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Simultaneous LC-HRMS determination of 28 benzodiazepines and metabolites in hair

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Abstract

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the simultaneous identification and quantification of 28 benzodiazepines, including 6 metabolites, in 50 mg of hair has been validated. Positive ion electrospray ionization and HRMS determination in full-scan mode were realized on an Orbitrap mass spectrometer at a nominal resolving power of 60,000. In-source collisional experiments were conducted to obtain additional information for a more reliable identification of the investigated drugs. HRMS in full-scan mode allowed the exact determination of molecular masses of all analytes eluting in the HPLC run, so that both the immediate and retrospective screening of results for drugs and their metabolites were available. Sample preparation consisted of an overnight incubation in phosphate buffer pH 8.4 and a subsequent liquid/liquid extraction with methylene chloride/diethyl ether (90:10). Gradient elution was performed on a Luna C18 analytical column and four deuterated analogues were used as internal standards (IS). Validation was performed using both spiked hair samples and hair samples from subjects treated with benzodiazepines. Selectivity was evaluated by analysis of 20 certified blank hair samples. Extraction efficiency and matrix effects were evaluated by analysis of true positive samples. The lowest limits of quantification (LLOQs) ranged from 1 to 10 pg/mg. Linearity was investigated in the range from LLOQ to 1,000 pg/mg, for each compound (R 2 0.998–0.999). Mean relative errors, calculated at three concentration levels, ranged from 1 to 20% (absolute value). Precision, at concentrations higher than the LLOQs, was always less than 15% expressed as percentage relative standard deviation. After validation, the procedure was applied to real samples collected for clinical and forensic toxicology purposes from subjects who were assumed to have taken benzodiazepines.

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References

  1. Drummer OH, Odell M (2001) Benzodiazepines and related drugs. In: The forensic pharmacology of drugs of abuse. Arnold, London, pp 103–175

  2. Villain M, Chèze M, Dumestre V, Ludes B, Kintz P (2004) Hair to document drug-facilitated crimes: four cases involving bromazepam. J Anal Toxicol 28:516–9

    CAS  Google Scholar 

  3. Goulle JP, Anger JP (2004) Drug-facilitated robbery or sexual assault: problems associated with amnesia. Ther Drug Monit 26:206–210

    Article  CAS  Google Scholar 

  4. Madea B, Musshoff F (2009) Knock-out drugs: their prevalence, modes of action, and means of detection. Dtsch Arztebl Int 106:341–7

    Google Scholar 

  5. Ghysel MH, Pépin G, Kintz P (1998) Drug submission. Toxicorama 10:126–127

    Google Scholar 

  6. Kintz P (2002) Soumission chimique: quels produits, quels prélèvements, quelles analyses? J Med Leg Droit Med 45:158–160

    Google Scholar 

  7. Cirimele V, Kintz P, Ludes B (1997) Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry. J Chromatogr B Biomed Sci Appl 700:119–129

    Article  CAS  Google Scholar 

  8. Negruz A, Moore C, Deitermann D, Lewis D, Kaleciak K, Kronstrand R, Feeley B, Niedbala RS (1999) Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair. J Anal Toxicol 23:429–435

    Google Scholar 

  9. Cordero R, Paterson S (2007) Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC-MS. J Chromatogr B Analyt Technol Biomed Life Sci 850:423–431

    Article  CAS  Google Scholar 

  10. Kronstrand R, Nyström I, Josefsson M, Hodgins S (2002) Segmental ion spray LC-MS-MS analysis of benzodiazepines in hair of psychiatric patients. J Anal Toxicol 26:479–484

    CAS  Google Scholar 

  11. Chèze M, Villain M, Pépin G (2004) Determination of bromazepam, clonazepam and metabolites after a single intake in urine and hair by LC-MS/MS. Application to forensic cases of drug facilitated crimes. Forensic Sci Int 145:123–130

    Article  Google Scholar 

  12. Chèze M, Duffort G, Deveaux M, Pépin G (2005) Hair analysis by liquid chromatography-tandem mass spectrometry in toxicological investigation of drug-facilitated crimes: report of 128 cases over the period June 2003–May 2004 in metropolitan Paris. Forensic Sci Int 153:3–10

    Article  Google Scholar 

  13. Laloup M, Del Mar Ramirez Fernandez M, De Boeck G, Wood M, Maes V, Samyn N (2005) Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of 26 benzodiazepines and metabolites, zolpidem and zopiclone, in blood, urine and hair. J Anal Toxicol 29:616–626

    CAS  Google Scholar 

  14. Villain M, Concheiro M, Cirimele V, Kintz P (2005) Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry. J Chromatogr B 825:72–78

    Article  CAS  Google Scholar 

  15. Ariffin MM, Miller EI, Cormack PAG, Anderson RA (2007) Molecularly imprinted solid-phase extraction of diazepam and its metabolites from hair samples. Anal Chem 79:256–262

    Article  CAS  Google Scholar 

  16. Irving RC, Dickson SJ (2007) The detection of sedatives in hair and nail samples using tandem LC-MS-MS. Forensic Sci Int 166:58–67

    Article  CAS  Google Scholar 

  17. Anderson RA, Ariffin MM, Cormack PAG, Miller EI (2008) Comparison of molecularly imprinted solid-phase extraction (MISPE) with classical solid-phase extraction (SPE) for the detection of benzodiazepines in post-mortem hair samples. Forensic Sci Int 174:40–46

