Rapid, Simultaneous Screen and Confirmation Analysis of Benzodiazepines in Biological Fluids by LC/MS/MS Using a C18 Core-shell Column Liming Peng, and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA PO86980511_L_1 Introduction Benzodiazepines, a class of psychoactive drugs known poor crossreactivity, resulting in low sensitivity for detecting for their broad range of therapeutic effects, such as low dosage benzodiazepines in biomatrices1. GC/MS is a sedative-hypnotic, anxiolytic, anesthetic, antidepressant, highly sensitive specific technique capable of accurately anticonvulsant, and anti-insomnia, are among the most identify of target analytes. However, derivatization is commonly prescribed drugs for clinical applications. required prior to analysis by GC/MS since GC is limited to However, as addictive drugs, benzodiazepines have compounds which are volatile at temperatures below 400 the potential for misuse, which can result in impaired °C. In comparison to GC/MS, LC/MS/MS 2,3,4,5 offers superior human performance or fatal cases of drug intoxication. sensitivity, selectivity, and robustness for simultaneously Benzodiazepines are also deliberately misused in sport detecting benzodiazepines and their metabolites in a competition. Consequently, they are classified as controlled complex mixture like biological fluids without any need substances that are included in doping control and routine for derivatization. Furthermore, due to its large dynamic employment screening. The development of adequate and range LC/MS/MS is well accepted as quantitative analytical rapid methods for their screening and confirmation analysis technique. therefore receives a great deal of attention in toxicological, In this work we present a rapid and simultaneous clinical, and forensic laboratories. screening, confirmation, and quantitative analysis of 38 Screening of benzodiazepines is commonly performed by benzodiazepines, non-benzodiazepine hypnotics, and their immunoassay. There are a wide variety of benzodiazepines metabolites in biological fluids - human plasma and urine which have common metabolic pathways and produce - by LC/MS/MS using a C18 core-shell column in a single multiple metabolites. This makes it difficult to develop a chromatographic run taking only 8 minutes. comprehensive and specific immunoassay suitable for all benzodiazepines. Furthermore, some benzodiazepines have Figure 1. Molecular Structures of Benzodiazepines and Metabolites N O Cl Cl Cl + N Br HO N N O N O Cl N N N N N N N N N N O O H O H Alprazolam Bromazepam Chlordiazepoxide Clobazam Clonazepam CH3 O O N Cl F + Cl N HO N N Cl O HO N Cl N N N N N O N N O N O O H Clorazepate Diazepam Demoxepam Estazolam Flunitrazepam F Cl Cl Cl N Cl Cl Cl Cl N N N N N O N HO HO O N F N N N F O H O F Flurazepam Halazepam Lorazepam Lormetazepam Medazepam F O Cl + N N N O N Cl N Cl N Cl N N N N O Cl N N N O H O Midazolam Nitrazepam Prazepam Triazolam Tetrazepam N O F N N F + N Cl N O O N O N N N N N N Cl N N HO N O N Cl O N N O O OH OH Zopiclone Zolpidem a-Hydroxyalprazolam 2-Hydroxyethylflurazepam 3-Hydroxyflunitrazepam OH O H H F + Cl F N N Cl N O H Cl H N N N N N N N N N O H O N O H O O H 7-Aminoflunitrazepam N-Desmethylflunitrazepam 7-Aminoclonazepam N-Ethylnordiazepam 4’-Hydroxynordiazepam Figure 2. The Metabolism of Diazepam CH3 O H O N N N-dealkylation Cl N Cl N Diazepam Nordiazepam 3-hydroxylation 3-hydroxylation CH3 O H O N N OH N-dealkylation OH Cl N Cl N Temazepam Oxazepam http://www.toxlab.co.uk/benzodia.htm Figure 3. Specific Pattern of Metabolism of Benzodiazepines Chlordiazepoxide Clorazepate Medazepam Normedazepam Demoxepam Ketazolam 3-Hydroxyprazepam Prazepam Nordiazepam Diazepam 4’-Hydroxydiazepam 3-Hydroxyhalazepam Halazepam Oxazepam 4’-Hydroxynordiazepam Temazepam Lormetazepam Clonazepam Triazolam Alprazolam Lorazepam 7-Aminoclonazepam a-Hydroxytrizolam a-Hydroxyalprazolam Experimental Conditions Benzodiazepines and metabolites were spiked into 200 µL of urine or human plasms at different concentration levels, along with 50 ng/mL internal standards (IS). To the plasma samples, 100 µL of 1M acetic acid was added and the sample was diluted to 1 mL with water. SPE Procedures HPLC Conditions Cartridge: Strata™-X Columns: As Noted Part No.: 8B-S100-TAK Dimensions: As Noted Condition: 1 mL Methanol Mobile Phases: A: 0.1 % Formic acid in Water Equilibrate: 1 mL Water B: 0.1 % Acid in Acetonitrile Load: Load above spiked sample Injection Volume: 2 mL onto SPE cartridge Gradient: 2.6 µm 1.7 µm Wash 1: 1 mL Water Time (min) % B Time (min) % B Wash 2: 1 mL to 15 % Methanol in Water Elute: 1 mL Methanol 0 20 0 20 Dry : Evaporate to dryness 5 70 5 70 Reconstitute : 200 µL Methanol/Water (1:1) 5.5 100 5.1 20 5.51 20 8.0 20 8.5 20 — — Sample: 1. Alprazolam 11. Flunitrazepam 21. Oxazepam 31. 