High-sensitivity simultaneous liquid chromatography-tandem mass spectrometry assay of ethinyl estradiol and levonorgestrel in human plasma

Abstract

A sensitive and simultaneous liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of ethinyl estradiol and levonorgestrel. The analytes were extracted with methyl-tert-butyl ether: n-hexane (50:50, v/v) solvent mixture, followed by dansyl derivatization. The chromatographic separation was performed on a Kinetex C₁₈ (50 mm×4.6 mm, 2.6 µm) column with a mobile phase of 0.1% (v/v) formic acid in water and acetonitrile in gradient composition. The mass transitions were monitored in electrospray positive ionization mode. The assay exhibited a linear range of 0.100-20.0 ng/mL for levonorgestrel and 4.00-500 pg/mL for ethinyl estradiol in human plasma. A run time of 9.0 min for each sample made it possible to analyze a throughput of more than 100 samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic and bioequivalence studies.

Keywords: Derivatization; Ethinyl estradiol; Human plasma; LC–MS/MS; Levonorgestrel.

Fig. 1

Product ion mass spectra of levonorgestrel (m/z 313.3→245.3, scan range 100–350 amu) in the positive ionization mode.

Fig. 2

Product ion mass spectra of dansyl chloride derivatized ethinyl estradiol (m/z 530.1→171.0, scan range 100–540 amu) in the positive ionization mode.

Fig. 3

MRM ion-chromatograms of (A) double blank plasma (without IS), (B) ethinyl estradiol at LLOQ (m/z 530.1→171.0) 4.00 pg/mL and IS, and (C) subject sample at 12.6 pg/mL concentration.

Fig. 4

MRM ion-chromatograms of (A) double blank plasma (without IS), (B) levonorgestrel at LLOQ (m/z 313.3→245.3) 0.100 ng/mL and IS, and (C) subject sample at 0.593 ng/mL concentration.

Fig. 5

Chromatographic separation of ethinyl estradiol and levonorgestrel from retention time of lysophospholipid.

Fig. 6

Injection of three extracted blank plasma samples during post-column infusion experiment of (A) derivatized ethinyl estradiol infusion and (B) levonorgestrel infusion with a chromatogram of the ULOQ sample.