Potentiometric and turbidimetric titrations were used to study the interaction between bovine serum albumin (BSA) and poly(diallyldimethylammonium chloride) (PDADMAC). Binding between BSA and PDADMAC, which takes place only above some critical initial pH (pHc), leads to a decrease in the pH of solution, indicating that the interactions enhance the dissociation constant Ka of the ionizable groups on BSA and result in an increase of the number of net negative charge on the protein. The pH difference caused by the interaction, ∆pH, decreases with added salt, which indicates that the effect of the interaction on the pKa of BSA increases with a decrease of ionic strength. Protein binding to PDADMAC imposes a stronger influence on the Ka of the carboxylic groups than on the Ka of the imidazolium and ammonium groups. The fraction of BSA bound (fb ) [BSA]b/Cpr) increases with polymer concentration Cp until all BSA are bound. The rate at which fb increases with added polymer at low Cp depends on the initial pH (pHi), consistent with an increase in the binding constant with pHi. Upon a further increase of pH, phase separation occurs at some well-defined point, pHφ, which increases with ionic strength. pHφ depends strongly on Cp at fixed BSA concentration, but only weakly on Cp at constant r ) Cpr/Cp. Phase separation may also be observed upon addition of polymer to BSA at pH > pHφ. In the range of pHc < pH < pHφ, pH titration and turbidimetry reveal the formation of soluble complexes. However, even when phase separation (coacervation) occurs, no corresponding change in the pH titration curve is observed, indicating that the protein's acid-base equilibria are unperturbed by phase separation.

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A simple, rapid and sensitive method using UPLC-MS/MS was established and validated for simultaneous determination of gelsemine and koumine in rat plasma after oral administration of Gelsemium elegans Benth extract. Plasma was performed with methanol precipitation and berberine was chosen as the internal standard. Plasma samples were separated on an Acquity UPLC® BEH C column (3.0 × 50 mm, 1.7 μm) with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase at a flow rate of 0.4 mL/min. Multiple reaction monitoring mode in positive ion mode was utilized for detection. The calibration curves were linear over the range of 0.2-100 ng/mL for gelsemine and 0.1-50 ng/mL for koumine, with the lower limits of quantification 0.2 and 0.1 ng/mL, respectively. The intra- and inter-precision and accuracy were well within the acceptable ranges. The developed method was successfully applied to an in vivo pharmacokinetic study in rat after oral administration of 10 mg/kg Gelsemium elegans Benth extract.