Desenvolvimento e validação de um novo método rápido de HPLC de fase reversa para quantifica-ção simultânea de melitina e apamina em apitoxina

Context: High performance liquid chromatography (HPLC) is a high sensitivity and precision to api-toxin samples analysis. However, it has the disadvantage of incurring high financial, environmental and time costs. Aims: The aim of this study is to develop a shorter HPLC method for the quantification of apitoxin samples. Methods: The method was performed and validated on an RP-C18 column, mobile phases A (water and trifluoroacetic acid (TFA) 0.1%) and B (acetonitrile and TFA 0.1 %), gradient mode, flow rate 1.5 mL/min and 220 nm absorbance. Key results: The gradient elution 5% - 70% phase B over 20 minutes, after a 4-minute isocratic elution with 5% B at 25°C. Results showed a linear in the range between 0.084 and 0.84 mg/mL (R2=0.995) for melittin and between 0.02 and 0.20 mg/mL (R2=0.995) for apamin, accurate method (recovery rate 101.11 ±9.47 % and 113.57 ±15.06 % respectively). Precision: 10.37 and 9.33 for melittin and apamin respectively, and result of a selective method in its usual matrix. The LOD was 0.00178 and 0.00117 mg/mL, and LOQ was 0.00541 and 0.00353 mg/mL, for melittin and apamin, respectively.