Screening of Medicinal Compounds in Blood by Atmospheric Pressure Laser Plasma Ionization Mass Spectrometry

—We proposed a procedure for screening drug compounds by high-resolution mass spectrometry with atmospheric pressure laser plasma ionization (APLPI). The analytical performance of the method was studied on examples of five medicinal compounds: Lidocaine, Bendazole, Papaverine, Chloropyramine, and Carbamazepine. Calibration dependences were obtained in the analysis of model solutions and blood samples; sensitivity coefficients were determined. The relative standard deviation of the results is in the range 20– 27%. The APLPI technique can detect promptly and reliably the studied medicinal compounds at concentrations 0.5–6 ng/mL in a sample. The sensitivity of APLPI exceeds that of atmospheric pressure electrospray ionization by 4–16 times.

In the mid-2000s, new methods of the ionization of organic compounds under atmospheric pressure entered the practice of mass spectrometry and since then have been intensively developed. Many methods have been developed, which are commonly referred to as "ambient ionization mass spectrometry" [1][2][3][4]. Their distinguishing feature is the ability of analyzing solid, liquid, and gaseous samples of complex composition in natural (laboratory or field) conditions without or with minimal sample preparation. During the analysis, the sample is outside the mass analyzer; ions of the compounds to be determined are generated in front of the device for introducing ions into the mass spectrometer, and the mass spectra are recorded in real time. The ionization of analytes is a sequence of ion-molecular reactions leading to the formation of analyte ions. The method of generation of primary reagents (ions or excited atoms and molecules) is one of the key factors determining the analytical characteristics of ionization methods in atmospheric air.
We demonstrated earlier [5] that laser-induced plasma, formed by the action of nanosecond and subnanosecond laser pulses on the surface of a metal target, can serve as an effective tool for creating a high concentration of primary reagents in the air. An estimate of the temperature of the plasma generated by a Nd:YAG laser (wavelength 1.06 μm, pulse duration 0.5 ns, pulse energy 0.25 mJ) in air showed that the plasma temperature exceeds 5.5 × 10 4 K in the first few nanoseconds [6]. High initial temperature, hard UV radiation of the plasma, and high electron density (up to 10 20 cm -3 ) [7,8] create conditions for the efficient generation of primary ions and excited molecules, which then provide "soft" ionization of a wide range of both polar and nonpolar organic compounds [5,6,9,10]. Depending on the chemical properties of the analytes, mass spectra of positive ions show protonated molecules, adducts with cations (usually with and H 3 O + ), and molecular ions (radical cations). In the negative ion detection mode, deprotonated molecules, adducts with anions, and molecular ions (radical anions) are observed [11]. The method based on the use of laser-induced plasma as an ion source for ambient mass spectrometry was called "atmosphericpressure laser plasma ionization" (APLPI) [12]. Such distinctive features of APLPI as versatility, simplicity, rapidity of analysis, and compatibility with serial mass analyzers equipped with an atmospheric-pressure ion source determine the high potential of the method for solving a wide range of analytical problems.
This article studies the analytical performance of APLPI in combination with high-resolution mass 4 NH + ARTICLES spectrometry and assesses a possibility of using the method for screening medicinal compounds in blood. The conventional approach to the mass spectrometric determination of physiologically active substances in biological fluids is based on the use of chromatography-mass spectrometry methods, ensuring high sensitivity and selectivity of the analysis. The main limitation of these methods is the time-consuming and laborious sample preparation procedure, which includes several successive stages (for example, hydrolysis, extraction, concentration, derivatization, and chromatographic separation) [13,14]. Conventional methods of analysis are increasingly being preceded by analytical screening methods capable, in the shortest possible time, answering the question whether a subsequent full-fledged quantitative analysis of the sample is necessary. The following requirements for screening methods can be distinguished in the case of medicinal compounds: -minimal sample preparation; -high productivity of analysis in comparison with the confirming method; -ease of interpretation of the results, because often the result must be obtained immediately after an analysis; -sensitivity of the determination, comparable to the confirming method; -possibility of detecting numerous components simultaneously in one sample.
This article presents the results of a study of the compliance of APLPI with these requirements using examples of the determination of medicinal compounds of Papaverine, Chloropyramine, Dibasol, Carbamazepine, and Lidocaine. EXPERIMENTAL Equipment. We used an Exactive mass spectrometer (Thermo Scientific, Germany) equipped with an orbital ion trap "Orbitrap". In experiments with laserinduced plasma ionization, the standard ion source with electrospray ionization was replaced by the in-lab developed APLPI source. The construction of the APLPI ion source is schematically shown in Fig. 1. Radiation of pulsed laser 1 was focused using tunable mirror 2 and lens 3 with a focal length of 50 mm on the surface of metal target 4 placed near the inlet of the atmospheric interface of mass spectrometer 5. Focusing radiation yields laser plasma near the target surface. Plasma was generated by a diode-pumped solidstate Nd:YAG laser RL-03/355 (ELS-94, Russia) with a wavelength of 1.06 μm, pulse duration of 0.5 ns, pulse energy of 400 μJ, and a pulse repetition rate of 100 Hz. Mirror 2 and lens 3 are mounted in holders that ensure their movement in such a way that it is possible to change the position of the focal spot on the target relative to the inlet of the mass spectrometer. The distance between the focal spot and the inlet in the experiments was approximately 5 mm. Target 4 is a stainless steel disk with a diameter of 12 mm; it is fixed on the shaft of an electric motor 6, which ensures its rotation at a frequency of about 1 Hz. Rotation minimizes the effect of target material erosion on the plasma formation.
The thermal desorption method was used to introduce an analyte into the ionization zone. Test liquid samples (2-10 μL) were placed in a stainless steel cell 7. After the evaporation of the solvent, the cell was placed at a distance of approximately 1 cm from the inlet of mass spectrometer 5. The position of the evaporation cell significantly affects the magnitude of the ion signal; therefore, at the preliminary stage of the study, we found the optimal geometric parameters at which the recorded ion current of the analytes took maximum values. To heat the evaporating cell rapidly, induction heater 8 was used, which can heat the cell to 270-280°C in about 20 s. The evaporating cell is a stainless steel tube through which a flow of a carrier gas can be fed. A Genius NM32LA nitrogen station (Peak Scientific, United Kingdom) was the carrier gas source. To saturate with water vapor, nitrogen was passed through a Drexler flask with bidistilled water.
Solutions were analyzed by electrospray using a standard HESI-II ion source. The following ionization conditions were used in the experiments: sputtering voltage, 3.8 kV; capillary temperature, 300°C; capillary voltage, 50.0 V; analyte flow rate, 10 μL/min; nitrogen pressure, 70 kPa. Formic acid at a concentration of 0.1% was added to the test samples.
All studies were carried out in the positive ion registration mode. Mass spectra were recorded in the m/z range 140-1000. The resolution of the mass analyzer in the experiments was 6 × 10 4 (for the peak at m/z 195). Before each series of experiments, the instrument was calibrated using four external standards in the mass range of 80-380 amu, which ensured the accuracy of m/z determination in this range at a level of 1 ppm. The ion injection time into the C-trap was set to 100 ms.

