Response surface methodology-based optimization of high-performance liquid chromatography for simultaneous detection...

Misuse of benzodiazepines in drug-facilitated crimes requires sensitive and selective analytical methods, yet their detection in beverages remains challenging due to matrix interferences and restriction by one-variable-at-time (OVAT) optimization approach. This study was aimed at applying response surface methodology (RSM) to optimize a high-performance liquid chromatography (HPLC) method for simultaneous detection of nimetazepam and diazepam in beverage samples. From the study, an 80% acetonitrile and wavelength of 210 nm for the detector were determined to be optimum through OVAT. On the other hand, the central composite design, an RSM approach, suggested that flow rate at 0.50 mL/min, ammonium formate buffer concentration at 0.04 M with a pH of 4.24 had produced a chromatogram with good separation and high sensitivity. The method showed good linearity (R² > 0.99) with limit of detection (LOD) and limit of quantification (LOQ) were 0.45 μg/mL and 1.50 μg/mL for nimetazepam, as well as 0.07 and 0.23 μg/mL for diazepam, respectively. Precision was determined in a range between 1.59% and 4.11% while accuracy was reported from 99.62% to 101.24% of percent recoveries with satisfactory selectivity. Recovery studies by spiking both analytes into beverages, resulting in recovery percentages ranged between 70.18% and 110.97%. Our study successfully demonstrated the enhanced performance of the HPLC method through the application of the RSM approach, particularly in recovering the target analytes from beverage samples and suitable for forensic analysis, providing a reliable and efficient simultaneous detection method.