HPLC Method for Determination and Optimization of 2,4-DNPH PreColumn Derivatization Effect of Reaction Conditions for Muscone in Natural Musk

Abstract

To establish a method for the determination of muscone in natural musk by pre-column derivatization HPLC with 2, 4-Dinitrophenyl hydrazine (DNPH) and the reaction conditions were optimized. The influencing factors of derivatization reaction were investigated by single factor, and the main influencing factors were optimized by orthogonal test. Under the optimum reaction conditions, muscone-DNPH derivatives were obtained and detected by 365 nm following the Alltima C18 column (5 μm, 150 mm × 4.6 mm) separation. Single factor investigation revealed: the suitable reaction medium and catalyst were ethanol and hydrochloric acid, respectively; the molar ratio of muscone to DNPH should not be less than 300 times, the appropriate reaction time was 20 min or more; the effect of reaction temperature on derivatization reaction was obvious and needs to be optimized. Furthermore, the orthogonal test of acid concentration, reaction time and temperature showed that the effect of temperature on reaction was significant (p 0.05); The optimum reaction conditions were: reaction temperature was 65°C, hydrochloric acid be added to derivatization solution was (1.25:98.75) (mL/mL) and the reaction time was 30 min. Under this conditions, there was good linearity in range of 0.04 μg•mL-1-30.00 μg•mL-1 for muscone (r=0.999 9), the average spiked recoveries were 98.37%-100.32% and their RSD were 0.57%-1.32%. All the RSD of repeatability of sample and standard treatment were less than 4.12%; the limits of detection (S/N=3) and quantitation (S/N=15) were 0.005 μg•mL-1 and 0.04 μg•mL-1 respectively. The established method for detecting is accurate with good reproducibility and can be used for determination of muscone in natural musk.