Reagents.
We used commercial medicinal preparations: Lidocaine (Organika, Russia), Suprastin (Egis, Hungary), Dibazol (Biosintez, Russia), Papaverine (injection solutions, Moskhimfarm Preparaty, Russia), and Carbamazepine (Finlepsin Retard, Teva, Poland) isolated from solid tablets. Solutions for injection were dissolved in a methanol-water mixture (1 : 1) in the required proportion. The solid tablets were preliminarily crushed, placed in distilled water, alkalized with sodium hydroxide to pH 14, and subjected to threefold extraction with diethyl ether. After phase separation, the upper ether layer with extracted Carbamazepine was separated, and the solvent was evaporated. The dry residue was redissolved in a methanol-water mixture (1 : 1), and a working solution was prepared.
The studied medicinal compounds are widely used in medical practice and can serve as good indicators of the applicability of APLPI to screening medicinal compounds.
Methanol (Merck, Germany), chloroform, diethyl ether, acetonitrile (Baum-Lyuks, Russia), formic acid (Arcos, Belgium), and twice-distilled water were used as solvents. All solvents used in the work were of cp grade or better. Model solutions of medicinal compounds were prepared by dilution in the chloroformacetonitrile (1 : 1) or methanol-water (1 : 1) system. Analytical ammonium chloride and sodium hydroxide (both of analytical grade, Baum-Lyuks, Russia) were also used.
Sample preparation. Venous blood samples (5 mL) were collected aseptically from unanesthetized rabbits from the marginal ear vein using a G21 infusion cannula (Vacuette) prefilled with heparin solution (100 U/kg) into chilled 5-mL hematological sterilized tubes with EDTA (K2/K3 2 mg) for whole blood. After sampling, the samples were immediately frozen in liquid nitrogen and stored at -20°C. All manipulations were performed according to the current requirements of the guidelines and ethical standards for working with laboratory animals.
Before the study, a known amount of an analyte was injected into thawed blood. In parallel, blood samples were analyzed without the additional introduction of the analyte. To isolate preparations from blood, we used the well-known sample preparation technique used in the chromatographic determination of amines and other highly basic compounds, based on the liquid extraction of analytes into the chloroformacetonitrile (1 : 1) system with the addition of 10 vol % of a saturated NH 4 Cl solution [15]. The procedure was as follows: 5 μL of a solution of analytes in a methanol-water mixture was added to 45 μL of a blood plasma sample, and the mixture was stirred on a laboratory shaker for 2 min. After that, 450 μL of the prepared extraction mixture was added to the blood. The samples were stirred in a shaker for 1 min and centrifuged at 15000 rpm for 3 min; the organic fraction was collected. The analytes were preconcentrated under a flow of dry nitrogen until the organic solvent was completely evaporated; the dry residue was dissolved in 50 μL of a methanol-water (1 : 1) mixture.

