Rapid and Sensitive Screening of Benzodiazepines in Serum Using Liquid Chromatography-APCI-Linear Ion Trap System

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1 T2007 Seattle, Washington Rapid and Sensitive Screening of Benzodiazepines in Serum Using Liquid hromatography-api-linear Ion Trap System Viktor Vorisek *, Vilma Habrdova, Pavel Zivny and Vladimir Palicka Institute of linical Biochemistry and Diagnostics, Department of linical and Forensic Toxicology, Medical Faculty of harles University and University Hospital Hradec Kralove, zech Republic Introduction The benzodiazepines are primarily administered for their sedative-hypnotic effects. They are used as anxiolytics, anticonvulsants and are administered preoperatively for their anterograde amnesia effects ( midazolam). However, hypotension, hypothermia, respiratory depression and death are associated with acute benzodiazepine toxicity. For the analysis of benzodiazepines in biological samples, a variety of spectroscopic, chromatographic and immunological methods are available. G/MS is the method of choice, but unfortunately not for all. Some of them show thermal instability.therefore, rapid and reliable screening of these substances is useful, especially for emergency cases. Aim We have developed the rapid screening and the appropriate quantitative method for fast identification of seven most frequent benzodiazepines in East zech territory as toxic agents ( alprazolam, flunitrazepam, diazepam, midazolam, oxazepam, bromazepam, clonazepam). Method Serum samples were extracted by liquid-liquid extraction using commercial Toxi-Tubes B from Toxi-Lab system or by SPE (mix phase MP1, Varian orp). A)for rapid screening: The HPL ( Rheos 2200 HPG high pressure gradient system, (Flux Instruments, AG) separation for this fast screening method was performed with 0.5% acetic acid/acetonitril gradient ( 60% H 3 OOH/ 40%H 3 to 80 % H 3 OOH/ 20%H 3 during 5 minutes) on Discovery 18, 20 x 4.6 mm, 5 µm ( Supelco). The total time of the analysis was 7 minutes. B)for quantitative purpose: The HPL ( Rheos 2200 HPG high pressure gradient system, Flux Instruments AG) separation for this fast screening method was performed with 0.5% acetic

2 acid/acetonitril gradient ( 80% H 3 OOH/ 20%H 3 to 5 % H 3 OOH/ 95%H 3 during 8 minutes) on Discovery 18, 20 x 4.6 mm, 5 µm ( Supelco). The total time of the analysis was 16 minutes. The mass spectrometer- linear ion trap LTQ XL ( ThermoElectron orp.) was operated in positive API or ESI mode. API The corona discharge voltage was set at 2.5 kv. The capillary temperature was 275 0, sheath gas flow was 40 arbitrary units, and auxiliary gas was set at 5 units. ESI The spray voltage was set at 5 kv. The capillary temperature was 320 0, sheath gas flow was 55 units, auxiliary gas flow 5 units. Full scan MS and MS n ( 30% normalized collision energy) spectra were acquired. Results and Discussion: Using a setting of 30% collision energy was convenient for all identified benzodiazepines: precursor ion for alprazolam 309 (M+H) + key product ions 281, 274, clonazepam , 288;bromazepam , 261, 236,181; diazepam , 228, 222, 182; flunitrazepam , 268; oxazepam ,259, midazolam , 276, 270, 264, 244 ( See Mechanisms of fragmentation part ) The achieved limits of detection were satisfactory in a range of 0.05 (for midazolam in spiked serum samples) 1 ng/ml (for the others) with S/ > 10. LL extraction recovery resulted in a range % for 0.5, 1, 5, 10 and 100 ng/ml spiked samples, SPE recovery was better than 85% for the same range of spiked serum samples ( See Table 1 and Table 2 ). Table 1 DRUG MEA LOD LOQ UMBER OF ASES ALPRAZOLAM 48,90 1,00 15,00 15,00 FLUITRAZEPAM 37,60 1,00 9,80 8,00 DIAZEPAM 658,00 1,00 10,00 32,00 MIDAZOLAM 292,50 0,05 1,00 5,00 OXAZEPAM 232,50 1,00 10,00 32,00 BROMAZEPAM 126,00 1,00 5,00 7,00 KLOAZEPAM 100,80 1,00 11,50 4,00 UITS G/ML

3 Table 2 DRUG spiked smpl o.of smpl SPE R LLE R ALPRAZOLAM 0,5;1;5; 3x5 65,00 60,00 FLUITRAZEPAM 0,5;1;5;100 4x5 60,00 72,00 DIAZEPAM 0,5;5;100 3x5 96,00 83,00 MIDAZOLAM 0,5;1;5;10;100 5x5 92,00 85,00 OXAZEPAM 0,5;1;5;10;100 5x5 95,00 81,00 BROMAZEPAM 0,5;1;5;10;100 5x5 91,00 76,00 KLOAZEPAM 0,5;1;5;10;100 5x5 88,00 83,00 UITS G/ML MEA % % smpl samples R - recovery of extraction process One of the specific key fragments of alprazolam is featured on Figure 1. Figure 2 presents the whole API-MS 2 spectrum of alprazolam from real sample. Figure 3 demonstrates the advantage of the use of linear ion trap with the possibility of MS n experiments for the evidence of some typical ions ( Fig 4: m/z 228, diazepam). However, these n - experiments in principle require a greater amount of an analyte, i.e. more originally neutral molecules in the space of linear ion trap in comparison with MS 2 dissociation. l H H H H H H Figure 1: m/z 281

4 Figure 2 :API-MS 2 spectrum of alprazolam from real sample ( male, age: 30y., intoxication,serum level: 61.2 ng/ml ) 8_30A_ # RT: AV: 217 L: 3.62 T: ITMS + c API corona Full ms @cid30.00 [ ] Relative Abundance m/z

5 Figure 3 MS 4 diazepam ( spiked serum sample, 250 ng/ml ) BZDZUKAZKA24 # RT: AV: 13 L: 4.12E1 T: ITMS + c API corona Full ms @cid @cid @cid17.00 [ ] Relative Abundance m/z

6 Figure 4 l H H H 3 H H m/z 228 onclusion: We have good and reliable screening method for timely identification of problematic benzodiazepines in serum matrix. Additional detection methods including precursor ion and neutral loss scanning with high sensitivity in fast cycle time are the outstanding merits of linear ion trap system. In addition, high quality MS 2 and MS 3 or, if need be, MS 4 spectra ( see diazepam ) and good mass isotopic resolution in molecular peak in full MS mode is useful tool for the correct identification of these analytes in real samples. ommercial available a programme package for the management, evaluation and interpretation of mass spectra and chromatograms ( Mass Frontier TM ) is the powerful implement of our instrument s software equipment. It was used successfully as the support for the prediction of fragmentation mechanisms. Although Mass Frontier TM offers many sophisticated features, doesn t calculate some important formatting benzodiazepine fragments in the event of soft ionization procedures. So it is necessary to control of advanced features. Acknowledgement: We thank all of our lab staff for their unique collaboration and help. This work has been financially supported by the grant of zech Ministery of Health o. MZO References: 1. Hopfgartner G., Husser., Zell M., Rapid Screening and haracterization of Drug Metabolites Using a ew Q-LIT-MS, J Mass Spectrom, 38 (2003) iessen, W.M.A.: Liquid hromatography-mass Spectrometry, 3 rd Ed., R, Taylor and Francis Group, Boca Raton, London, ew York, 2006, 608 pp.