Application of an LC–MS/MS method for reliable determination of amodiaquine, N-desethylamodiaquine, artesunate and dihydroartemisinin in human plasma for a bioequivalence study in healthy Indian subjects

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 124; pp. 67 - 78
• Main Authors: Rathod, Dhiraj M., Patel, Keyur R., Mistri, Hiren N., Jangid, Arvind G., Shrivastav, Pranav S., Sanyal, Mallika
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 30.05.2016
• Subjects: Amodiaquine; Amodiaquine - analogs & derivatives; Amodiaquine - blood; Amodiaquine - pharmacokinetics; Artemisinins - blood; Artemisinins - pharmacokinetics; Artesunate; Chromatography, Liquid - methods; Dihydroartemisinin; Human plasma; Humans; India; LC–MS/MS; N-desethylamodiaquine; Tandem Mass Spectrometry - methods; Therapeutic Equivalency; Dihydroartemisinin; Amodiaquine; Human plasma; LC–MS/MS; Artesunate; N-desethylamodiaquine
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2016.02.021

[Display omitted] •A reliable method for determination of amodiaquine, artesunate and their metabolites in plasma.•Thorough investigation of extraction conditions and chromatography for simultaneous analysis.•Extensive evaluation of stability of analytes and matrix effects.•Bioequivalence study with fixed dose formulations in healthy Indian subjects.•Method reproducibility is established by incurred sample reanalysis. A sensitive and high throughput bioanalytical method has been developed for reliable determination of amodiaquine (AQ), N-desethylamodiaquine (DEAQ), artesunate (AS) and dihydroartemisinin (DHA) in human plasma by LC–MS/MS. The method employs a solid phase extraction procedure without an evaporation step and with optimum use of organic solvents to circumvent degradation of artemisinin derivatives. The analytes and their deuterated internal standards (ISs) were analyzed on Hypersil Gold (100mm×4.6mm, 5μm) column using acetonitrile and 2.0mM ammonium formate (pH 2.50) in 80:20 (v/v) ratio as the mobile phase. A triple quadrupole mass spectrometer equipped with an electrospray ionization interface was used to detect and quantify the analytes. The method was established over the concentration range of 0.250–30.0ng/mL, 1.50–180ng/mL, 2.00–600ng/mL and 5.00–1400ng/mL for AQ, DEAQ, AS and DHA respectively using 250μL human plasma. The intra-day and inter-day accuracy and precision (% CV) across quality controls varied from 93.3–105.0% and 1.7–8.3 respectively for all the analytes. The stability was assessed in whole blood as well as in plasma samples under different conditions. All four analytes were stable in whole blood up to 2h on melting ice. The long term stability in plasma was ascertained up to 90 days. IS-normalized matrix factors ranged from 0.988–1.023 for all the analytes. The method was successfully applied to a bioequivalence study using 50mg artesunate and 135mg amodiaquine fixed dose formulation in 14 healthy subjects.