RESULTS AND DISCUSSION
Analytical performance of APLPI in the analysis of model solutions. The evaluation of reproducibility in the determination of medicinal compounds in laboratory air showed that the relative standard deviation (RSD) of the ion signal of analytes in all experiments exceeded 70% (for five consecutive measurements of the same solution). An obvious reason for the low reproducibility is the uncontrolled effect of interfering compounds present in air on the processes of ionization and the subsequent transport of analyte ions to the detector. The relatively high efficiency and versatility of ionization under the action of laser-induced plasma in air dictate a high background ion signal, which also deteriorates the sensitivity of the analysis. When the Orbitrap mass spectrometer detects various ions, the limiting factor becomes the capacity of the C-trap used to focus and inject a packet of ions into the orbital ion trap [16]. Rapid filling of the C-trap with ions of interfering compounds decreases the accumulation time of analyte ions and, consecutively, the magnitude of its ion signal [17,18]. Matrix effects and, mainly, suppressing the ionization of analyte molecules deteriorate the analytical performance.
In the developed ion source with thermal desorption sampling, analytes are fed into the ionization zone in a nitrogen flow with saturated water vapor (Fig. 1). This solution can significantly decrease the background signal and improve the reproducibility of the analysis results compared to ionization in the laboratory air. We added water vapor to the carrier gas because water molecules play a key role in the formation of ions of organic compounds in APLPI, being one of the main sources of protons [5]. Experiments have shown that the addition of saturated water vapor to pure nitrogen intensified the ion signal of analytes by more than one order of magnitude. The intensity of the ion signal also depended on the flow rate of the carrier gas. The dependence of sensitivity on the nitrogen flow rate showed that, with an increase in flow rate from 0 to 30 mL/min, the ion signal of analytes grew approximately threefold, and in the range of flow rates of 30-100 mL/min, the signal changed insignificantly. Further experiments were performed at a nitrogen flow rate of 30 mL/min.
The main ionization channel for all the compounds studied in this work is protonation with the formation of [M + H] + ions. An example is the mass spectra of Lidocaine and Chloropyramine (the active substance of Suprastin) (Fig. 2). Despite the rather "hard" conditions at the initial stages of its development, atmospheric pressure laser-induced plasma ensures "soft" ionization of organic compounds with a low degree of ion fragmentation. Among the studied compounds, only the mass spectra of Chloropyramine contained fragment ions. The fragmentation of protonated Chloropyramine molecules is caused by the cleavage of the C-N bond with the elimination of the amine C 2 H 6 NH (Fig. 2b). Fragmentation is much lower compared to such methods as electron ionization [19] or SALDI [20], while APLPI and atmospheric pressure electrospray ionization (ESI) give similar mass spectra. Figure 3 shows calibration dependences of the ion current of protonated analyte molecules on their concentration in the model solution. Each point in the plots represents the average value for three consecutive measurements in the analysis of one and the same solution. The volume of the sample introduced into the microcell was 5 μL. The dependences are linear in the investigated range of concentrations. The sensitivity coefficients, defined as the slope of calibration curves, were different for different compounds. In a series of studied medicinal substances, the sensitivity coefficient had a maximum value in the determination of Lidocaine and a minimum value in the determination of Chloropyramine. The relative standard deviation of the ion signal of analytes, calculated by the results of three successive measurements in different series of experiments, lied in the range 20-27%. In screening methods, one of the main analytical characteristics was the cut-off concentration с min (cut-off concentration), that is, the minimum concentration of an analyte in a sample, the probability of obtaining a false negative result for which does not exceed a predetermined value, usually, 0.05 [21]. The data obtained by estimating the cut-off concentration of the studied compounds (Table 1) suggest that the c min values are in the range 0.4-5 ng/mL.

