Quantification of the major metabolites of bromhexine in human plasma using RRLC-MS/MS and its application to pharmacokinetics.

(E)-4-hydroxydemethylbromhexine (E-4-HDMB) and (E)-3-hydroxydemethylbromhexine (E-3-HDMB) were found as major metabolites, while (Z)-4-hydroxydemethylbromhexine and (Z)-3-hydroxydemethylbromhexine as minor metabolites of bromhexine in human plasma. These compounds were identified in comparison with synthetic authentic samples. A sensitive and selective rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) method was developed to quantify the concentration of bromhexine and its two major metabolites (E-4-HDMB and E-3-HDMB) in human plasma. Following solid phase extraction, the analytes were separated on a Zorbax 1.8microm particle size reversed-phase C(18) column, using a gradient elution program with solvents consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in 5mM ammonium acetate at a flow rate of 0.7mL/min. Detection was carried out with an Agilent 6460 triple-quadrupole mass spectrometer operated with an electrospray ionization source mode operated in the positive ion mode. The recovery of bromhexine, E-4-HDMB, E-3-HDMB, and internal standard (IS) was 63.1-70.9%, 60.5-68.4%, 57.0-63.5%, and 87.8%, respectively. The matrix factors of bromhexine, E-4-HDMB, E-3-HDMB, and IS were 89.9-96.7%, 89.6-94.8%, 90.4-91.4%, and 103%, respectively. After an oral administration of 8.0mg bromhexine to five healthy male subjects, AUC(0-24h) values of bromhexine, E-4-HDMB, and E-3-HDMB were found to be 93.5+/-31.9, 34.0+/-14.5, and 15.8+/-6.89ngh/mL, respectively; while C(max) values were 24.6+/-5.16, 3.11+/-1.13, and 5.36+/-2.55ng/mL, respectively. Plasma concentration of bromhexine, E-4-HDMB, and E-3-HDMB declined with t(1/2) which gave 3.6+/-0.5, 8.4+/-2.7, and 6.4+/-2.5h, respectively.