ISSN 1061-9348, Journal of Analytical Chemistry, 2017, Vol. 72, No. 14, pp. 1446–1450. © Pleiades Publishing, Ltd., 2017. Original Russian Text © M.V. Ovcharov, S.S. Barsegyan, S.A. Kovaleva, L.N. Kulikova, R.S. Borisov, 2017, published in Mass-spektrometriya, 2017, Vol. 14, No. 1, pp. 28–32.
ARTICLES
New Approaches to the Application of DART Mass Spectrometry Coupled with Planar Chromatography for the Analysis of Mixtures of Organic Compounds M. V. Ovcharova, b, S. S. Barsegyana, S. A. Kovalevaa, L. N. Kulikovac, and R. S. Borisovc, d, * aShared
Research and Educational Center, Peoples’ Friendship University of Russia, Moscow, 117198 Russia b JSC Bruker, Moscow, 119017 Russia c Peoples’ Friendship University of Russia, Moscow, 117198 Russia d Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Moscow, 119991 Russia *e-mail:
[email protected] Received February 27, 2017; in final form, March 6, 2017
Abstract⎯A desorption cell for TLC/DART mass spectrometry is developed. An analysis of mixtures of pharmaceutical substances has shown that the proposed approach can improve the sensitivity of the method and achieve the reproducibility of the recorded mass spectra and selected ion chromatograms. Keywords: mass spectrometry, DART, thin-layer chromatography, desorption, ionization DOI: 10.1134/S106193481714009X
INTRODUCTION DART (Direct Analysis in Real Time] mass spectrometry is widely used for the analysis of mixtures of organic compounds [1–4]. The main advantage of this method is its rapidity and no necessity in any sample preparation procedure. However, the method of analysis usually does not include the preliminary separation of components. As a result, the total mass spectrum of a sample is recorded, which significantly complicates its interpretation, reduces the probability of the detection and identification of minor components of complex mixtures. Therefore, it is of considerable interest to develop methods that would ensure the separation of components of the mixtures to be analyzed while preserving the main advantages of the DART technique. It is obvious that thin-layer (planar] chromatography (TLC) the best corresponds to the requirements: the method is sufficiently rapid, it is not strict for sample preparation, and is widely used in analytical chemistry [5, 6]. Currently, the TLC/DART method is used for the analysis of plant raw materials [7] and quality control of pharmaceutical preparations [8]. Initially, in the work of this type, the elution zones of the analytes were cut from plates for planar chromatography and placed in the ionization region of a DART mass spectrometer [9]. In some cases, for the desorption of analytes from the plate surface, a laser placed in an ion source was used [10] or extraction of the compounds under the investigation from TLC plates was carried out, followed by analysis of the solutions obtained [11]. The evolution of the DART tech-
nique was the production of mass spectrometers that ensure the variation of the angle of bombardment of analyzed samples, which offers certain advantages in recording mass spectra of analytes from TLC plates [12], but may be accompanied by the strong ion scattering in the atmosphere and a drop in sensitivity. Of considerable interest is the development of a procedure, which ensures the achievement of the direct bombardment of the plate with metastable helium atoms in a narrow ionization region without intercepting the path of ions to the skimmer’s aperture, which will make possible, on the one hand, the achievement of a better “chromatographic resolution”, and, on the other hand, the attainment of sufficient sensitivity in recording the mass spectra of components with a high signal-to-noise ratio. The present paper deals with the solution of this problem. EXPERIMENTAL For registering DART mass spectra, we used a JEOL JMS-T100LP mass spectrometer (Japan) (resolution at half-height of the peak R = 7000, determination accuracy m/z ±5 ppm) equipped with DART ION SENSE ion detector (USA) (flow of ionization gas 2 L min–1, voltage on the discharging electrode 4000 V). To apply a predetermined amount of a substance to a TLC plate, CAMAG LINOMAT-5 autosprayer (Switzerland) was used. A drive of a Pump-11 HARVARD APPARATUS syringe dispenser (USA) was used as an auto-feeder of the chromatogram. For
1446
NEW APPROACHES TO THE APPLICATION
O
O
O
O
O
1447
O
2-(prop-1-en-2-yl)-2H2-isopropyl-7Hfuro[3,2-g]chromene-7(3 H)-one furo[3,2-g]chromene-7-one Anmarine
Fig. 1. Anmarine, a mixture of two isomers.
