10, respectively. An LOQ value ranged from 0.01 ng/ml (a-hydroxyalprazolam) to 0.05 (oxazepam). In this study, LOQ was used as LOD. Urine is not the only biological specimen that we analyze. We also detect benzodiazepines and their metabolites in blood, hair and organs for different forensic aims (i.e., DFSA, DUID, and death). Eleven different benzodiazepine structures were found in all urine specimens: diazepam, nordiazepam, oxazepam, temazepam, delorazepam, lorazepam, midazolam, a-hydroxymidazolam, alprazolam, a-hydroxyalprazolam and lormetazepam (Table VI). This result was possible because, as mentioned above, the specimens were formed by mixing urines in different proportions from patients under addiction treatment. Thus, the interpretation of the results regarding the determination of parent drugs and their metabolites is complicated. In fact, nordiazepam, oxazepam and temazepam belong to the diazepam metabolic pathway, but they are drug substances in their own right, as well as lorazepam, which is a metabolite of delorazepam and lormetazepam (Figure 1). Furthermore, the enzymatic hydrolysis procedures, used to de-conjugate benzodiazepine glucuronides during urine drug testing, are known to induce reductive transformation of oxazepam (into nordiazepam), temazepam (into diazepam) and lorazepam (into delorazepam) (17, 18). Thus, with Sample CEDIA EMITs FPIA KIMS IMC LC MS-MS S I.L. D B A 1 a (19.5) a (27.2) a (3.5) (5.8) 44.1 (18.1) 2 a 5,117.5 (38.9) a 4,818.9 (44.0) a a (7.2) 1,640.0 (25.7) (15.1) 3 a a a Neg 4 a (24.9) (31.4) (26.9) a a (9.7) (11.9) a (9.7) 5 a (19.0) (28.9) (28.2) a a a (10.2) (5.4) 6 a 1,168.6 (27.5) 1,337.2 (38.5) 1,038.2 (18.1) a a a (6.6) (13.2) 7 a (27.2) (35.5) (27.1) a (8.8) (7.3) 8 a (24.8) 1,103.4 (28.4) (22.8) a (7.7) (14.8) (18.3) (11.7) (16.4) (9.6) a (6.7) 74.9 (17.5) 10 a 2,132.5 (40.0) a 2,002.9 (25.9) a a (9.2) (4.3) 11 a 1,657.4 (29.8) 2,039.4 (5.3) 1,512.2 (25.5) a a (8.7) (6.9) 12 2,074.1 (47.6) (28.0) (24.6) (39.7) a a (7.7) (3.6) Not all participating laboratories provided a quantitative result. a Insufficient number of quantitative replies for a reliable statistical data. IA Tests Versus LC MS-MS for Benzodiazepines Detection 661

4 Table VI LC MS-MS analysis results Benzodiazepine Sample Diazepam Nordiazepam Oxazepam Temazepam Delorazepam Lorazepam Midazolam a-oh-midazolam Alprazolam ,LOQ a,loq a-oh-alprazolam ,LOQ,LOQ Lormetazepam Concentrations are expressed in ng/ml. Sample 3 was negative. a LOQ: limit of quantification; values: 0.02 ng/ml for alprazolam and 0.01 ng/ml for a-hydroxyalprazolam. Figure 1. Structures and transformations of the detected benzodiazepines (m, metabolic pathway; r, enzymatic reduction). the exception of alprazolam, midazolam and lormetazepam, the other molecules may be both drugs and biotransformation products (metabolism and reductive activity of b-glucuronidase). The total concentration range was ng/ml and the most present benzodiazepine was lorazepam with an average of ng/ml. Alprazolam and its metabolite a-hydroxyalprazolam were under LOQs in Samples 9 and 10. Low concentrations of diazepam ( ng/ml), midazolam ( ng/ml) and delorazepam ( ng/ml) can be primarily explained by their extensive metabolism. Discussion The objective of the present study was to examine the performance of the main IAs in comparison with reference analysis by LC MS-MS. Our results show that there is a very high SENS (72 100%), but a low SPEC (14 42%, EMIT-A. excluded). This leads to a high FP rate, which in turn requires a high number of confirmation analyses. On the other hand, the possibility of FNs is a critical aspect that can generate errors of great severity: an unconfirmed FN case remains as negative data, leading to mistaken evaluations and potentially serious consequences, 662 Bertol et al.

