Quantification of Tafenoquine and 5,6-Orthoquinone Tafenoquine by UHPLC-MS/MS in Blood, Plasma, and Urine, and Application to a Pharmacokinetic Study

Abstract

Analytical methods for the quantification of the new 8-aminoquinoline antimalarial tafenoquine (TQ) in human blood, plasma and urine, and the 5,6-orthoquinone tafenoquine metabolite (5,6-OQTQ) in human plasma and urine have been validated. The procedure involved acetonitrile extraction of samples followed by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Chromatography was performed using a Waters Atlantis T3 column with a gradient of 0.1% formic acid and acetonitrile at a flow rate of 0.5 mL per minute for blood and plasma. Urine analysis was the same but with methanol containing 0.1% formic acid replacing acetonitrile mobile phase. The calibration range for TQ and 5,6-OQTQ in plasma was 1 to 1200 ng/mL, and in urine was 10 to 1000 ng/mL. Blood calibration range for TQ was 1 to 1200 ng/mL. Blood could not be validated for 5,6-OQTQ due to significant signal suppression. The inter-assay precision (coefficient of variation %) was 9.9% for TQ at 1 ng/mL in blood (n = 14) and 8.2% for TQ and 7.1% for 5,6-OQTQ at 1 ng/mL in plasma (n = 14). For urine, the inter-assay precision was 8.2% for TQ and 6.4% for 5,6-OQTQ at 10 ng/mL (n = 14). TQ and 5,6-OQTQ are stable in blood, plasma and urine for at least three months at both -80 °C and -20 °C. Once validated, the analytical methods were applied to samples collected from healthy volunteers who were experimentally infected with Plasmodium falciparum to evaluate the blood stage antimalarial activity of TQ and to determine the therapeutic dose estimates for TQ, the full details of which will be published elsewhere. In this study, the measurement of TQ and 5,6-OQTQ concentrations in samples from one of the four cohorts of participants is reported. Interestingly, TQ urine concentrations were proportional to parasite recrudescence times post dosing To our knowledge, this is the first description of a fully validated method for the measurement of TQ and 5,6-OQTQ quantification in urine.

Keywords: 5,6-orthoquinone tafenoquine; analytical method; blood; malaria; plasma; tafenoquine; urine.

Figure 1

The predicted fragmentation patterns and the MS/MS spectra of tafenoquine (TQ) and 5,6-orthoquinone tafenoquine (5,6-OQTQ). The predicted fragmentation patterns of (A) TQ; and (B) 5,6-OQTQ. The MS/MS spectra of (C) TQ showed the key fragments at m/z 379.13 (predicted 379.1265, observed 379.1260, mass error −1.0551), m/z 447.19 (predicted 447.1890, observed 447.1885, mass error −1.1181), and m/z 391.13 (predicted 39.1264, observed 391.1253, mass error −2.8124); and (D) 5,6-OQTQ showed the key fragments at m/z 287.14 (predicted 287.1390, observed 287.1387, mass error −1.0448), m/z 259.14 (predicted 259.1441, observed 259.1439, mass error −0.7718) and m/z 219.08 (predicted 219.0764, observed 219.0762, mass error −0.9129). These MS/MS scans were from the FTMS (Fourier transform mass spectrometer) QExactive Plus (Thermo Fisher, Waltham, MA, USA) positive mode scans at 70,000 resolution.

Figure 2

Chromatograms of 5,6-OQTQ, SIL-TQ and TQ in human plasma and urine. (A) Blank plasma with internal standard (SIL-TQ, blue peak). (B) Spiked plasma at low limit of quantification (LLOQ) for TQ (green peak) and 5,6-OQTQ (red peak). (C) Spiked plasma at 50 ng/mL TQ and 15 ng/mL 5,6-OQTQ. (D) Spiked plasma at 1200 ng/mL TQ and 200 ng/mL 5,6-OQTQ. (E) A clinical trial participant plasma sample. (F) Blank urine with internal standard (SIL-TQ). (G) Spiked urine at LLOQ for TQ and 5,6-OQTQ. (H) Spiked urine at 150 ng/mL TQ and 5,6-OQTQ. (I) Spiked urine at 1000 ng/mL TQ and 5,6-OQTQ. (J) A clinical trial participant urine sample.

Figure 3

TQ concentration—time profiles in human plasma (A) and human blood (B) from three subjects given 200 mg TQ orally; Subject 1, (circle line); Subject 2, (square line); Subject 3, (triangle line).

Figure 4

TQ in human urine (n = 3). (A) The amount of TQ excreted (AE) versus time profile; (B) The cumulative amount of TQ excreted (CAE) versus time profile. Time was the endpoint of each 24 h urinary collection period.

Figure 5

5,6-OQTQ in human urine (n = 3). (A) AE versus time profile; (B) CAE versus time profile. Time was the endpoint of each 24 h urinary collection period.