Study And Application Of Simultaneously Determination Of Seven Benzodiazepines By High-Performance Liquid Chromatography

OBJECTIVE: Benzodiazepines (BZDs) are central nerval system inhibitors, whichs are phenobarbital. These drugs are frequently abused even if result in poisoning, for they could be acquired easily. Furthermore they are usually used as criminal tool by reason of its little dosage and fast effect. So it is significative to detect their content in vivo. Seven benzodiazepines are in common used in clinic, including diazepam, clonazepam, nitrazepam, triazolam, alprazolam, estazolam and midazolam. The content of drugs in vivo is very low and the concentration is between ng/mL and μg/mL. Methods to analysis benzodiazepines in vivo include immunoassay, spectrographic method, chromatography, and so on. Of the total, high-performance liquid chromatography (HPLC) is widely used, which is rapid, selective and accurate. Now the reports about the HPLC methods mostly aim at one, two or three drugs. We have not seen some reports using HPLC gradient elution to determine benzodiazepines at one time. This study is to build a sensitive, accurate, simple HPLC method for the determination of 7 benzodiazepines in serum and urine simultaneously using gradient elution by ultraviolet detector, which could rapidly identify if someone has taken benzodiazepines and which one has been taken. METHODS: Seven benzodiazepines in serum and urine were separated and detected by RP-HPLC method using gradient elution. Exteral standardization was applied for quantitative analysis. The method was carried out on the Kromasil C18 column (4.6×250mm i.d,5μm). Two kinds of mobile phases were adopted in this study. Mobile phase Ⅰ was consisted of methanol (component A) and 30mmoL· L-1 of NH4Ac buffer (pH 5.6)(component B). The programme of gradient elution was: from beginning to 20 min, the proportion of component A was 55% and component B was 45%; later the proportion of component A was steppes up to 85% and component B was debased to 15%, then this mode was keeped until the end of analytic process. Mobile phase Ⅱwas consisted of acetonitrile and 30mmoL · L-1 of NH4Ac buffer (pH 5.6)(33:67). The detective wavelength was set at 230nm, the flow rate was 1mL·min-1, the column temperature was room temperature, and injection volume was 20μL. RESULTS: In serum and urine sample, precision (RSD) varied from 1% to 10%and recoveris were between 94% and 110%. Peak areas presented good linear relationship with concentration in wider ranges, correlation coefficients were more than 0.9992. The limit of detection (LOD) of diazepam was 2ng·mL-1, LOD of nitrazepam was 7ng·mL-1, LOD of estazolam was 5.5ng·mL-1, LOD of alprazolam was 6ng·mL-1, LOD of clonazepam was 5ng·mL-1, as well as triazolam and midazolam. CONCLUSION: A gradient RP-HPLC method was established, which could separate and determine 7 benzodiazepines simultaneously within 40 minutes. The method is simple, specific, reliable, and suitable for the quantitative determination of these compounds in serum and urine samples.