Triboelectric nanogenerator (TENG) mass spectrometry of falsified antimalarials

Rationale An epidemic of low‐quality medicines continues to endanger patients worldwide. Detection of such ‘medicines’ requires low cost, ambient ionization sources coupled to fieldable mass spectrometers for optimum sensitivity and specificity. With the use of triboelectric nanogenerators (TENGs), the charge required to produce gas‐phase ions for mass analysis can be obtained without the need for high‐voltage electrical circuitry, simplifying and lowering the cost of next‐generation mass spectrometry instruments. Methods A sliding freestanding (SF) TENG was coupled to a toothpick electrospray setup for the purposes of testing if falsified medicines could be fingerprinted by this approach. Extracts from both genuine and falsified medicines were deposited on the toothpick and the SF TENG actuated to generate electrical charges, resulting in gas‐phase ions for both active pharmaceutical ingredients and excipients. Results Our previous work had shown that direct analysis in real time (DART) ambient mass spectrometry can identify the components of multiple classes of falsified antimalarial medicines. Experiments performed in this study show that a simple extraction into methanol along with the use of a SF TENG‐powered toothpick electrospray can provide similar detection capabilities, but with much simpler and rugged instrumentation, and without the need for compressed gases or high‐voltage ion source power supplies. Conclusions TENG toothpick MS allows for rapid analyte ion detection in a safe and low‐cost manner, providing robust sampling and ionization capabilities.

chains). 3 The term 'counterfeit' , very commonly used to describe PQM, is not adequate to describe falsified medicines and is only preferred when describing trademark infringement. 4 PQM have been found in both low-and middle-income countries (L/MICs), as well as in wealthier countries with well-functioning medicine regulatory agencies. 5 Despite ongoing efforts and interventions worldwide, falsification of medicines remains a lucrative and prevalent activity. 6 Pharmaceutical forensics can significantly gain from advances in portable analytical instrumentation for field use: detection of PQM at point of sale, clinics, or pharmacies could effectively prevent patients being exposed to these 'products'. Chemical analysis techniques typically used for PQM field detection include Raman spectroscopy, NIR spectroscopy, thin-layer chromatography, colorimetry, dissolution assays, paper analytical devices, and others. 7 However, field implementation of these techniques usually forces a compromise between cost and performance, necessarily reducing sensitivity and specificity. For this reason, field detection and forensic characterization of PQM typically rely on the use of simpler field techniques for screening purposes, in combination with tiered laboratory approaches. 8 Mass spectrometry (MS), once a complex technique available only in centralized facilities, has progressively shown better performance in field applications through portable, many times handheld, mass spectrometers, 9 achieving the right balance between resolution, sensitivity, power consumption and cost. The performance of such mass spectrometers is now sufficient to meet the challenges posed by pharmaceutical forensics, which include the detection of falsified medicines in pass/fail scenarios, the identification of 'wrong active ingredients' present in fakes, and semi-quantitation of the correct active pharmaceutical ingredients (APIs) to distinguish high-quality samples from degraded and substandards, the latter sometimes being more prevalent than falsified medicines. [10][11][12] In order to take advantage of advances in portable mass spectrometers, a necessary condition is the ability to sample and ionize neutral analytes with minimal sample preparation 13 as well as simple high-voltage supply apparatus. Here, we present the first application of the recently developed TENG MS ion source 14 to pharmaceutical forensics using a dry wooden toothpick to sustain electrospray ionization (ESI). 15 Samples of both genuine and falsified antimalarial tablets were subjected to a quick extraction in methanol, and a small volume of the extract was deposited on the toothpick tip.
Manual (human-powered) actuation of a SF TENG connected to the toothpick readily led to mass spectra that provided reproducible, qualitative information that was comparable with that obtained from conventional and resource-intensive ionization approaches, such as direct analysis in real time (DART). 16 2 | EXPERIMENTAL 2.1 | Materials, supplies, and sample preparation World Health Organization (WHO) pre-qualified artemether-lumefantrine tablets (10 mg/tablet co-formulated with 120 mg/tablet, respectively) were obtained directly from a reputable provider and were used as genuine comparators. ACT samples collected in parts of Sub-Saharan Africa during active law-enforcement operations were used to test the TENG-MS approach. These samples were suspected of being falsified through initial Raman spectroscopy screening. Follow-up fingerprinting studies on these samples through DART-MS experiments confirmed the absence of the expected APIs, and in some cases the presence of wrong APIs. 16 Solvents used for tablet extraction included Nanopure water Glucose, mannitol, chloramphenicol, and ciprofloxacin standards were purchased from Sigma-Aldrich and used to confirm chemical assignments for wrong active ingredients found in falsified antimalarials.