Screening of medicinal compounds in blood samples.
Preliminary studies have shown that the sensitivity and reproducibility of a direct blood test are much lower compared to an analysis of model solutions. Therefore, it is advisable to use the APLPI screening of blood samples without sample preparation only for relatively high concentrations of drugs (over 1 μg/mL). Testing samples with lower concentrations requires a sample preparation procedure. We used a well-known technique based on the extraction of analytes into a chloroform-acetonitrile (1 : 1) system with the addition of 10 vol % of a saturated NH 4 Cl solution.
Extraction causes the dilution of the sample, increasing the minimum determined concentration of the analyte in the blood. Sensitivity can be improved by adding an analyte preconcentration stage with  Table 1 shows data on the assessment of the cut-off concentration in the determination of medicinal compounds in blood after extraction and preconcentration. A comparison with the results of analyses of model solutions showed that the cut-off concentration in the analysis of blood samples was higher, but not more than 1.5 times. An assessment of the reproducibility suggests that the relative standard deviations of blood test results are approximately in the same range as for model solutions. The data obtained indicate that the APLPI technique can detect rapidly studied medicinal compounds in the sample at their concentrations 6 ng/mL and lower.
Currently, gas chromatography-atmospheric pressure electrospray ionization mass spectrometry is one of the main methods of confirmatory quantitative analysis in the determination of drug compounds in biological fluids. We compared the analytical performances of two ionization methods, APLPI and ESI, in analyzing the same samples on the same mass spectrometer. The dependences of the ion signal of Lidocaine on its concentration in blood (Fig. 4), measured in blood extracts (without preconcentration) by the APLPI and ESI, show that for the same amounts of the analyte introduced into the device, the ion signal in APLPI is approximately four times higher. However, the relative standard deviation of the ion signal in ESI did not exceed 5%, which is more than four times lower than in APLPI. For other studied compounds, the sensitivity of APLPI was also higher, from 4 to 16 times. The difference between the ion signals of analytes is the largest in the determination of Bendazole (Fig. 5).
An additional advantage of APLPI over ESI is the small sample volume required for analysis, which can be essential for solving many problems in medicine, pharmaceutics, and other disciplines. The small sample volume also implies a possibility of preconcentrating the analyte at the stage of sample preparation.
We should note that the high resolution of the mass spectrometer is fundamental in the APLPI analysis of samples of complex composition. In the region of mass spectrum near the peak of protonated Bendazole molecules (Fig. 5, inset), eight peaks of individual compounds are recorded in addition to the analyte. The high resolution of the mass spectrometer, combined with the ability of determining the molecular weights of ions with high accuracy, avoids erroneous, false positive results in a blood test.

CONCLUSIONS
The ionization of organic compounds by laserinduced plasma under atmospheric pressure is studied in application to the problem of drug screening. The instrumental implementation of the method is based on its combination with high-resolution mass spectrometry. To introduce the analyte to the ion source, a thermal desorption sample injection in a nitrogen flow saturated with water vapor is developed. Analytical performance in the determination of Lidocaine, Ben-   Concentration, ng/mL Ion signal, arb. units ×10 7 dazole, Papaverine, Chloropyramine, and Carbamazepine in model solutions and blood proved that highresolution mass spectrometry with atmospheric pressure laser plasma ionization is an effective method for screening medicinal compounds. The method is characterized by high analytical throughput, ease of the interpretation of the results, the ability of detecting various components simultaneously in a single sample, and high sensitivity exceeding that of the confirmation method with electrospray ionization.

COMPLIANCE WITH ETHICAL STANDARDS
Conflict of interest. The authors declare that they have no conflicts of interest.
Statement of the welfare of animals. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.