Selective and rapid determination of raltegravir in human plasma by liquid chromatography-tandem mass spectrometry in the negative ionization mode

Abstract

A selective and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard (IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 µL human plasma. The chromatographic separation was achieved on a Chromolith RP-18e endcapped C₁₈ (100 mm×4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1→316.1 for raltegravir and m/z 446.1→319.0 for IS. The linearity of the method was established in the concentration range of 2.0-6000 ng/mL. The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.

Keywords: Bioequivalence study; Human plasma; LC–ESI–MS/MS; Negative ionization mode; Raltegravir.

Fig. 1

Product ion mass spectra of (A) raltegravir (m/z 443.1→316.1, scan range 100–500 amu) and (B) internal standard, raltegravir-d3 (m/z 446.1→319.0, scan range 100–500 amu), in the negative ionization mode.

Fig. 2

MRM chromatograms of (A) a mixture of raltegravir and raltegravir glucuronide and (B) raltegravir glucuronide.

Fig. 3

MRM ion-chromatograms of (A) double blank plasma (without analyte and IS), (B) blank plasma with raltegravir-d3 (IS), (C) raltegravir at LLOQ and IS and (D) subject sample at C_max after administration of 400 mg dose of raltegravir.

Fig. 4

Injection of four extracted blank plasma samples during post-column infusion of raltegravir with a chromatogram of raltegravir at HQC level.

Fig. 5

Mean plasma concentration-time profile of raltegravir after oral administration of 400 mg (test and reference) tablet formulation to 18 healthy Indian subjects.