    Article  CAS  Google Scholar 

  18. Xiang P, Sun Q, Shen B, Chen P, Liu W, Shen M (2010) Segmental hair analysis using liquid chromatography-tandem mass spectrometry after a single dose of benzodiazepines. Forensic Sci Int (in press)

  19. Toyo’oka T, Kanbori M, Kumaki Y, Nakahara Y (2001) Determination of triazolam involving its hydroxy metabolites in hair shaft and hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis. Anal Biochem 295:172–179

    Article  Google Scholar 

  20. Concheiro M, Villain M, Bouchet S, Ludes B, Lopez-Rivadulla M, Kintz P (2005) Windows of detection of tetrazepam in urine, oral fluid, beard, and hair, with a special focus on drug-facilitated crimes. Ther Drug Monit 27:565–570

    Article  CAS  Google Scholar 

  21. Kintz P, Villain M, Chèze M, Pépin G (2005) Identification of alprazolam in hair in two cases of drug-facilitated incidents. Forensic Sci Int 153:222–226

    Article  CAS  Google Scholar 

  22. Hegstad S, Khiabani HZ, Kristoffersen L, Kunoe N, Lobmaier PP, Christophersen AS (2008) Drug screening of hair by liquid chromatography-tandem mass spectrometry. J Anal Toxicol 32:364–372

    CAS  Google Scholar 

  23. Miller EL, Wylie FM, Oliver JS (2008) Simultaneous detection and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolite, and opiates in hair by LC-ESI-MS-MS using a single extraction method. J Anal Toxicol 32:457–469

    CAS  Google Scholar 

  24. Pelander A, Ristimaa J, Rasanen I, Vuori E, Ojanperä I (2008) Screening for basic drugs in hair of drug addicts by liquid chromatography/time-of-flight mass spectrometry. Ther Drug Monit 30:717–24

    Article  CAS  Google Scholar 

  25. Nielsen MK, Johansen SS, Dalsgaard PW, Linnet K (2010) Simultaneous screening and quantification of 52 common pharmaceuticals and drugs of abuse in hair using UPLC-TOF-MS. Forensic Sci Int 196:85–92

    Article  CAS  Google Scholar 

  26. EURACHEM Working Group (1998) The fitness for purpose of analytical methods: a laboratory guide to method validation and related topics. LGC, Teddington

  27. Peters FT, Maurer HH (2002) Bioanalytical method validation and its implications for forensic and clinical toxicology: a review. Accred Qual Assur 7:441–449

    Article  CAS  Google Scholar 

  28. Shah VP, Midha KK, Findlay JWA, Hill HM, Hulse JD, McGilveray IJ, McKay G, Millar KJ, Patnaik RN, Powell ML, Tonelli A, Viswanathan CT, Yacobi A (2000) Bioanalytical method validation–a revisit with a decade of progress. Pharm Res 17:1551–1557

    Article  CAS  Google Scholar 

  29. ISO 5725-1:1994. Accuracy (trueness and precision) of measurement methods and results. Part 1: General principles and definitions

  30. Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 75:3019–3030

    Article  CAS  Google Scholar 

  31. Favretto D, Stocchero G, Vogliardi S, Frison G, Trevisanuto D, Castagna F, Ferrara SD (2010) Neonatal hair analysis by liquid chromatography-high-resolution mass spectrometry to reveal gestational exposure to venlafaxine. Ther Drug Monit 32:30–39

    Article  CAS  Google Scholar 

  32. Ojanperä S, Pelander A, Pelzing M, Krebs I, Vuori E, Ojanperä I (2006) Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/Time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 20:1161–1167

    Article  Google Scholar 

  33. Nielen MWF, van Engelen MC, Zuiderent R, Ramaker R (2007) Screening and confirmation criteria for hormone residue analysis using liquid chromatography accurate mass time-of-flight, Fourier transform ion cyclotron resonance and orbitrap mass spectrometry techniques. Anal Chim Acta 586:122–129

    Article  CAS  Google Scholar 

  34. Virus E, Sobolevsky T, Rodchenkov G (2008) Introduction of HPLC/Orbitrap mass spectrometry as screening method for doping control. J Mass Spectrom 43:949–957

    Article  CAS  Google Scholar 

  35. van der Heeft E, Bolck YJC, Beumer B, Nijrolder AWJM, Stolker AAM, Nielen MWF (2009) Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis. J Am Soc Mass Spectrom 20:451–463

    Article  Google Scholar 

  36. European Commission (2002) Commission Decision 2002/657/EC. Off J Eur Commun L221:8

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Authors and Affiliations

  1. Forensic Toxicology and Antidoping, Medical School, University Hospital of Padova, Via Falloppio 50, 35121, Padova, Italy

    Susanna Vogliardi, Donata Favretto, Marianna Tucci, Giulia Stocchero & Santo Davide Ferrara

Authors
  1. Susanna Vogliardi
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  2. Donata Favretto
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  3. Marianna Tucci
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  4. Giulia Stocchero
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  5. Santo Davide Ferrara
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Correspondence to Donata Favretto.

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Published in the special issue Forensic Toxicology with Guest Editors Frank T. Peters, Hans H. Maurer, and Frank Musshoff.

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Vogliardi, S., Favretto, D., Tucci, M. et al. Simultaneous LC-HRMS determination of 28 benzodiazepines and metabolites in hair. Anal Bioanal Chem 400, 51–67 (2011). https://doi.org/10.1007/s00216-011-4742-8

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  • DOI: https://doi.org/10.1007/s00216-011-4742-8

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