Paper spray mass spectrometry - A potential complementary technique for the detection of polar compounds in sports drug testing.

In this proof-of-concept study, paper spray mass spectrometry was investigated as a high-throughput and fully automated technique for the initial testing of particularly polar compounds that are prohibited in sports. The technique allows the ionization of analytes from complex sample matrices such as blood and urine when spotted onto a paper strip. By minimizing sample preparation and omitting chromatographic separation, paper spray mass spectrometry benefits from considerable cost- and time-savings compared to conventional high performance liquid chromatography/tandem mass spectrometry, especially in cases where conventional reversed-phase liquid chromatography offers limited applicability. All but one of the investigated model compounds fulfilled the World Anti-Doping Agency's (WADA's) requirements regarding the applicable minimum required performance limits for initial testing procedures. In addition, the combination of paper spray mass spectrometry and ion mobility separation results in enhanced selectivity and sensitivity and is a particularly valuable analytical configuration.

a paper strip. By minimizing sample preparation and omitting chromatographic separation, paper spray mass spectrometry benefits from considerable cost-and time-savings compared with conventional high performance liquid chromatography/ tandem mass spectrometry, especially in cases where conventional reversed-phase liquid chromatography offers limited applicability. All but one of the investigated model compounds fulfilled the World Anti-Doping Agency's (WADA's) requirements regarding the applicable minimum required performance limits for initial testing procedures. In addition, the combination of paper spray mass spectrometry and ion mobility separation results in enhanced selectivity and sensitivity and is a particularly valuable analytical configuration.

K E Y W O R D S
human urine, ion mobility, paper spray mass spectrometry, sports drug testing, VeriSpray

| j INTRODUCTION
High performance liquid chromatography coupled to mass spectrometry (HPLC-MS) is used widely in sports drug testing, enabling high-throughput initial testing procedures. 1-6 With the development of increasingly powerful instrumentation, a tendency towards simplified assays has been observed in doping controls, where sample pretreatment has been reduced to a minimum, leading eventually to so called "dilute-and-inject" assays. [7][8][9][10][11] However, extraordinary hydrophilic compounds still represent a challenging task in HPLC-MS due to their divergent chromatographic behavior and, consequently, limited compatibility with commonly employed reversed-phase HPLC-based test methods.
In this pilot study, paper spray mass spectrometry (PS-MS) was investigated as an alternative technique for the initial testing of highly polar compounds that are considered as prohibited in sports. This technique, first described in 2010, 12,13 ionizes analytes directly from complex sample matrices such as blood, dried blood spots, biological tissues, or urine that are spotted onto a paper strip. 14,15 Over the years, PS-MS has been explored further in forensic and toxicological drug screening. [16][17][18] By minimizing sample preparation and omitting chromatographic separation, mass spectrometric results can be obtained in less than 2 minutes.
Experiments were carried out using the Thermo Scientific VeriSpray ion source on a Thermo Scientific TSQ Altis triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA). The paper spray workflow, displayed in Figure 1, typically involves the following steps.
In the first step, a defined volume of the urine sample is spotted onto a single use paper strip in a 24 strip VeriSpray sample plate. After  Prior to the analysis of the aforementioned compounds, paper spray experiments to optimize solvents as well as the applied spray voltage were performed and tuned to achieve the highest ionization efficiency by comparison of signal-to-noise (S/N) ratios between blank and spiked urine specimens. In Table 2, the analytical performance of the assay is summarized for selected polar stimulants. For these compounds, the assay provides the highest sensitivity using pure acetonitrile as the rewet and spray solvent. The addition of water or 0.1% aqueous acetic acid as proton donor negatively affected the S/N ratio. Moreover, to further investigate the sensitivity of the assay for all model compounds, calibration curves were determined in spiked urine specimens, and the limit of quantification (LOQ) was estimated at a S/N ratio of 4, %CV < 15% and ± 20% accuracy.

| j Polar compounds
All of the aforementioned hydrophilic model compounds except for ITPP were successfully determined in human urine using paper spray mass spectrometry at relevant concentrations. The coefficient of determination for linear regression (R 2 ) for each calibration curve was greater than 0.98, indicating good linearity with detection limits that meet the criteria of sports drug testing ( Figure S1). 19 Table 1 provides the results of all tested compounds and specifies the selected analytical parameters, e.g. spray voltage, ionization mode, paper spray solvents, quantifier precursor-product ion transition, and internal standard. from the set of compounds tested due to its chromatographic and mass spectrometric behavior. Routine analytical applications targeting ITPP utilize hydrophilic-interaction liquid chromatography mass spectrometry (HILIC-MS) combined with dilute-and-inject or solid-phase extraction protocols using weak-anion-exchange cartridges. 20 In this study using PS-MS, the negative ionization mode was used to determine ion transitions derived from the 2-fold deprotonated precursor ion. In addition, the Na-adduct of ITPP was investigated. As quantifier precursor/product ion pairs, m/z 301.9 ! 524.9 (ITPP) and m/z 312.9 ! 546.9 (ITPP Na-adduct) were chosen.
To verify the ability of the method effectively to ionize ITPP from the paper strip, the first step was to analyze the compound in pure methanol. Both ITPP itself and its Na-adduct provided an LOQ of According to the manufacturer, bemitil is known to improve both physical performance and resistance to stress. 25  To verify the analytical performance of the assay a calibration curve in the range 0.5 to 250 ng/mL was prepared in urine. The coefficient of determination for linear regression (R 2 ) was greater than 0.99 indicating good linearity with a detection limit of 10 ng/mL with a S/N > 5.

| j Internal standards
The use of appropriate internal standards is of utmost importance in paper spray mass spectrometry, and optimal results are typically achieved when an isotopically labeled internal standard is used as it has the same extraction efficiency from the paper and ionization efficiency as the matching analyte. The importance of choosing an appropriate internal standard was exemplified when studying metformin, a metabolic modulator. As a first attempt, meldonium-D 3 was chosen as the internal standard for metformin because isotopically labeled metformin was not available. However, using meldonium-D 3 as an ISTD resulted in moderate linearity (R 2 = 0.94) and excluding the ISTD resulted in improved results (R 2 > 0.99). Due to the absence of any chromatographic separation, omitting ISTDs is not recommended to allow for compensating unpredictable matrix effects.

| j Ion mobility/paper spray MS
Ion mobility spectrometry offers separation based on a combination of compound-specific factors, such as molecular shape, conformation, charge state, and size of gas phase ions. Although the use of internal standard in paper spray can lead to successful quantitation, the high background from the paper, minimal sample cleanup, and lack of chromatographic separation could limit its detection capability. The combination of ion mobility with paper spray mass spectrometry can help to overcome the above-mentioned limitations and makes it possible to have short turnaround times with increased levels of quantitation.
FAIMS refers to (high) field asymmetric ion mobility spectrometry and is an atmospheric pressure separation technique based on differences in ion mobility by applying alternating low and high electric T A B L E 2 Optimization of the S/N for polar stimulants by adjusting the rewet and spray solvent