| TENG wooden-tip MS analysis of antimalarial tablets
Dry wooden toothpicks supplied from Haioreum (South Korea) were initially 8 cm in length, 3 mm in diameter, and cut to 4 cm to enable placement onto a clip connected directly to the SF TENG power source.
The toothpick was not altered in any other way. Figure 1  on an acrylic support. The movable SF TENG part was made of Cu foil (55 × 65 mm 2 ), also mounted onto an acrylic board. To operate the TENG, the movable part was placed on top of the static portion, which was fixed onto a stage placed underneath the ion source. Both parts were oriented so that the two metal layers were separated by the FEP layer ( Figure 1). The movable part was slid between the two positions corresponding to the two Cu-coated regions of the static part, with a travel distance of 60 mm.

| DART analysis of antimalarial tablets
Analysis of the solid ACT tablets was performed with a commercial DART-SVP ion source (IonSense Inc., Saugus, MA, USA), directly sampling powder from crushed antimalarial tablets, as previously described. 16 Ultra-grade 99.999% pure helium gas (Airgas, Atlanta, GA, USA) flowing at 2.2 L min −1 was used to generate the metastable helium plasma. A grid electrode voltage of +250 V was used, with a gas temperature of 500°C. A gas-ion separator tube (GIST) provided additional pumping of the heated helium gas preventing it from overloading the vacuum system of the mass spectrometer. The DART source was placed 1 cm away from the GIST opening. Tablet powder was introduced between the GIST and the DART nozzle using a borosilicate glass capillary, and held in place for 5 s to generate DART-MS spectra.   Figures 2B and 2C). Experiments were also carried out with a DART ion source in positive ion mode using exactly the same mass spectrometer for comparison purposes (Figure 2A). DART ionization  It was also observed that with a single sliding movement of the TENG top electrode, the overall ion abundance was comparable with that seen by sampling~1 mg of crushed solid tablet by DART for~5 s. The higher abundance of lumefantrine observed by TENG-MS was also observed in our previous experiments using ESI. 16 The opposite was true for artemether, and this is probably because this compound has no easily protonated basic site unlike the tertiary amine on lumefantrine. An observed advantage of TENG was the ability to form sodiated and potassiated artemether adduct ions without the excessive fragmentation seen in DART, while also avoiding the need for compressed gases.  Finally, the negative ion mode mass spectrum for a type #3 falsified ACT ( Figure 3I) revealed a major set of species related to chloramphenicol, indicating that, unlike type #2 falsified samples that were richer in sugars, this type of sample contained higher concentration of this wrong active ingredient. Clearly, TENG wooden-tip MS enabled fingerprinting this type of poor quality pharmaceutical successfully, providing information to source its origins based on chemical composition, and providing a tool for finding Despite using manual TENG actuation, the spectra collected were highly reproducible, as shown in Figure S3 (supporting information) using a chloramphenicol-containing tablet. This reproducibility makes qualitative comparisons simpler without the need for complex equipment that would void the simplicity of the TENG ion source.

| CONCLUSIONS
The triboelectric nanogenerator wooden-tip MS method offer a simple, affordable way to generate ions, producing information that is comparable with, or even richer than, that produced with ion sources such as DART. Even if manually actuated, this new type of triboelectric ion source yields satisfactory results in terms of producing chemical fingerprints of falsified medicines lacking the expected active ingredient, with the additional advantage of allowing identification of any 'wrong' active ingredients that might be present.
It is expected that, when coupled to fieldable or miniature mass spectrometers, triboelectric ion sources will lead to a new generation of less costly instrument platforms that can be used for routine medicine quality monitoring in the field and the clinic. Although positive and negative ion mode were tested independently for TENG wooden-tip MS in the experiments described here, the ability of triboelectric sliding freestanding devices to generate charges of opposite polarity in any given actuation cycle could be further leveraged to improve analytical performance. Along these lines, experiments with TENG and rapid MS polarity switching modes should further increase sample throughput, yielding information about both active ingredients and excipients in a single experiment.