Abstract
A 2D liquid chromatography (LC) system using hydrophilic interaction chromatography (HILIC) and reversed phase columns has been employed for comprehensive (LC × LC) separation of rat muscle tissue micro-dialysate. Incorporation of an on-line reverse-phase solid phase extraction (SPE) enrichment column in front of the first dimension enabled aqueous samples with high salt concentrations to be injected directly without compromising the chromatographic performance of the HILIC column. Since the SPE enrichment column allowed injection of large sample volumes (e.g. 450 μL), a capillary HILIC column (inner diameter 0.3 mm) could be employed instead of a larger column which is often used in the first dimension to load sufficient amounts of sample. The two chromatographic dimensions were connected using a column selector system with 18, 1.0 mm I.D. C18 “transition” SPE columns. A PLRP C18 column was used in the second dimension. The 2D LC system’s performance was evaluated with a tryptic digest mixture of three model proteins. Good trapping accuracy (HILIC→transition SPE→RP recovery >95%) and repeatability (within-and between day retention time RSDs of first and second dimension chromatography >1%) was achieved. A dialysis sample of rat muscle tissue was separated with the 2D system, revealing complexity and large differences in concentrations of the various compounds present, factors which could potentially interfere with the quantification and monitoring of two target analytes, arg-bradykinin and bradykinin. Subsequently, “Heart-cut” 2D LC-electrospray–mass spectrometry (ESI–MS) with post-column on-line standard injection was employed to monitor arg-bradykinin and bradykinin levels as a function of various muscle conditions. The method’s quantification precision was RSD = 3.4% for bradykinin.
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References
Evans CR, Jorgenson JW (2004) Anal Biochem 378(8):1952–1961
Shellie RA, Haddad PR (2006) Anal Biochem 386(3):405–415
Linke TR, Catharine A, Harrison EH (2006) J Chromatogr A 1123(2):160–169
Dugo P, Kumm T, Chiofalo B, Cotroneo A, Mondello L (2006) J Sep Sci 29(8):1146–1154
Schoenmakers PJ, Vivo-Truyols G, Decrop WMC (2006) J Chromatogr A 1120(1–2):282–290
Saber AL, Wilson SR, Elmosallamy MAF, Lundanes E, Greibrokk T (2007) J Liq Chromatogr Relat Technol 30:393–403
Holm A, Storbraten E, Mihailova A, Karaszewski B, Lundanes E, Greibrokk T (2005) Anal Biochem 382(3):751–759
Chen J, Balgley BM, DeVoe DL, Lee CS (2003) Anal Chem 75(13):3145–3152
Hemstrom P, Irgum K (2006) J Sep Sci 29:1784–1821
Gilar M, Olivova P, Daly AE, Gebler JC (2005) Anal Chem 77(19):6426–6434
Boix F, Roe C, Rosenborg L, Knardahl S (2004) J Appl Physiol 98:534–540
Ray WS, Boix F, Holm A, Molander P, Lundanes E, Greibrokk T (2005) J Sep Sci 28(14):1751–1758
Acknowledgements
G. Vivo-Truyols is supported by EU Project MEIF-CT-2005-024198 (“CHEVAR”, Marie Curie Intra-European Fellowships).
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Wilson, S.R., Jankowski, M., Pepaj, M. et al. 2D LC Separation and Determination of Bradykinin in Rat Muscle Tissue Dialysate with On-Line SPE-HILIC-SPE-RP-MS. Chroma 66, 469–474 (2007). https://doi.org/10.1365/s10337-007-0341-4
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DOI: https://doi.org/10.1365/s10337-007-0341-4