Christina Y. Hampton

Characterization of Solid Counterfeit Drug Samples by Desorption Electrospray Ionization and Direct- analysis-in-real-time Coupled to Time-of-flight Mass Spectrometry

The search for more versatile, sensitive, and robust ionization methods is a recurring theme in mass spectrometry (MS). Since the discovery of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), many developments such as atmospheric pressure MALDI, nanospray ionization, Venturi-assisted electrospray, and ion-funnel atmospheric pressure interfaces, have paved the way to improved characterization of small molecules and biomolecules. One of the bottlenecks in achieving high sample throughput with both ESI and MALDI is the need to dissolve, extract, and/or filter the sample prior to analysis. Moreover, vacuum-incompatible materials cannot be easily investigated by MS without disturbing their innate structure. Recently, two novel methods for the direct ionization of solid samples under atmospheric pressure by MS were reported: desorption electrospray ionization (DESI) and direct analysis in real time (DART). More recently, McEwen et al. described a modified atmospheric pressure chemical ionization (APCI) technique for the direct analysis of solids which they named atmospheric pressure solids analysis probe (ASAP). DESI makes use of a high-speed liquid spray directed at a sample held or deposited on a surface at atmospheric pressure. Ions generated during this process are sampled by a mass spectrometer. Several DESI applications such as the mapping of analytes separated by thin-layer chromatography, the detection of explosives, and the screening of pharmaceutical tablets and illicit drugs quickly followed the proof-of-principle description of the method. DART involves an ionizing beam of metastable He atoms (S1, 19.8 eV) generated by a corona discharge. The DART ionization mechanism is still not completely understood. In negative ion mode, the metastable He atoms generate electrons that produce negatively charged oxygen–water clusters, which then form the corresponding adducts. In positive ion mode, metastable He atoms generate protonated gaseous water clusters by Penning ionization. Then, by proton exchange, these clusters form [M+H] ions, which are generally the predominant species. DART’s high throughput coupled with the high mass accuracy now attainable with modern time-of-flight mass (TOF) analyzers and accurate isotopic abundance measurements make it especially suitable for the rapid identification of unknown species in solid materials. One particularly relevant example is counterfeit drug samples. Counterfeit drugs are defined as those that are “deliberately and fraudulently mislabeled with respect to identity and/or source”. They may include products with the “wrong” ingredient(s), without active ingredient(s), or with an insufficient amount of active ingredient(s). In recent years, a particularly alarming case of drug counterfeiting has been reported by field researchers who have detected counterfeit products that mimic the vital antimalarial, artesunate. The consumption of fake antimalarials has resulted in the death of many patients. Evidence suggests that the production of counterfeit artesunate tablets is on an industrial scale. For example, one health care organization in southeast Asia unwittingly purchased 100,000 artesunate tablets which were later shown to be counterfeit. Classic hyphenated analysis methods, such as liquid chromatography–mass spectrometry (LC–MS), lack the required sample throughput to survey such large numbers of samples in a reasonable amount of time. Figure 1a shows a schematic of the DART TOF MS setup used to screen 52 representative samples of a database containing more than 400 artesunate-based antimalarial tablets. Figure 1b and 1c show the negative ion mode DART TOF MS data of genuine and counterfeit artesunate (M) tablets, respectively. The spectrum shown in Figure 1b has signals corresponding to the diagnostic [M H] artesunate anion (experimental m/z=383.1702, calculated m/z=383.1711) and palmitic acid, a ubiquitous contaminant. Artesunate fragment ions due to dissociation of the highly labile artesunate carboxylic acid side [a] Prof. Dr. F. M. Fern ndez, C. Y. Hampton School of Chemistry and Biochemistry Georgia Institute of Technology 770 State St. Atlanta, GA 30332 (USA) Fax: (+1)404-385-6447 E-mail : facundo.fernandez@chemistry.gatech.edu [b] Dr. R. B. Cody JEOL USA, Inc. 11 Dearborn Road, Peabody, MA 01960 (USA) [c] Dr. M. D. Green Division of Parasitic Diseases, National Center for Infectious Diseases Center for Disease Control and Prevention 1600 Clifton Road, Mailstop F12, Atlanta, GA 30333 (USA) [d] Dr. R. McGready Shoklo Malaria Research Unit Mae Sot Tak (Thailand) [e] Dr. S. Sengaloundeth Food and Drug Department Ministry of Health, Government of the Lao PDR Vientiane (Lao PDR) [f] Prof. N. J. White, Dr. P. N. Newton Microbiology Laboratory, Mahosot Hospital Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Vientiane (Lao PDR) and Centre for Clinical Vaccinology and Tropical Medicine Churchill Hospital, Oxford University, Oxford, OX37LJ (UK) [] Prof. White is also affiliated with: Wellcome Trust–Mahidol University–Oxford Tropical Medicine Research Programme, Faculty of Tropical Medicine Mahidol University, Bangkok, 10400 (Thailand)

Transmission-mode direct analysis in real time and desorption electrospray ionization mass spectrometry of insecticide-treated bednets for malaria control

