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Home , Adinazolam, Camazepam, Chlordiazepoxide, Climazolam, Clorazepate, Clotiazepam

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Order No. A91DX-0701526-UC1-4A00 | Printed in USA | © 2009 Siemens Healthcare Diagnostics Inc. Syva has been R1 R2 a leading developer N and manufacturer of AB R3 X N drugs-of-abuse tests R4 for more than 30 years. R2 C

Now part of Siemens Healthcare ® Diagnostics, Syva boasts a long and have as their basic chemical structure successful track record in drugs-of-abuse a ring fused to a seven-membered diazepine ring. testing, and leads the industry in the All important benzodiazepines contain a 5-aryl substituent ring (ring C) and a 1,4–diazepine ring. production of enzyme immunoassays. In addition to drugs-of-abuse assays, Syva Introduction has been a key player in the development When researchers synthesized the earliest and manufacture of therapeutic drug benzodiazepines in the 1960s, the medical monitoring assays. community was hoping for an effective Syva products are sold in more than anti- agent that would not produce tolerance, dependence, or withdrawal symptoms. 45 countries worldwide. Nor would the hoped-for possess recreational value. While the benzodiazepines were an improvement over the widely-used , the medical community is still waiting for an anti- anxiety agent free of addictive qualities. Librium1 () was the first , and the immensely successful Valium2 () followed just a few years later.

1 Librium is a registered trademark of Hoffman-LaRoche, Inc. 2 Valium is a registered trademark of Hoffman-LaRoche, Inc. Today pharmaceutical companies market more patient to normal functioning. Because subjects than 35 brands of benzodiazepines worldwide. rapidly develop a tolerance towards the mood- Among these structurally-similar compounds exists altering effects of benzodiazepines, these drugs a wide variance in selectivity, dosage, metabolic work best as short-term treatment. Over the half-life, treatment indication, and potency. long- term, dependence can, and does develop. Dependence on prescription benzodiazepines Benzodiazepines Possess Unique is well documented. Antianxiolytic Properties Benzodiazepines are also used as , The immense success of benzodiazepines is largely , , muscle relaxants, attributable to their unique antianxiolytic properties. anesthesia, and pre-anesthesia. They are used to They provide relief from anxiety, and their degree of treat , disorders, muscle spasms, is much less than that of the barbiturates. and withdrawal. One benzodiazepine, Because of their pharmacological action, they , appears to work as an anti-. are safer than both barbiturates and dicarbamates. Studies have revealed significant residual Unlike barbiturates, benzodiazepines do not effects after the administration of -doses cause general neuronal depression by themselves. of benzodiazepines. In one study evaluating They are, therefore, almost incapable of causing driving performance, residual effects following respiratory or circulatory collapse. Deaths involving two nightly 30-mg doses were at least as great benzodiazepine use are almost always the result as the acute effects of 100 mg/dL blood of interactions with one or more drugs. alcohol concentration—the legal intoxication Benzodiazepines are —hypnotic central level in most states. nervous system (CNS) . They work with the γ-aminobutyric acid (GABA) receptors of the CNS. Screening for Benzodiazepines GABA is a neurotransmitter stored in the nerve cells Syva Company has designed the EMIT® and released by the to calm the body. Benzodiazepine Assays to detect the presence of nearly 30 benzodiazepines and their metabolites. In state of anxiety, polypeptides stimulate the The EMIT® II and EMIT® d.a.u.™ Benzodiazepine nerve cells and suppress the release of GABA. Assays detect the compounds and metabolites Benzodiazepines interact with receptors to allow listed in Table 1. Also listed are the concentrations its release. Once GABA is released, the stimulation at which the compounds produce results by the polypeptides ceases, and the state of anxiety approximately equal to the cutoff calibrator ends. Benzodiazepines produce an additional (200 ng/mL ).3 calming effect with GABA. Syva Company has developed a more sensitive Effective, Relatively Safe, Often Abused antibody, and has changed the cut off for the Medical practitioners generally consider EMIT d.a.u. Benzodiazepine Assay from 300 benzodiazepines safe and effective in appropriate ng/mL to 200 ng/mL; equal to that of the EMIT II therapeutic circumstances. Because of their relative Benzodiazepine Assay. These new assays detect medical safety, they have effectively displaced the the widely-used alprazolam, , and barbiturates as antianxiolytics. at concentrations 3 to 5 times lower than formerly possible. Benzodiazepines are most often used to treat acute anxiety rather than chronic depression. Physicians prescribe benzodiazepine treatment to restore the Table 1: Benzodiazepines and Concentrations that Produce a Result Approximately Equivalent to EMIT® Calibrator A Level 1 (200 ng/mL Oxazepam)