the visual control of the quality of separation, an UFS 254/365 OOO “IMID” chromatographic irradiator (Russia) was used. Mixtures of organic compounds were separated on plates for planar chromatography (ZAO Sorbpolymer Sorbfil UF-254 10 × 15 cm on an aluminum substrate (Russia) and Macherey-Nagel DC-Fertigplatten SILGUR-25 UV254 10 × 20 (Germany)); solvents of reagent grade (“Khimmed”, Russia) were used for elution. Pharmaceutical substances of the purity at least 95% or pharmacopoeial purity were used as model compounds. RESULTS AND DISCUSSION At the first stage of the work, a possibility of using the DART technique with no modifications for the analysis of pharmaceutical substance anmarine (Fig. 1), a mixture of two isomers (isopropenyl- and anhydro-isopropylpsoralenes), was tested. To simulate the chromatographic separation of a mixture of compounds, a substance solution was applied to a TLC plate at six points on the same straight line, after which the plate moved to the ionization region of the mass spectrometer. As the recorded mass spectra contained a significant number of ion peaks formed as a result of ionization of the compounds present in the laboratory air, the chromatograms were built by the
Intensity, arb.units
103 20
10
1.0 Time, min
2.0
Fig. 2. Chromatogram by m/z 229 ion obtained from the surface of a TLC plate with an anmarine substance applied to it. JOURNAL OF ANALYTICAL CHEMISTRY
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mass number of the protonated [M + H]+ molecule (m/z 229) characteristic of anmarine. The chromatogram obtained in this way (Fig. 2) contained asymmetric peaks, the envelope of which was unstable, and the area varied considerably from one experiment to another. The experiment demonstrated that in future it reproducible chromatographic results cannot be obtained in this way. To solve this problem, we fabricated a cell, representing a polytetrafluoroethylene (PTFE) tube with a diameter of 6 mm with rectangular apertures, which, on one hand, allowed us to fix the plate for planar chromatography in the course of analysis, and, on the other hand, localized the ionization place on it and prevented the scattering of ions in the atmosphere (Fig. 3). At that apertures a served to move the TLC plate, and apertures b provided the directed movement of the gas flow along the surface of the chromatogram to the skimmer apertures and prevented overloading of the mass spectrometer vacuum system (their total cross section was about 10 mm2). The desorption– ionization of the analyte in such a cell took place in a narrow region of 4 mm. Among the important advantages of this approach are the partial isolation of the system from atmospheric air (pressure in the cell was higher than atmospheric); its constant purge, which reduced chemical memory, characteristic of the DART method; and the insulation of the plate from the metal parts of the device (Fig. 4). As standard means for supplying a TLC plate to the ion source are not suitable for the cell, such a system was made from a programmable syringe drive. The efficiency of the proposed modification was evaluated using an example of a TLC plate with an anmarine substance applied to it in 6 points. The experiments showed that the overall appearance of the chromatographic peaks significantly improved (Fig. 5), their height and area increased 10- to 20-fold, and the reproducibility of the obtained areas increased. Peaks of ions associated with the ionization of the cell material (polytetrafluoroethylene) were not observed. It should be noted that the absence of pronounced “shoulders” on the right side of the chro-
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a
b
Fig. 4. A method of installing the cell and positioning the TLC plate.
Fig. 3. PTFE tubes with rectangular apertures used in the work.
Intensity, arb.units
Intensity, arb.units
103 100
50
(a)
105 2
229 H+ O
1
105
220
230 229
O
240
(b)
H+
4 –
2 1.0
O
O
O
O
2.0 Time, min
220
Fig. 5. Chromatogram by m/z 229 ion obtained from the surface of TLC plate with anmarine substance applied to it, using a desorption cell.
matographic peaks indicates that there was no noticeable condensation of analyte vapors on the cell material and their subsequent ionization d not take place as the plate moved. The next series of experiments was carried out with analytes separated on the plate by TLC, which were isomeric compounds composing the anmarin substance. Note that these isomers have identical DART spectra and cannot be distinguished by this method (Fig. 6). A comparison of the total areas of chromatographic peaks on the obtained chromatograms showed that, despite a considerable variation of their absolute values from one experiment to another, the ratio of these values for the isomers was close within the limits of one experiment (Fig. 7, Table 1). The further verification of the applicability of the proposed approach was carried out using an artificially prepared mixture of six pharmaceutical substances: clopidogrel, anmarine, phenylbutazone, amiodarone, ibuprofen, and norethisterone. All these substances,
230 m/z
240
Fig. 6. DART mass spectra of: (a) isopropenyl- and (b) anhydroisopropyl psoralenes separated on TLC plates.
with the exception of anmarine, are individual compounds. Fig. 8 shows the resulting total ion current chromatogram. A comparison of the ratio of chromatographic peak areas for each of analyte in the chromatogram by the characteristic ion (Table 2) shows that the response factors for these compounds may differ by more than two orders of magnitude, Table 1. Retention times (T1 and T2), peak areas (S1 and S2) of psoralen isomers, and ratios of peak areas (S2/S1) No.