5 Table VII Percentage mean relative errors (%MRE) for IA tests, using LC MS-MS concentrations values as actuals Sample CEDIA EMIT FPIA KIMS Syva I.L. Dimension 1 1,773 1, Average Median particularly in forensic cases, such as DFSA, DUID or drug-related deaths. For these reasons, drawing conclusions based on screening alone is improper and should be strongly discouraged. When the circumstances of a case suggest benzodiazepine use, negative results should also be investigated by instrumental techniques such as LC MS-MS to reduce the incidence of false negativity. FPIA seems to be mainly affected by this problem, presenting the lowest SENS (72%) and NPV (7%), with only one TN in all the negative test results. FPIA also presents a low ACC (65%). IMC tests present the lowest ACC (57%) and PPV (51%) due to a high FP rate. Remarkable values of SENS, ACC, PPV and NPV (above 80%) were shown by CEDIA, EMITs and KIMS methods. Observing the quantitative results obtained by the IAs, a general overestimation is noted compared with LC MS-MS; frequently, the percentage of mean relative errors (%MRE) is.100% (Table VII). Single cases excluded, KIMS and FPIA gave closer results to LC MS-MS values than the other IAs. Since the IA responses depend on the affinity of the analyte to the antibody, it stands to reason that it is necessary to consider the cross-reactivity when evaluating semi-quantitative datum, bearing in mind that the result corresponds to a combined concentration. These considerations are very important in cases such as ours, where various structures are present in a single specimen, each with its own contribution to the combined concentration. It must be said that the molecules analyzed by the LC MS-MS are different from those analyzed by the IAs, due to enzymatic hydrolysis effects. In fact, b-glucuronidase hydrolysis releases molecules unavailable for the antibodies already bound to glucuronic acid (oxazepam, temazepam, lorazepam, lormetazepam, a-hydroxyalprazolam and a-hydroxymidazolam), influencing in this way the results of the analysis. With the present study, we have succeeded in explaining the great differences that result from the different types of tests, and especially in comparison with the confirmation analyses. Conclusions Currently, IAs represent the main screening test for benzodiazepines and other molecules in many analytical environments, providing information about both positivity or negativity and about semi-quantitative determination. However, evaluation of the outcomes must consider the possibility of incorrect results, especially when classes of drugs with large structural variability are analyzed (i.e., benzodiazepines, amphetamine designer drugs). In the present study, CEDIA, EMITs and KIMS tests provided quite good levels of SENS, PPV and ACC, while FPIA presented the least reliable results for ACC, SENS and NPV. It should be noted that the SPEC values were very low for all the methods (except for EMIT-A.), with high FP rates. In light of all these aspects, the role of the confirmatory analysis is essential and it must be performed also for negative cases when an FN is probable. This study has also confirmed that semi-quantitative data from IAs are only indicative and they cannot replace instrumental techniques. Furthermore, we wish to warn about the indiscriminate use of these tests without confirmation in forensic cases, i.e., cases involving the use of benzodiazepines in DUID, DFSA or other circumstances. References 1. Garretty, D.J., Wolff, K., Hay, A.W.M., Raistrick, D. (1997) Benzodiazepine misuse by drug addicts. Annals of Clinical Biochemistry, 34, Drummer, O.H., Randon, D.L. (1996) Sudden death and benzodiazepines. The American Journal of Forensic Medicine and Pathology, 17, Mari, F., Defraia, B., Rensi, R., Gualco, B., Vaiano, F., Bertol, E. (2013) Fatal poisoning due to co-ingestion of benzodiazepines and alcohol: a case report. Indian Journal of Forensic Medicine and Toxicology, 7, Gueye, P.N., Lofaso, F., Borron, S.W., Mellero, F., Vicaut, E., Harf, A. et al. (2002) Mechanism of respiratory insufficiency in pure or mixed drug-induced coma involving benzodiazepines. Clinical Toxicology, 40, Adams, R.H. (1986) An accident and emergency department s view of self-poisoning: a retrospective study from United Norwich Hospitals. Human Toxicology, 5, Kadjalainen, K., Lintonen, T., Impinen, A., Ma kel, P., Rahkonen, O., Lillsunde, P. et al. (2010) Mortality and causes of death among drugged drivers. Journal of Emidemiology and Community Health, 64, Schwartz, R.H., Milteer, R., LeBeau, M.A. (2000) Drug-facilitated sexual assault. Southern Medical Journal, 93, ElSohly, M.A., Gul, W., Murphy, T.P., Avula, B., Khan, I.A. (2007) LC-(TOF) MS analysis of benzodiazepines in urine from alleged victims of drug-facilitated sexual assault. Journal of Analytical Toxicology, 1, Dowd, S.M., Strong, M.J., Janicak, P.G., Negrusk, A. (2002) The behavioral and cognitive effects of two benzodiazepines associated with drug-facilitated sexual assault. Journal of Forensic Sciences, 47, Steward, S.H., Westra, H.A. (2002) Benzodiazepine side-effects: from the bench to the clinic. Current Pharmaceutical Design, 8, Mandrioli, R., Mercolini, L., Raggi, M.A. (2008) Benzodiazepine metabolism: an analytical perspective. Current Drug Metabolism, 9, Spiehler, V. (2000) Hair analysis by immunological methods from the beginning to Forensic Science International, 107, Mearrick, P.T., Chahl, J.S. (1985) Screening for benzodiazepines in urine after hydrolysis of glucuronide conjugates. Clinical Chemistry, 31, Meatherall, R. (1994) Benzodiazepine screening using EMIT II and TDx: urine hydrolysis pretreatment required. Journal of Analytical Toxicology, 18, IA Tests Versus LC MS-MS for Benzodiazepines Detection 663

6 15. Bertol, E., Vaiano, F., Furlanetto, S., Mari, F. Cross-reactivities and structure-reactivity relationships of six benzodiazepines to EMIT w immunoassay. Journal of Pharmaceutical and Biomedical Analysis, De Rienz, R.T., Holler, J.M., Manos, M.E., Jemionek, J., Past, M.R. (2008) Evaluation of four immunoassay screening kits for the detection of benzodiazepines in urine. Journal of Analytical Toxicology, 32, Fu, S., Lewis, J., Wang, H., Keegan, J., Dawson, M. (2010) A novel reductive transformation of oxazepam to nordiazepam observed during enzymatic hydrolysis. Journal of Analytical Toxicology, 34, Fu, S., Molnar, A., Bowron, P., Lewis, J., Wang, H. (2011) Reduction of temazepam to diazepam and lorazepam to delorazepam during enzymatic hydrolysis. Analytical and Bioanalytical Chemistry, 400, Bertol et al.