Determination of Benzodiazepines in Forensic Samples by HPLC with Photo-Diode Array Detection

Abstract

A high-performance liquid chromatographic (HPLC) method has been developed for the analysis of several benzodiazepines and some of their metabolites in blood, plasma and urine. The method included a liquid-liquid extraction with n-hexane:ethylacetate, a gradient elution on a C8 reversed phase column with a non-electrolyte eluent and a photo diode array detection. This allowed a rapid detection, a purity check, and identification as well as quantitation of the eluting peaks. The detection limit was 10 to 30 ng and the limit of quantitation was 0.05 μg/mL, using 1 mL of blood, plasma or urine. The procedure is applied routinely in forensic toxicological analyses involving blood, stomach content, urine and organ samples. About 30 positive cases are reported. The avoidence of the use of an electrolyte buffer in the eluent resulted in a robust procedure, free of technical problems and of long rinsing periods, suitable for routine use in forensic toxicology analysis involving blood, urine, stomach content and tissue samples.

Polarographic determination of diazepam with its parallel catalytic wave in the presence of persulfate

“…Liquid chromatography and non-aqueous titrimetry were recommended by the Pharmacopeia [1,2]. Derivative UV spectroscopy [3,4], HPLC [5][6][7][8], potentiometric [9], polarographic [10][11][12] and voltammetric [13] methods had been reported. Among these methods, the differential plus polarographic and adsorptive stripping voltammetric methods based on the reduction wave of diazepam were of higher analytical sensitivity.…”

Separation and HPLC analysis of 15 benzodiazepines in human plasma

“…Precision and sensitivity are also better than those reported for other existing HPLC-UV methods which analyse a similar number of BZDs [22,23] for forensic purposes. The method described by He et al [22] has much higher LOQ values (50 ng/mL vs. 2.5 -7.5 ng/mL) and uses larger volumes of sample (1 mL instead of 500 lL) as well as a more complicated HPLC procedure (gradient elution) and a solvent-intensive sample pretreatment by LLE. The method described by McIntyre et al [23] uses again gradient elution and LLE, with a twostep procedure; sensitivity (LOD 10 -100 vs. 1 -2.5 ng/ mL), extraction yields (14 -79 vs. 93 -100%) and precision values (interday RSD = 10 -15 vs. 2.0 -7.9%) are also less satisfactory than those obtained in our experiments.…”

“…Furthermore, this kind of procedure is surely more feasible and less time consuming than the liquid -liquid extraction (LLE) procedures often adopted for the same purpose [12,14,23]. Precision and sensitivity are also better than those reported for other existing HPLC-UV methods which analyse a similar number of BZDs [22,23] for forensic purposes. The method described by He et al [22] has much higher LOQ values (50 ng/mL vs. 2.5 -7.5 ng/mL) and uses larger volumes of sample (1 mL instead of 500 lL) as well as a more complicated HPLC procedure (gradient elution) and a solvent-intensive sample pretreatment by LLE.…”

“…Other methods based on HPLC with UV or photodiode array (PDA) detection, which simultaneously analyse only a few BZDs (usually four to eight), can be found in the literature [13 -21]. Two HPLC methods with diode array or UV detection, which allow the determination of more than ten BZDs, have been described [22,23]; however, they were only applied to forensic analysis: their sensitivity is too low for application to therapeutic drug monitoring (TDM) and method validation requirements do not match those of TDM. In contrast, the aim of this study was the development of a feasible and reliable HPLC-UV method for the simultaneous determination of 15 BZDs in patients' plasma for TDM purposes.…”

Experimental design for optimization of microwave-assisted extraction of benzodiazepines in human plasma

“…Although high-performance liquid chromatography (HPLC) with mass spectrometry detection [5][6][7] is highly selective, the cost of the instrumentation makes it less suitable for clinical or toxicology laboratories. In contrast, HPLC-UV detection or diode array detection (DAD) instrumentation is widely available in most analytical laboratories [8][9][10][11][12].…”