A comparison of the phenolic compound fingerprints derived from selected sage (Salvia) species with use of thin-layer chromatography directly and indirectly coupled with mass spectrometry   M. Sajewicz1, D. Staszek1, M. Natić1,2, M. Waksmundzka-Hajnos3, T. Kowalska1 1Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland 2Faculty of Chemistry, University of Belgrade, Studentski Trg 12-16, 11000 Belgrade, Serbia 3Department of Inorganic Chemistry, Medical University of Lublin, 6 Staszica Street, 20-081 Lublin, Poland I. Introduction  In our earlier studies [1-4], we presented the results of investigations focused on a comparison of phenolic compounds and essential oils originating from twenty different sage (Salvia) species by means of fingerprints obtained by TLC/densitometry, HPLC/DAD, and HPLC/ELSD. In paper [4], a comparative analysis of chromatographic fingerprints recorded for the different sage (Salvia) species with use of chemometrics was also given. In this study, we compare fingerprinting performance of the TLC-MS system earlier described in paper [5] with the performance of the new TLC-LC-MS separation combined with fingerprinting. With use of this new approach, the preliminary TLC fractionation of phenolic acids and flavonoids derived from the several sage species is followed by the high-performance liquid chromatographic separation and fingerprinting of individual fractions with use of the LC-MS system. In that way, multidimensional fingerprints are obtained, with an enhanced content of analytical information. Abundant chromatographic evidence is provided and relevant conclusions are drawn. II. Extraction Ekstrakty przygotowano z 1 g średnio sproszkowanego surowca. Do materiału roślinnego dodano 20 mL acetonu, 2 mL HCl (281 g/L), 1 mL metenaminy (5 g/L) i utrzymywano 30 min we wrzeniu na łaźni wodnej pod chłodnicą zwrotną. Hydrolizat przesączono do kolby miarowej (100 mL). Odsączony materiał roślinny ponownie ekstrahowano 20 mL acetonu i utrzymywano 10 min we wrzeniu. Ekstrakcję powtórzono jeszcze raz. Wyciągi sączono do tej samej kolby miarowej i uzupełniono acetonem do 100 mL. Następnie odmierzono 20 mL roztworu do rozdzielacza, dodano 20 mL wody i ekstrahowano octanem etylu, porcjami 15 mL i trzy razy po 10 mL. Połączone warstwy organiczne przemywano dwukrotnie po 40 mL wody. Warstwę organiczną przesączono do kolby miarowej o pojemności 50 mL i uzupełniono octanem etylu. III. Thin Layer Chromatography As stationary phase, silica gel was used (the commercial Si 60 F254 coated glassplates 10 cm x 20 cm in size, Merck, Darmstadt, Germany; cat. #1.05729). As mobile phase, a mixture of ethyl acetate + toluene + formic acid, 70:30:1 (v/v). The 20-μL aliquots of the sage extracts obtained from the dried plant material were spotted to the plates. Chromatograms were developed in the horizontal DS model chromatographic chambers (Chromdes, Lublin, Poland). The obtained chromatograms were scanned with use of scanning densitometer CD 60 (Desaga, Heidelberg, Germany). IV. Mass Spectrometric Analysis of Chromatographic Bands The developed chromatograms were densitometrically scanned in order to localize the separated chromatographic bands. Then the bands were selected for mass spectrometric analysis. To this effect, the TLC-MS Interface appliance (Camag, Muttenz, Switzerland) was used, which enabled direct elution of a given band from the chromatographic plate and its on-line introduction to mass spectrometer. In our study, elution was carried out at ambient temperature with use of methanol (its flow rate equal to 0.2 mL min-1). The eluate was introduced to the Varian 500-MS model mass spectrometer (Varian, Harbor City, CA, USA) and the samples were analyzed in the ESI mode (full ESI-MS scan, positive ionization, spray chamber temperature 45oC, drying gas temperature 150oC, drying gas pressure 25 psi, capillary voltage 70 V, needle voltage 5 kV). Varian MS Workstation v. 6.9.1 software was used for data acquisition and processing. Fig.1. Densytogram V. Conclusions In this study, we compare two interesting fingerprinting possibilities, both involving thin-layer chromatography (TLC) and mass spectrometry (MS). These two strategies are applied to one and the same natural mixture of botanical origin (flavonoids extracted from the sage species). Possibility 1 is a combination of the TLC group separation with the MS group fingerprinting of the separated fractions (TLC-MS). Possibility 2 is a combination of the TLC group separation, direct transport of the separated fractions to the LC column, LC separation of the TLC fractions to individual chemical species, and finally MS fingerprinting of the individual separated species (TLC-LC-MS). More abundant fingerprinting data are derived from the TLC-LC-MS system than from the TLC-MS one. Multidimensional TLC-LC-MS fingerprints seem a very welcome option for further chemometric processing, meant for plant identification, chemotaxonomy, etc. VI. References [1] Grygierczyk G., Sajewicz M., Staszek D., Wojtal Ł., Waksmundzka-Hajnos M., and Kowalska T. J. Liq. Chromatogr. Relat. Technol. 2009, 32, 1223-1240. [2] Cieśla Ł., Hajnos M., Staszek D., Wojtal Ł., Kowalska T., and Waksmundzka-Hajnos M. J. Chromatogr. Sci. 2010 (in press) [3] Sajewicz M., Hajnos M., Staszek D., Wojtal Ł., Waksmundzka-Hajnos M., and Kowalska T. J AOAC Int. 2010 (in press) [4] Daszykowski M., Sajewicz M., Rzepa J., Hajnos M., Staszek D., Wojtal Ł., Kowalska T., Waksmundzka-Hajnos M., and Walczak B. Acta Chromatogr. 2009, 21, 513-530. [5] Sajewicz M., Wojtal Ł., Hajnos M., Waksmundzka-Hajnos M., and Kowalska T.: J. Planar Chromatogr. – Modern TLC 2010 (in press)