T1 min
T2 min
S1
S2
S2/S1
1
2.03
2.30
410387
993596
2.42
2
2.05
2.31
549237 1506392
2.74
3
2.00
2.26
1242290 3156187
2.54
4
2.02
2.27
1090 977 3164891
2.90
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×103
(a)
16
100
8
50
0
0
0.8
300
0.4
150
Intensity, arb.units.
Intensity, arb.units.
×106
0 10
1449
(b)
0 700
5
350
0
0
0.90
700
0.45
350
0
0 1.0
2.0
1.0
Time, min
2.0 Time, min
Fig. 7. Chromatograms obtained using plates for planar chromatography with separated isopropenyl- and anhydro isopropyl psoralenes: (a) by total ion current, and (b) by ion with m/z 229.
Intensity, arb.units
×106
which obviously does not permit quantitative analysis without calibration and introducing standards into the mixture. Probable reasons for such a difference are, probably, not only differences in the efficiency of analyte ionization, but also in their thermal stability and probability of their desorption from the adsorbent surface.
10
5
0 1.0
CONCLUSIONS
2.0 Time, min
Fig. 8. Total ion current of the mixture of pharmaceutical substances after their separation by thin-layer chromatography. JOURNAL OF ANALYTICAL CHEMISTRY
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The method of “focusing” gas flow with metastable helium atoms on a TLC plate, proposed in this study, ensures a substantial increase in the yield of ions and reproducibility of the obtained DART mass spectra. Chromatograms obtained using such an approach No. 14
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Table 2. Parameters obtained for a mixture of pharmaceuticals Area Main ion in the mass Amount of applied Release time, min of chromatographic Response factor spectrum, Da substance, nmol peak, arb. units
Substance Clopidogrel
321 [M + H]+
1.03
6.0
2108302
35.2
Phenylbutazone 309 [M + H] Anmarine 229 [M + H]+
1.36
4.5
257552
5.5
1.31; 1.46
3.5
2576807
73.4*
1.74
8.0
2255725
28.1
2.49
4.4
876308
20.0
2.49
155.0
450584
+
Norethysteron Amiodarone Ibuprofen
299 [M + H] 546
+
[M-C6H13N]+ +
161 [M-HCO2]
0.29
* For the total area of chromatographic peaks of two isomers.
are suitable for estimating relative concentrations of components in mixtures by characteristic ions in corresponding calibration procedures using an internal standard. ACKNOWLEDGMENTS We are grateful to E. S. Chernetsova for methodological assistance in the initial study of DART mass spectrometry. REFERENCES 1. Ebert, B.L., Galili, N., Tamayo, P., Bosco, J., Mak, R., Pretz, J., Tanguturi, S., Ladd-Acosta, C., Stone, R., Golub, T.R., and Raza, A., PLoS Med., 2008, vol. 5, no. 2, p. e35. 2. Petucci, C., Diffendal, J., Kaufman, D., Mekonnen, B., Terefenko, G., and Musselman, B., Anal. Chem., 2007, vol. 79, no. 13, p. 5064. 3. Rehakova, J., Rehurkova, I., Ruprich, J., Kalvodova, J., and Matulova, D., Chem. Listy, 2008, vol. 102, p. 777.
4. Chernetsova, E.S., Abramovich, R.A., and Revel’skii, I.A., Pharm. Chem. J., 2012, vol. 45, no. 11, p. 598. 5. Kim, H.J., Jee, E.H., Ahn, K.S., Choi, H.S., and Jang, Y.P., Arch. Pharmacal Res., 2010, vol. 33, no. 9, p. 1355. 6. Kim, H.J., Oh, M.S., Hong, J., and Jang, Y.P., Phytochem. Anal., 2011, vol. 22, no. 3, p. 258. 7. Smith, N.J., Domin, M.A., and Scott, L.T., Org. Lett., 2008, vol. 10, no. 16, p. 3493. 8. Wood, J.L. and Steiner, R.R., Drug Test. Anal., 2011, vol. 3, no. 6, p. 345. 9. Morlock, G. and Schwack, W., Anal. Bioanal. Chem., 2006, vol. 385, p. 586. 10. Hea, X.N., Xiea, Z.Q., Gaoa, Y., Hua, W., Guoa, L.B., Jiangc, L., and Lua, Y.F., Spectrochim. Acta, Part B, 2012, vol. 67, p. 64. 11. Howlett, S.E. and Steiner, R.R., J. Forensic Sci., 2011, vol. 56, no. 5, p. 1261. 12. Chernetsova, E.S., Revelsky, A.I., and Morlock, G.E., Rapid Commun. Mass Spectrom., 2011, vol. 25, p. 2275.
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Translated by V. Kudrinskaya
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