Simultaneous Determination of Six Benzodiazepines in Spiked Soft Drinks by High Performance Liquid Chromatography with Ultra Violet Detection (HPLC-UV)

Document Type : Research article


1 Department of Toxicology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Forensic Toxicology, Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran

3 Iran Polymer and Petrochemical Research Institute, Tehran, Iran


A high performance liquid chromatographic method with ultra violet detection for simultaneous analysis of six benzodiazepines (BZDs) (chlordiazepoxide, diazepam, clonazepam, midazolam , flurazpam and lorazepam) has been developed for forensic screening of adulterated non-alcoholic drinks. Samples were analyzed after a simple procedure for preparation using pH adjustment and filtering. Isocratic elution on a C18 column (250mm × 4.6 mm, 5μm) in the temperature 45ºC with a mobile phase consisting of 15mM phosphate buffer: methanol (50:50 v/v) at a flow rate 1.4 mL/min has been done. The column eluent was monitored with a UV detector at 245 nm. This allowed a rapid detection and identification as well as quantization of the eluting peaks. Calibration curves for all drugs in the range of 0.5- 10 µg/ mL that all the linear regression and has more than 0.996. Recovery rates for the BZDs were in the range 93.7- 108.7%. The limits of detection were calculated between 0.01- 0.02 µg/ mL. Also, the limits of quantification were 0.03- 0.05 µg/mL. Within-day and between -day coefficient of variation for all BZDs at all concentrations in the range of 0.45 - 7.69 % was calculated. The procedure can provide a simple, sensitive and fast method for the screening of six BZDs in adulterated soft drinks in forensic analysis.


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