Benzodiazepine Trade EMIT II EMIT d.a.u. Benzodiazepine Trade EMIT II EMIT d.a.u. Compounds and Name Concentration Concentration Compounds and Name Concentration Concentration Metabolics (ng/mL) (ng/mL) Metabolics (ng/mL) (ng/mL) 200 300 200 300 200 300 200 300 Alprazolam Xanax 100 150 50 100 * Anseren, 210 250 50 100 α-Hydroxyalprazolam* 120 160 60 100 Ansieten, Anxon, Lectopam, 340 800 400 550 Contamex, Lexotanil Larpaz, Chlordiazepoxide Librium 800 1890 500 1400 Loftran, ** Frisium 180 290 100 270 Marcen, Clonopin, 250 1000 500 640 Parcil, Rivotril Sedotime, Solatran, Tranxene † † † † Unakalm ** Clozan, 400 580 100 250 Ativan 750 1400 1000 1300 Distensan, Rize, Rizen, Lormetazapam* Ergocalm, 230 350 200 280 Ticnor, Lembrol, Trecalmo, Loramet, Veratran Minias, Noctamid, ** Demoxepam 500 1050 900 1000 Pronoctan N-Desalkyl-* 230 320 100 180 Nobrium 130 180 100 140 Diazepam Valium 110 150 40 80 Hypnovel, 100 200 120 180 ** Rohypnol 220 450 100 230 Versed Flurazepam Dalmane 110 230 100 130 Mogodan 200 350 200 260 1-N-Hydroxyethyl- 130 160 60 100 Norchlor-diazepoxide** 670 1850 1800 1800 flurazepam* Nordiazepam** 100 150 60 100 Paxipam 140 210 80 160 Oxazepam Serax 200 300 200 300 Verstran 110 170 80 100 3 These concentrations apply only to the EMIT d.a.u. (Centrax) Benzodiazepine Assay. Temazepam Restoril 190 250 70 190 * Benzodiazepine metabolite. * Musaril, 150 250 100 100 ** Not sold in U.S. Myolastan † Clorazepate degrades rapidly in stomach to nordiazepam Triazolam Halcion 120 250 70 110 which hydroxylates to oxazepam. α-Hydroxy-triazolam* 120 160 100 140