Transmission-mode direct analysis in real time (TM-DART) is presented as an alternative sampling strategy to traditional methods of sample introduction for DART MS analysis. A custom-designed sample holder was fabricated to rapidly and reproducibly position insecticide-treated nets normal to the ionizing metastable gas stream, enabling transmission of desorbed analyte ions through the holder cavity and into the MS. Introduction of the sample at this fixed geometry eliminates the need for optimizing sample position and allows spectra based on factors such as metastable gas temperature and flow to be systematically evaluated. The results presented here, supported by computational fluid dynamic simulations, demonstrate the effects of these factors on the resulting mass spectra and the potential of this sampling strategy to be used for qualitative and quantitative analyses. Transmission-mode desorption electrospray ionization (TM-DESI) experiments on similar insecticide-treated nets were performed for comparison purposes.

Comparison of the internal energy deposition of direct analysis in real time and electrospray ionization time-of-flight mass spectrometry.

The internal energy (Eint) distributions of a series of p-substituted benzylpyridinium ions generated by both direct analysis in real time (DART) and electrospray ionization (ESI) were compared using the “survival yield” method. DART mean Eint values at gas flow rates of 2, 4, and 6 L min−1, and at set temperatures of 175, 250, and 325 °C were in the 1.92–2.21 eV range. ESI mean Eint at identical temperatures in aqueous and 50% methanol solutions ranged between 1.71 and 1.96 eV, and 1.53 and 1.63 eV, respectively. Although the results indicated that ESI is a “softer” ionization technique than DART, there was overlap between the two techniques for the particular time-of-flight mass spectrometer used. As a whole, there was an increase in Eint with increasing reactive and drying gas temperatures for DART and ESI, respectively, indicating thermal ion activation. Three dimensional computational fluid dynamic simulations in combination with direct temperature measurements within the DART ionization region revealed complex inversely coupled fluid-thermal phenomena affecting ion Eint values during atmospheric transport. Primarily, that DART gas temperature in the ionization region was appreciably less than the set gas temperature of DART due to the set gas flow rates. There was no evidence of Eint deposition pathways from metastable-stimulated desorption, but fragmentation induced by high-energy helium metastables was observed at the highest gas flow rates and temperatures.

Peptidyl α-Ketoamides with Nucleobases, Methylpiperazine, and Dimethylaminoalkyl Substituents as Calpain Inhibitors

A series of peptidyl alpha-ketoamides with the general structure Cbz-L-Leu-D,L-AA-CONH-R were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. Nucleobases, methylpiperazine, and dimethylaminoalkyl groups were incorporated into the primed region of the inhibitors to generate compounds that potentially cross the blood-brain barrier. Two of these compounds (Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-adenin-9-yl and Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-(4-methylpiperazin-1-yl) have been shown to have useful concentrations in the brain in animals. The best inhibitor for calpain I was Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-2-methoxyadenin-9-yl (K(i) = 23 nM), and the best inhibitor for calpain II was Cbz-Leu-D,L-Phe-CONH-(CH(2))(3)-adenin-9-yl (K(i) = 68 nM). On the basis of the crystal structure obtained with heterocyclic peptidyl alpha-ketoamides, we have improved inhibitor potency by introducing a small hydrophobic group on the adenine ring. These inhibitors have good potential to be used in the treatment of neurodegenerative diseases.

Comparison of the internal energy deposition of Venturi-assisted electrospray ionization and a Venturi-assisted array of micromachined ultrasonic electrosprays (AMUSE).

The internal energy deposition of a Venturi-assisted array of micromachined ultrasonic electrosprays (AMUSE), with and without the application of a DC charging potential, is compared with equivalent experiments for Venturi-assisted electrospray ionization (ESI) using the “survival yield” method on a series of para-substituted benzylpyridinium salts. Under conditions previously shown to provide maximum ion yields for standard compounds, the observed mean internal energies were nearly identical (1.93–2.01 eV). Operation of AMUSE without nitrogen flow to sustain the air amplifier focusing effect generated energetically colder ions with mean internal energies that were up to 39% lower than those for ESI. A balance between improved ion transfer, adequate desolvation, and favorable ion energetics was achieved by selection of optimum operational ranges for the parameters that most strongly influence the ion population: the air amplifier gas flow rate and API capillary temperature. Examination of the energy landscapes obtained for combinations of these parameters showed that a low internal energy region (≤1.0 eV) was present at nitrogen flow rates between 2 and 4 L min−1 and capillary temperatures up to 250°C using ESI (9% of all parameter combinations tested). Using AMUSE, this region was present at nitrogen flow rates up to 2.5 L min−1 and all capillary temperatures (13% of combinations tested). The signal-to-noise (S/N) ratio of the intact p-methylbenzylpyridinium ion obtained from a 5 µM mixture of thermometer compounds using AMUSE at the extremes of the studied temperature range was at least fivefold higher than that of ESI, demonstrating the potential of AMUSE ionization as a soft method for the characterization of labile species by mass spectrometry.