2-Hydroxyethylflurazepam 2. Bromazepam 12. Flurazepam 22. Prazepam 32. 3-Hydroxyflunitrazepam 3. Chlordiazepoxide 13. Halazepam 23. Temazepam 33. 4’-Hydroxynordiazepam 4. Clobazam 14. Lorazepam 24. Triazolam 34. 7-Aminodesmethylflunitrazepam 5. Clonazepam 15. Lormetazepam 25. Tetrazepam 35. 7-Aminoclonazepam 6. Clorazepate 16. Medazepam 26. Zopiclone 36. 7-Aminoflunitrazepam 7. Diazepam 17. Midazolam 27. Zolpidem 37. N-Desmethylflunitrazepam 8. Demoxepam 18. Nitrazepam 28. a-Hydroxyalprazolam 38. N-Ethylnordiazepam 9. Desalkylflurazepam 19. Nordiazepam 29. a-Hydroxymidazolam 10. Estazolam 20. Norfludiazepam 30. a-Hydroxytriazolam Instrumentation HPLC System: Agilent 1200 SL Pump: G1312B (Binary Pump) Autosampler: G1337C HP-ALS-SL MS Detector: AB Sciex API4000™ LC/MS/MS Turbo V™ Source with ESI probe MS Detection Tem. CUR Gas 1 Gas 2 IS CAD 550 ºC 20 psi 55 psi 45 psi 5000 V 4.0 CUR: curtain gas; Gas 1: nebulizer gas; Gas 2: turbo gas; IS: IonSpray voltage; CAD: Collision Gas Scheduled MRM: MRM detection window: 20 s; Target scan time: 1 Figure 4. XIC of 57 Benzodiazepines, Metabolites and Internal Standards Columns: Kinetex® 2.6 µm XB-C18 Dimensions: 100 x 2.1 mm Part No.: 00D-4496-AN Flow Rate: 0.5 mL/min Temperature: 30 °C Backpressure: 382 bar Injection Concentration: 50 ng/mL 2.6e5 2.4e5 2.2e5 s p 2.0e5 c , y t i 1.8e5 s n e t n I 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 App ID 19763 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min Figure 5. XIC of a Mix of 57 Benzodiazepines, Metabolites and Internal Standards spiked into Human Plasma or Urine (1) Columns: Kinetex 2.6 µm XB-C18 Dimensions: 100 x 2.1 mm Part No.: 00D-4496-AN Flow Rate: 0.5 mL/min Temperature: 35 °C Backpressure: 307 bar Injection Concentration: 50 ng/mL 4.0e5 In human plasma matrix 3.5e5 3.0e5 2.5e5 2.0e5 Intensity, cps 1.5e5 1.0e5 5.0e4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 4.0e5 In urine matrix 3.5e5 3.0e5 2.5e5 2.0e5 Intensity, cps 1.5e5 1.0e5 5.0e4 App ID 19765 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Figure 6. XIC of a Mix of 57 Benzodiazepines, Metabolites and Internal Standards spiked into Human Plasma or Urine (2) Columns: Kinetex 1.7 µm XB-C18 Dimensions: 50 x 2.1 mm Part No.: 00B-4498-AN Flow Rate: 0.4 mL/min Temperature: 35 °C Backpressure: 253 bar Injection Concentration: 50 ng/mL 3.5e5 In human plasma matrix 3.0e5 2.5e5 2.0e5 1.5e5 Intensity, cps 1.0e5 5.0e4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min 3.5e5 In urine matrix 3.0e5 2.5e5 2.0e5 1.5e5 Intensity, cps 1.0e5 5.0e4 App ID 19766 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min Results and Discussion LC separation and MS/MS detection for screening In general, drug-protein binding in human plasma is not of identification general concern with all benzodiazepines - only with specific ones. The absolute recoveries of flurazepam, midazolam, Separations of benzodiazepines were carried out on a -hydroxymidazolam, and 7-aminoflunitrazepam from Kinetex 2.6 µm XB-C18 100 x 2.1 mm and a Kinetex 1.7 µm a human plasma were initially very low (<50 %, data not XB-C18 50 x 2.1 mm column in gradient elution mode, using presented). Significant improvement in recoveries of these 0.1 % Formic acid in Water and Acetonitrile as mobile phase. compounds was observed after adding 0.1 M Acetic acid to ESI in positive ion mode, with multiple reaction monitoring plasma samples before SPE with the purpose of disrupting (MRM) and scheduled MRMs were used for screening, analyte-protein binding. confirmation, and quantification of benzodiazepines and their metabolites. Quantification Instead of monitoring all MRM transitions during the entire Method linearity was studied in the concentration range acquisition period, scheduled MRMs monitor only appropriate 1-300 ng/mL (at 1, 5, 10, 50, 100, and 300 ng/mL) with MRM transitions within the expected chromatographic 50 ng/mL of specified deuterated benzodiazepines and elution window.