Simplified and Rapid Determination of Primaquine and 5,6-Orthoquinone Primaquine by UHPLC-MS/MS: Its Application to a Pharmacokinetic Study.

The method for the determination of primaquine (PQ) and 5,6-orthoquinone primaquine (5,6-PQ), the representative marker for PQ active metabolites, via CYP2D6 in human plasma and urine has been validated. All samples were extracted using acetonitrile for protein precipitation and analyzed using the ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) system. Chromatography separation was carried out using a Hypersil GOLDTM aQ C18 column (100 × 2.1 mm, particle size 1.9 µm) with a C18 guard column (4 × 3 mm) flowed with an isocratic mode of methanol, water, and acetonitrile in an optimal ratio at 0.4 mL/min. The retention times of 5,6-PQ and PQ in plasma and urine were 0.8 and 1.6 min, respectively. The method was validated according to the guideline. The linearity of the analytes was in the range of 25-1500 ng/mL. The matrix effect of PQ and 5,6-PQ ranged from 100% to 116% and from 87% to 104% for plasma, and from 87% to 89% and from 86% to 87% for urine, respectively. The recovery of PQ and 5,6-PQ ranged from 78% to 95% and form 80% to 98% for plasma, and from 102% to from 112% to 97% to 109% for urine, respectively. The accuracy and precision of PQ and 5,6-PQ in plasma and urine were within the acceptance criteria. The samples should be kept in the freezer (-80 °C) and analyzed within 7 days due to the metabolite stability. This validated UHPLC-MS/MS method was beneficial for a pharmacokinetic study in subjects receiving PQ.

An increase in urinary primaquine and a reduction in urinary primaquine-5,6-orthoquinone in the Thai population with CYP2D6 reduced enzyme function.

Objectives: Primaquine is metabolized by the cytochrome P450-2D6 enzyme (CYP2D6) to an active primaquine-5,6-orthoquinone (POQ). No relationships of CYP2D6 polymorphisms with the pharmacokinetics of primaquine and POQ were reported in the Thai population. Methods: We evaluated the genetic distribution of CYP2D6 in 345 Thai army populations together with the pharmacokinetic profiles of primaquine and POQ in plasma and urine (n = 44, descriptive data are presented in median (range)). All dose-related pharmacokinetic parameters were normalized by primaquine dose per body weight before statistical analysis. Results: CYP2D6*10 was the allele observed with the highest frequency (56.62%) corresponding to CYP2D6*10/*10 (32.94%) and CYP2D6*1/*10 (27.94%) genotypes. CYP2D6 intermediate metabolizers (CYP2D6 IM) were found in 44.41% of the cohort and had an increase in the cumulative amount of primaquine excreted (CAE) in urine compared to normal metabolizers of CYP2D6 (CYP2D6 NM); (CYP2D6 IM vs. CYP2D6 NM: 2444 (1697-3564) vs. 1757 (1092-2185) µg/mg/kg, p = 0.039), a reduction in urine CAE of POQ (CYP2D6 IM vs CYP2D6 NM: 115 (46-297) vs. 318 (92-498) µg/mg/kg, p = 0.047) and a reduction in the POQ/primaquine CAE ratio in urine (CYP2D6 IM vs. CYP2D6 NM: 0.06 (0.01-0.11) vs. 0.16 (0.06-0.26), p = 0.009). No significant differences were found in the pharmacokinetic profiles of plasma primaquine and POQ. Conclusions: The CYP2D6 polymorphisms influenced the changes in primaquine and POQ that were noticeable in the urine, supporting the role of the CYP2D6 gene testing before drug administration.