Simultaneous quantification of four benzodiazepines from whole blood by highperformance liquid chromatography in forensic toxicological analysis

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Abstract

A rapid high performance liquid chromatography method, using a monolithic column, was developed for quantitative determinations of benzodiazepines (diazepam, clonazepam, lorazepam, midazolam) in whole blood. A liquid-liquid extraction step with n-chlorobutane isolates the drugs from alkalinized blood. The separation was carried out in reversed phase conditions using a Chromolith Performance (RP-18 100x4.6 mm) column. For the mobile phase, a mixture of a phosphate buffer (pH= 2.5)/acetonitrile (65/35 v/v), in isocratic mode at 2 mL/min. An ultraviolet spectrophotometer was used as the detector at the wavelength of 220 nm. The total run time of the analytical method is less than 4-6 minutes. The calibration curves showed linearity and the correlation coefficient of each individual curve was greater than 0.995. The method was linear over a concentration range of 0.03-0.6 μg/mL for clonazepam, lorazepam and midazolam. For diazepam of linearity was over the range 0.04-5.0μg/mL. Quantification limits ranged from 0.03-0.04μg/mL and the accuracy were from 80% to 105% for the recovery test.

The results indicate that this analytical method is simple, specific, accuracy, sensitive, demonstrating from the validation data and a higher robustness. The proposed method is applied routinely in forensic toxicological analysis involving blood.

[1]. O.H. Drummer, Forensic Sci. Int., 142(2), 101-113 (2004).

[2]. F. Barbone, A.D. McMahon, P.G. Davey, A.D. Morris, I.C. Reid, D.G. McDevitt, and T.M. MacDonald, Lancet, 352 (9137), 1331-1336 (1998).

[3]. J.L. Ferguson and D. Couri, J. Anal. Toxicol., 1(4), 171-174 (1977).

[4]. A.J.H. Louter, E. Bosma, J.C.A. Schipperen, J.J. Vreuls and U.A.Th. Brinkman, J. Chromatogr. B, 689(1), 35-43 (1997).

[5]. C. Moore, G. Long and M. Marr, J. Chromatogr. B Biomed. Appl. 655(1), 132-137 (1994).

[6]. S.M. Sultan and A.E. El-Mubarak, Talanta, 43(4), 569-576 (1996).

[7]. A. Bugey and C. Staub, J. Pharm. Biomed. Anal. 35(3), 555-562 (2004).

[8]. W. He and N. Parissis, J. Pharm. Biomed. Anal. 16(4), 707-715 (1997).

[9]. K.K. Akerman, J. Jolkkonen, M. Parviainen and I. Penttila, Clin. Chem., 42(9), 1412-1416 (1996).

[10]. C. Kratzsch, O. Tenberken, F.T. Peters, A.A. Weber, T. Kraemer and H.H. Maurer, J. Mass Spectrom., 39(8), 856-872 (2004).

[11]. B.E. Smink, J.E. Brandsma, A. Dijkhuizen, K.J. Lusthof, J.J. De Gier, A.C.G. Egberts and D.R.A. Uges, J. Chromatogr. B., 811(1), 13-20 (2004).

[12]. H.M. Rivera, G.S. Walker, D.N. Sims and P.C. Stockham, Eur. J. Mass Spectrom. 9(6), 599-607 (2003).

[13]. A.M. Jimenez Moreno, M. Jose Navas and A.G. Asuero, Crit. Rev. Anal. Chem., 44, 68-106, (2014)

[14]. M. Nakamura, Biomed. Chromatogr., 25, 1283-1307, (2011)

[15]. H. al-Hawasli, M.A. al-Khayat and M.A. al- Mardini, J. Pharm. Anal., 2(6), 484-491 (2012)

[16]. A. Bugey and C. Straub, J. Sep Sci. 30(17), 2967-2978 (2007).

[17]. A. Bugey, S. Rudaz and C. Straub, J. Chromatogr. B. 832(2), 249-255 (2006).

[18]. D.R. Bunch and S. Wang, J. Sep. Sci., 34, 2003-2012, (2011)

[19]. ***Guidance for Industry - Bioanalytical Methods Validation, Draft Guidance, Revision 1, FDA 2013.

[20]. ***Guideline on validation of bioanalytical methods, EMEA/CHMP/EWP/192217/2009.

Ovidius University Annals of Chemistry

Analele Universitatii "Ovidius" Constanta - Seria Chimie

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