Simultaneous analysis of seven benzodiazepines in dietary supplements as adulterants using high performance liquid chromatography and its application to an identification system for diazepam

A high performance liquid chromatography (HPLC) method for the simultaneous analysis of the seven benzodiazepines (BZDs), oxazolam, nitrazepam, oxazepam, tofisopam, triazolam, clotiazepam and diazepam (DIA) is presented for application in the screening examination of dietary supplements as adulterants. Samples were analyzed after extraction with methanol. HPLC analysis was performed with on a column of Wakosil 5C18 (4.6 x 150 mm, 5 mu m) with the mobile phase consisting of 1-heptanesulfonic acid sodium salt 5 mM dissolved in water/acetonitrile 1000 ml (13 : 7, v/v), adjusted with phosphoric acid to pH 2.4. The column eluent was monitored with a photodiode array detector, and the quantitative analysis of BZDs was performed at 225 nm. The calibration curves of the seven BZDs showed good linearity and the correlation coefficients were better than 0.999 in all cases. When this procedure was applied to commercial supplements, an DIA-adulterated capsule-type supplement was detected. Finally, DIA was identified and determined using a combination of three different analytical methods: HPLC/photodiode array, thin-layer chromatography (TLC), and LC/MS. DIA was present at a concentration of 1.9 mg/capsule. The procedure described here can provide a broad analysis in a single run within 17 min and is available for the screening of seven BZDs in adulterated supplements with minimal sample preparation.