Determination of unbound piperaquine in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry

Abstract

Piperaquine (PQ) is an antimalarial drug that is highly protein-bound. Variation in plasma protein contents may affect the pharmacokinetic (PK) exposure of unbound drug, leading to alteration of clinical outcomes. All published methods for determination of PQ in human plasma measure the total PQ including both bound and unbound PQ to plasma proteins. There is no published method for unbound PQ determination. Here we report an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for determination of PQ in human plasma filtrate prepared by filtering human plasma through Millipore Microcon® centrifugal filters (10k NMWL). The filter cup had to be treated with 5% benzalkonium chloride to reduce non-specific binding to the filter devices before filtration of plasma samples. Multiple reactions monitoring (MRM) of the ion pairs m/z 535/288 for PQ and m/z 541/294 for the internal standard (IS) was selected for quantification. When electrospray ionization (ESI⁺) was used, paradoxical matrix effect was observed despite the structure similarity of the deuterated IS: Ion suppression for PQ versus ion enhancement for the PQ-d₆, even though they were closely eluted: 0.62 min versus 0.61 min. Separation was achieved on Evo C₁₈ column (50 × 2.1 mm, 1.7 μm, Phenomenex Inc.) eluted with 10 mM NH₄OH and MeCN. When atmospheric pressure chemical ionization in positive mode (APCI⁺) was used for ion source, matrix effect diminished. Separation was achieved on a PFP column (30 × 2.1 mm, 1.7 μm, Waters, Corp.) eluted with aqueous 20 mM ammonium formate 0.14% trifluoroacetic acid (A) and methanol-acetonitrile (4:1, v/v) containing 0.1% trifluoroacetic acid (B) at 0.8 mL/min flow rate in a gradient mode: 30-30-80-80-30-30%B (0-0.1-1.0-1.40-1.41-1.50 min). The retention time was 0.67 min for both PQ and the IS. The method was validated with a linear calibration range from 20 to 5,000 pg/mL and applied to clinical samples.

Keywords: Piperaquine; Plasma; Plasma filtrate; UHPLC-MS/MS; Unbound; free.

Fig. 1.

Representative product ion spectra of piperaquine (upper panel) and the IS (lower panel).

Fig. 2.

Paradoxical matrix effects for PQ and the IS (PQ-d₆) in plasma filtrate. A sample with 600 pg/mL PQ in 10%MeCN 0.5%FA solution (dash line) and plasma filtrate (solid line) was analyzed with ESI⁺ as the ion source.

Fig. 3.

Chromatograms of PQ (solid line) and IS (dash line) from a clinical sample collected at 2 h post last dose. The PQ was separated from the interfering peak after mobile phase B switched to MeOH-MeCN (4:1, v/v) 0.1%TFA (lower panel). Flow rate was 1.2 mL/min (upper panel) or 0.8 mL/min (lower panel).

Fig. 4.

Representative chromatograms of PQ (upper panel) and PQ-d₆ (IS) (lower panel) from blank and LLOQ samples: gray dash line, blank plasma filtrate; black solid line, PQ at LLOQ (20 pg/mL) level.

Fig. 5.

Concentration-time profile of PQ from a pregnant women under dihydroartemisinin-PQ chemoprevention therapy.