This study aimed to analyze a high-performance liquid chromatography (HPLC) separation using a pentafluorophenyl column of parent drug hydroxychloroquine (HCQ) and its active metabolite, desethylhydroxchloroquine (DHCQ) applying to determine bioequivalence of two different formulations administered to patients. A rapid, simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for bioanalysis of HCQ and its metabolite DHCQ in human whole blood using deuterium derivative hydroxychloroquine-D4 as an internal standard (IS). A triple-quadrupole mass spectrometer was operated using electrospray ionization in multiple reaction monitoring (MRM) mode. Sample preparation involves a two-step precipitation of protein techniques. The removed protein blood samples were chromatographed on a pentafluorophenyl (PFP) column (50 mm × 4.6 mm, 2.6 μm) with a mobile phase (ammonium formate solution containing dilute formic acid) in an isocratic mode at a flow rate of 0.45 mL/min. The standard curves were found to be linear in the range of 2 – 500 ng/mL for HCQ; 2 – 2,000 ng/mL for DHCQ in spite of lacking a highly sensitive MS spectrometry system. Results of intra- and inter-day precision and accuracy were within acceptable limits. A run time of 2.2 min for HCQ and 2.03 min for DHCQ in blood sample facilitated the analysis of more than 300 human whole blood samples per day. Taken together, we concluded that the assay developed herein represents a highly qualified technology for the quantification of HCQ in human whole blood for a parallel design bioequivalence study in a healthy male.

Introduction
 Materials and Methods
  Chemicals and reagents
  Pharmaceutical formulation
  LC-MS and LC-MS/MS analyses
  Standard curve and sample preparation
 Validation
  Precision, accuracy and recovery
  Matrix effect
  Stability experiments
  Parallel bioequivalence study and statistical analysis
 Results and Discussion
  Optimization of sample preparation and chromatographicconditions
  Method validation
  Stability verification
  Application of parallel design to a bioequivalence study
 References

• Jung Youl Park(Department of Applied Chemistry, Daejeon University)
• Hyun Ho Song(Clinical & Bio Research Center, Korea Pharmaceutical Test & Research Institute (KPTR))
• Young Ee Kwon(Clinical & Bio Research Center, Korea Pharmaceutical Test & Research Institute (KPTR))
• Seo Jin Kim(Department of Marine Molecular Bioscience, College of Life Science, Gangneung-Wonju National University)
• Sukil Jang(Department of Marine Molecular Bioscience, College of Life Science, Gangneung-Wonju National University)
• Seong Soo Joo(Department of Marine Molecular Bioscience, College of Life Science, Gangneung-Wonju National University) Corresponding author