A tiered analytical approach for investigating poor quality emergency contraceptives

Abstract

Reproductive health has been deleteriously affected by poor quality medicines. Emergency contraceptive pills (ECPs) are an important birth control method that women can use after unprotected coitus for reducing the risk of pregnancy. In response to the detection of poor quality ECPs commercially available in the Peruvian market we developed a tiered multi-platform analytical strategy. In a survey to assess ECP medicine quality in Peru, 7 out of 25 different batches showed inadequate release of levonorgestrel by dissolution testing or improper amounts of active ingredient. One batch was found to contain a wrong active ingredient, with no detectable levonorgestrel. By combining ultrahigh performance liquid chromatography-ion mobility spectrometry-mass spectrometry (UHPLC-IMS-MS) and direct analysis in real time MS (DART-MS) the unknown compound was identified as the antibiotic sulfamethoxazole. Quantitation by UHPLC-triple quadrupole tandem MS (QqQ-MS/MS) indicated that the wrong ingredient was present in the ECP sample at levels which could have significant physiological effects. Further chemical characterization of the poor quality ECP samples included the identification of the excipients by 2D Diffusion-Ordered Nuclear Magnetic Resonance Spectroscopy (DOSY 1H NMR) indicating the presence of lactose and magnesium stearate.

Figure 1. TLC analysis.

Results for a levonorgestrel standard, sample 1 (positive control), and sample 2a observed under 254 nm light.

Figure 2. HPLC-DAD analysis.

HPLC chromatograms from assay / content uniformity analysis for a levonorgestrel standard, sample 1 (positive control), and sample 2a.

Figure 3. UHPLC-IMS-TOF-MS analysis.

Extracted ion chromatogram (A) and extracted ion mobility chronogram (B) for a levonorgestrel standard with m/z 313.2168±0.005. Extracted ion chromatogram (C) and extracted ion mobility chronogram (D) for sulfamethoxazole standard with m/z 254.0599±0.005. Extracted ion chromatogram (E) and extracted ion mobility chronogram (F) for sulfamethoxazole in the poor quality contraceptive tablet with m/z 254.0599±0.005. Mass spectra for the target compounds are displayed as insets.

Figure 4. DART MS and MS/MS analysis using a collision energy of 20 eV.

(A): Mass spectrum of a contraceptive tablet containing levonorgestrel. (B): MS/MS spectrum for the precursor ion with m/z 313.2419 shown in A. (C): Mass spectrum of levonorgestrel standard. (D): MS/MS spectrum for the precursor ion with m/z 313.2190 shown in C. (E): Mass spectrum of sample 2a. (F): MS/MS spectrum for the precursor ion with m/z 254.0646 shown in E. (G): Mass spectrum of sulfamethoxazole standard. (H): MS/MS spectrum for the precursor ion with m/z 254.0630 shown in G.

Figure 5. 2D DOSY ¹H NMR analysis.

Spectra (log D vs. chemical shift) for levonorgestrel-containing (A) and sample 2a (B) contraceptive tablets dissolved in CD₄O:D₂O (80∶20 v/v). Signals observed at 3.3 ppm and in the range 4.6–5 ppm are due to the deuterated solvent mixture.