* Not sold in U.S. ** Benzodiazepine metabolite Short-Acting Compounds Are More Table 2: Benzodiazepines by Structure Difficult to Detect Group I Group II For testing purposes, we can divide the benzodiazepines into two general groups. The Three-Ring Structures: Four-Ring Structures: longer-acting, higher dosages shorter-acting, lower dosages first grouping consists of longer-acting compounds, 1,4-Benzodiazepines: 1,5-Benzodiazepines: characterized by a three-ring chemical structure. (diazolobenzodiazepines) clobazam These are the 1,4-benzodiazepines, or diazolos. bromazepam Imidazo-1,4-benzodiazepines: The diazolos metabolize slowly, breaking down chlordiazepoxide midazolam clonazepam to biologically active compounds (compounds clorazepate : that have CNS effects). The phase in which active diazepam alprazolam metabolites form is quite slow; the half time is flunitrazepam triazolam 40 to 50 hours. This long half time allows for the flurazepam : halazepam clotiazepam build-up of active metabolites that are responsible ketazolam for the long-acting effects. Finally, the metabolites lorazepam attach to glucuronides and are eliminated. medazepam nitrazepam The second grouping consists of shorter-acting, oxazepam low-dosage benzodiazepines characterized by a four-ring chemical structure. The prazepam 1,5-benzodiazepines, triazolobenzodiazepines, imidazo-1,4-benzodiazepines, and thienodiazepines temazepam are included in this grouping. The short-acting benzodiazepines have more rapid antibody does not recognize a metabolite once and direct metabolic routes. These benzodiazepines it has conjugated with a glucuronide. Only a small are more potent and require much lower doses. fraction of benzodiazepines pass through the They metabolize and conjugate with glucuronides urinary tract unchanged. so rapidly that active metabolite build-up does Since most benzodiazepines are excreted in the form not occur. Since active metabolites are not in the of unrecognizable glucuronides, the immunoassay body for an extended period, action is shorter must be able to recognize the very small amounts of and less complicated by residual effects. Detecting unchanged parent drug and metabolites. these compounds in urine is more difficult, but very important. Short-acting compounds alprazolam (Xanax), and triazolam (Halcion), are widely used and their use is growing steadily. The EMIT reagent antibody is highly specific and recognizes a drug or metabolite by its particular shape. The kidneys excrete most benzodiazepines as conjugated metabolites. The EMIT reagent Confirming Positive Benzodiazepine Test Results The following methods all are capable, in Many factors combine to present a challenge to varying degrees, of confirming the presence the clinician confirming benzodiazepine screening of benzodiazepines, identifying specific results. Quantifying and identifying benzodiazepine benzodiazepines, and quantifying benzodiazepines compounds is a complex, analytical task. in the sample: Thirty-five compounds are prescribed all over the • Thin Layer Chromatography (TLC) world and billions of pills are produced annually. • Gas Chromatography (GC) The low-dosage levels of the newer benzodiazepines • Gas Chromatography/Mass Spectrometry (GC/MS) leave smaller concentrations in the urine. Some of the benzodiazepines are thermally degradable • High Performance Liquid Chromatography (HPLC) and damaged by procedures that use heat. Also, • Liquid Chromatography/Mass Spectrometry (LC/MS) the major urinary metabolites are often very polar and difficult to detect by gas chromatography. Chromatography and Mass Spectrometry Further, many benzodiazepines share metabolic GC, HPLC, and other chromatographic methods pathways and produce the same metabolites. base identification on retention times relative For example, oxazepam, in addition to being a to known standards. parent drug, is a metabolite of camazepam, Chromatography procedures usually employ chlordiazepoxide, clorazepate, diazepam, hydrolysis to separate the benzodiazepine halazepam, ketazolam, medazepam, prazepam, compounds and metabolites from the and temazepam. glucuronide. Two kinds of hydrolysis are Fortunately, most screening laboratories available—acid and enzyme. can seek the services of specialty laboratories Acid hydrolysis uses hydrochloric acid (HCl) for sophisticated confirmation procedures. to quickly break three-ring benzodiazepine structures Also, the literature covering benzodiazepine down to two-ring structures called benzophenones. confirmation is extensive. Acid hydrolysis will not convert alprazolam, midazolam, and triazolam, to benzophenones. Laboratories’ Needs Determine These compounds have four-ring structures, and Confirmation Method they tend to be more stable than the higher-dosage The level of detail the laboratory needs to three-ring diazolo compounds. derive from confirmation varies. In the criminal justice, employment, or military arenas, a laboratory One advantage of acid hydrolysis is its rapidity, may only need corroborating test evidence that a which makes it more suitable for emergencies. benzodiazepine or benzodiazepines are in the The identification of two or more benzophenones sample. However, circumstances may call for can often provide evidence of a particular the forensic toxicologist to find the exact identity benzodiazepine or its metabolites, but not always. and quantity of the compound or compounds Sometimes identifying benzophenones merely the sample contains. reveals which benzodiazepines cannot be present. Gas chromatography plus mass spectra analysis accurately quantifies and specifically identifies compounds according to molecular structure. GC/MS is usually considered the gold standard of References confirmation methods. However, when confirming 1. Gilman AG, Rall TW, Nies AS, Taylor P (eds): Goodman benzodiazepines, the same cautions that apply and Gilman’s The Pharmacological Basis of Therapeutics. to GC, apply to GC/MS. Extensive literature on GC/MS Elmsford NY, Pergamon Press, 1990. methods is available. 2. Baselt RC, Cravey RH: Disposition of Toxic Drugs and Chemicals in Man, ed 3. Chicago IL, Year book Medical High-performance liquid chromatography (HPLC) Publishers Inc, 1990. does not employ heat, and is an excellent method 3. Schuckit, MA: Drug and Alcohol Abuse. New York, Plenum for confirming the presence of benzodiazepines. Medical Book Company, 1989. Like GC, it does not unequivocally identify 4. Olsen RW, Venter JC (eds): Receptor Biochemistry and Methodology, Volume 5 Benzodiazepine/GABA Receptors and the parent compound, however, adding mass Chloride Channels Structural and Functional Properties. spectrometry should help with this. Information New York, Alan R. Liss Inc, 1986. on successful HPLC and HPLC/MC confirmations 5. Woods JH, Katz JL, Winger G: Use and abuse of is also readily available. benzodiazepines, issues relevant to prescribing. JAMA 1988;23:3476–3479. The liquid chromatographic procedure used in 6. Woods JH, Katz JL, Winger G: Abuse liability of LC/MS does not destroy heat degradable compounds, benzodiazepines. Pharmacological Reviews 1987; and mass spectrometry accurately identifies and Vol 39;No 4:290–319. quantifies the compounds by molecular structure. 7. Fraser AD, Bryan W, Isner AF: Urinary screening for LC/MS is a less well-known method that is gaining midazolam and its major metabolites with the Abbott increased acceptance. Since it is a relatively new ADx and TDx analyzers and the EMIT dau Benzodiazepine Assay with confirmation by GC/MS. JAT 1991;15:8–12. procedure, instrumentation is not yet widely 8. Sioufi A, Dubois JP: Chromatography of benzodiazepines. J available. Literature on the method is available, Chrom 1990;531:459–480. though sparse for benzodiazepines. Notes: