HPLC with Simultaneous UV and Reductive Electrochemical Detection at the Hanging Mercury Drop Electrode: A Highly Sensitive and Selective Tool for the Determination of Benzodiazepines in Forensic Samples

Abstract

A versatile, sensitive, and selective high-performance liquid chromatographic (HPLC) procedure for the determination of common benzodiazepines and some of their most frequently occurring metabolites in forensic samples was developed and optimized with respect to effective and rapid sample preparation and high selectivity of the analytical assay. The optimized method includes liquid-liquid extraction of the drugs with chloroform followed by isocratic reversed-phase chromatography on a LiChrospher-100 RP-8ec column (150 x 4.6-mm i.d.) with a mobile phase consisting of 0.03 mol/L acetate buffer (pH 4.6)/acetonitrile (55:45, v/v). The use of dual-mode detection made up of UV-detection (250 nm) in series with reductive electrochemical detection (-1.4 V vs. Ag/AgCl) at the hanging mercury drop electrode permits the detection and quantitation of benzodiazepines even in degraded samples with higher selectivities than usually reached with conventional HPLC techniques. Depending on the actual benzodiazepine species, detection limits are in the range of 2.0 to 14.1 ng/mL. Mean recovery values of the drugs from blood range from 82 to 92%; within-day and day-to-day repeatabilities typically lie between 3 and 9%. Several case work examples demonstrate the high selectivity and remarkably low matrix sensitivity of the described assay.

Development and validation of an HPLC method for the determination of benzodiazepines and tricyclic antidepressants in biological fluids after sequential SPE

“…For the routine analysis of BZDs and antidepressants various techniques have been used, such as: spectrophotometry and spectrofluorimetry [14,15], potentiometry [16], indirect kinetic catalytic method by stopped flow technique [17], nonaqueous CE [18], modified carbonpaste electrodes as sensors [19], immunoassay [20,21], radioreceptor assay [22], separation techniques like TLCdensitometry [23,24] and RPLC-ESI MS [25,26], Ultra Performance LC-MS/TOF (UPLC-MS/TOF) [27], micellar EKC (MEKC) [28,29], GC-nitrogen phosphorus detection (GC/ NPD) [30], GC-electron capture detection (GC-ECD) [31], GC-MS detection [32,33], GC-FID [34] and HPLC coupled with fluorescence detector [35], chemiluminescence detector [36], UV -Vis detector or HPLC-DAD [37,38], HPLC-ECD [39], HPLC-MS [40 -42], or HPLC-MS/MS [43].…”

Forensic intoxication with clobazam: HPLC/DAD/MSD analysis

“…Although gas chromatography [9] is much more sensitive and specific, it requires derivatization procedures and thermolabile benzodiazepines are not suited to this technique. As a consequence high-performance liquid chromatography (HPLC) [10][11][12][13][14][15][16][17][18][19] and more recently, the coupling of HPLC with mass spectrometry (MSD) [20,21] provides a useful and robust technique for the analysis of benzodiazepines.…”

Electrochemical Detection of Benzodiazepines, Following Liquid Chromatography, for Applications in Pharmaceutical, Biomedical and Forensic Investigations

“…Pre-2006 applications of liquid chromatography coupled with electrochemical detection for the determination of benzodiazepines These redox processes have been shown to occur with a variety of electrode materials and as a result a range of different electrochemical techniques, including polarography and voltammetry have been successfully utilised. These have been shown to be sensitive, economic and flexible and have been exploited for the determination of a range of parent benzodiazepines and their metabolites in complicated matrices such as blood [28][29][30], and forensic samples [31][32][33][34]. Utilising techniques such as adsorptive stripping voltammetry [35] detection limits in the nM to µM range are readily obtainable.…”