Multi-analyte HPLC–DAD Method for Concurrent Analysis of Six Antimicrobials and Three Proton Pump Inhibitors Frequently used in Management of Helicobacter pylori Infection: Application to Simulated Intestinal Fluid Samples

Abstract

The present work deals with the optimization, validation and application of a versatile HPLC–DAD method for concurrent estimation of nine antimicrobials and proton pump inhibitors, namely amoxicillin (AMX), doxycycline (DOX), furazolidone (FRZ), lansoprazole (LNS), levofloxacin (LVF), metronidazole (MTZ), omeprazole (OMZ), pantoprazole (PNZ) and tinidazole (TNZ). The selected nine drugs are frequently included in various treatment regimens of Helicobacter pylori infection. Successful separation was accomplished using the analytical column Agilent Zorbax Eclipse plus-C18 (250 × 4.6 mm, 5 µm particle size) and a mobile phase prepared from phosphate buffer pH 5 and acetonitrile pumped at a flow rate 1 mL/min using a gradient elution program. The gradient elution started with buffer/acetonitrile ratio 90:10, then it was altered in 15 min to reach 40:60 by volume. Quantification of the analytes was based on measuring peak areas of AMX at 230 nm, LVF, LNS and PNZ at 290 nm, OMZ at 300 nm, MTZ and TNZ at 320 nm, and DOX and FRZ at 360 nm. The separated compounds eluted at retention times 5.68, 6.43, 7.82, 8.84, 9.42, 10.75, 12.82, 13.74 and 14.90 min for AMX, MTZ, LVF, TNZ, DOX, FRZ, OMZ, PNZ and LNS respectively. Validation of the proposed HPLC procedure was carefully studied according to the ICH items: ranges, precision, accuracy, linearity, robustness and limits of detection and quantitation. The linear dynamic ranges were 5–100, 5–50, 2–40, 10–100, 10–100, 5–50, 2.5–30, 3–30 and 2–30 µg/mL for AMX, MTZ, LVF, TNZ, DOX, FRZ, OMZ, PNZ and LNS, respectively with correlation coefficients > 0.9993. Application fields of the validated method included analysis of laboratory-prepared binary dosage forms along with analysis of several ternary mixtures in spiked simulated intestinal fluid.