Combining two-dimensional diffusion-ordered nuclear magnetic resonance spectroscopy, imaging desorption electrospray ionization mass spectrometry, and direct analysis in real-time mass spectrometry for the integral investigation of counterfeit pharmaceuticals

Abstract

During the past decade, there has been a marked increase in the number of reported cases involving counterfeit medicines in developing and developed countries. Particularly, artesunate-based antimalarial drugs have been targeted, because of their high demand and cost. Counterfeit antimalarials can cause death and can contribute to the growing problem of drug resistance, particularly in southeast Asia. In this study, the complementarity of two-dimensional diffusion-ordered (1)H nuclear magnetic resonance spectroscopy (2D DOSY (1)H NMR) with direct analysis in real-time mass spectrometry (DART MS) and desorption electrospray ionization mass spectrometry (DESI MS) was assessed for pharmaceutical forensic purposes. Fourteen different artesunate tablets, representative of what can be purchased from informal sources in southeast Asia, were investigated with these techniques. The expected active pharmaceutical ingredient was detected in only five formulations via both nuclear magnetic resonance (NMR) and mass spectrometry (MS) methods. Common organic excipients such as sucrose, lactose, stearate, dextrin, and starch were also detected. The graphical representation of DOSY (1)H NMR results proved very useful for establishing similarities among groups of samples, enabling counterfeit drug "chemotyping". In addition to bulk- and surface-average analyses, spatially resolved information on the surface composition of counterfeit and genuine antimalarial formulations was obtained using DESI MS that was performed in the imaging mode, which enabled one to visualize the homogeneity of both genuine and counterfeit drug samples. Overall, this study suggests that 2D DOSY (1)H NMR, combined with ambient MS, comprises a powerful suite of instrumental analysis methodologies for the integral characterization of counterfeit antimalarials.

Figure 1

Analyses of formulation 2 via (A) 2D DOSY 1H NMR in DMSO-d6, with TMPS as internal reference standard (where SDMSO represents DMSO satellite signals), (B) DART MS in positive-ion mode, and (C) DESI MS in positive-ion mode.

Figure 2

Analyses of formulation 11 using (A) 2D DOSY 1H NMR in DMSO-d6, with TMPS as internal reference standard (SDMSO represents DMSO satellite signals); and (B) DESI MS in positive-ion mode. (C) DESI spectrum, in positive-ion mode, of a sucrose standard (10 μL, 1 mg/mL), (D) DESI spectrum, in positive-ion mode, of a lactose standard (10 μL, 1 mg/mL). Standards were deposited onto polytetrafluoroethane (PTFE) and analyzed after air drying. The insets in panels (B), (C), and (D) represent the corresponding DESI MS2 spectra generated from the ion at m/z 365.3.

Figure 3

Analyses of formulation 4 by (A) 2D DOSY 1H NMR in DMSO-d6, with TMPS as internal reference standard, (B) DART MS in positive-ion mode, and (C) DESI MS in positive-ion mode.

Figure 4

DESI MS images of a genuine artesunate tablet (formulation 15) constructed using the spatial relationship between various spectral features and their intensity, with data acquisition in full-scan MS mode: (A) sodiated artesunic acid monomer (m/z 407.2), (B) sodiated artesunic acid dimer (m/z 791.2), (C) sodiated lactose (m/z 365.3), and (D) sodiated acetaminophen (m/z 174.1). All images are shown in false color scale and pixilated format.

Figure 5

DESI MS images of a counterfeit artesunate sample (formulation 16) constructed based on the spatial relationship between various spectral features and their intensity, with data acquisition in full-scan MS mode: (A) sodiated acetaminophen (m/z 174.1), (B) sodiated acetaminophen dimer (m/z 325.1), (C) sodiated lactose (m/z 365.3), and (D) sodiated artesunic acid monomer (m/z 407.2). All images are shown in false color scale and pixilated format.

Scheme 1

Fragment Ions Observed for the Isomeric Species: (A) [Sucrose + Na]+ and (B) [Lactose + Na]+ Generated from the Reagentless DESI MS2 Analysis of Sucrose and Lactose Standards (10 μL, 1 mg/mL) Respectively, Deposited on PTFE and Analyzed after Solvent Evaporation