Metabolic Profiling of Ultrasmall Sample Volumes with GC/MS: From Microliter to Nanoliter Samples Maud M. Koek,* ,† Floor Bakels, ‡ Willem Engel, § Arn van den Maagdenberg, ‡,| Michel D. Ferrari, ‡ Leon Coulier, † and Thomas Hankemeier § Analytical Science Department, TNO Quality of Life, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands, Department of Neurology, Leiden University Medical Centre, Leiden, The Netherlands, LACDR Analytical Biosciences, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands, and Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands Profiling of metabolites is increasingly used to study the functioning of biological systems. For some studies the volume of available samples is limited to only a few micro- liters or even less, for fluids such as cerebrospinal fluid (CSF) of small animals like mice or the analysis of individual oocytes. Here we present an analytical method using in-liner silylation coupled to gas chromatography/mass spectrom- etry (GC/MS), that is suitable for metabolic profiling in ultrasmall sample volumes of 2 μL down to 10 nL. Method performance was assessed in various biosamples. Deriva- tization efficiencies for sugars, organic acids, and amino acids were satisfactory (105-120%), and repeatabilities were generally better than 15%, except for amino acids that had repeatabilities up to about 35-40%. For endogenous sugars and organic acids in fetal bovine serum, the response was linear for aliquots from 10 nL up to at least 1 μL. The developed GC/MS method was applied for the analysis of different sample matrixes, i.e., fetal bovine serum, mouse CSF, and aliquots of the intracellular content of Xenopus laevis oocytes. To the best of our knowledge, we present here the first comprehensive GC/MS metabolite profiles from mouse CSF and from the intracellular content of a single X. laevis oocyte. The aim in metabolomics research is to measure as many metabolites as possible in body fluids, tissue samples, or even cells, in order to gain insights in the functioning of biological systems. Gas chromatography coupled to mass spectrometry (GC/MS) is a very suitable technique for this purpose, and several GC/MS based metabolomics methods have been reported. 1-7 Most of these methods rely on derivatization with a silylation reagent prior to analysis to convert the polar functional groups that are problematic in GC/MS analysis to less polar groups. Previously, the development and validation of a one-dimensional GC/MS method 8 and a comprehensive two-dimensional gas chro- matography time-of-flight mass spectrometry (GC × GC/TOF-MS) method 9 were described using oximation and subsequently silylation prior to analysis. Both methods allowed the measurement of a broad range of small, medium-polar to polar metabolites. These methods are very suitable for the analysis of cell extracts, body fluids, and tissues when reasonable amounts of sample are available. Usually approximately 100 μL of cell extract or body fluids and 5-10 mg of tissue are needed for sample workup, in order to obtain good and repeatable profiles of the metabolites. In some cases, this amount of sample is not available. Cerebrospinal fluid (CSF) is an optimal body fluid to study pathophysiology of neurological diseases, and mouse models have become available to study human diseases such as Alzheimer’s, 10 Parkinson’s, 11 or migraine, 12 but the amount of CSF is limited to a few microliters. The acquisition of metabolite profiles in mouse CSF sample is potentially very promising for translational studies, and the application of proven methods such as LC/MS and GC/MS is very desirable. An even more demanding drive behind miniaturization of the sample workup is the growing interest to measure differences in metabolite concentrations at the level of individual cells or even in cellular compartments. To acquire metabolite profiles from sample volumes of a few microliters down to nanoliters, or ultimately from a single cell, is an enormous challenge for analytical chemists. For example, a human oocyte has a diameter of about 100 μm with a volume of only approximately 1 μL. Most reports on the analysis and separation of biomolecules from single cells involve the use of capillary electrophoresis or * To whom correspondence should be addressed. E-mail: maud.koek@tno.nl. † TNO Quality of Life. ‡ Department of Neurology, Leiden University Medical Centre. § Leiden University. | Department of Human Genetics, Leiden University Medical Centre. (1) Fiehn, O.; Kopka, J.; Trethewey, R. N.; Willmitzer, L. Anal. Chem. 2000, 72 (15), 3573–3580. (2) Jonsson, N.; Gullberg, J.; Nordstrom, A.; Kusano, M.; Kowalczyk, M.; Sjostrom, M.; Moritz, T. Anal. Chem. 2004, 76 (6), 1738–1745. (3) Pierce, K. M.; Hope, J. L.; Hoggard, J. C.; Synovec, R. E. Talanta 2006, 70 (4), 797–804. (4) Roessner, U.; Wagner, C.; Kopka, J.; Trethewey, R. N.; Willmitzer, L. Plant J. 2000, 23 (1), 131–142. (5) Shellie, R. A.; Fiehn, O.; Welthagen, W.; Zimmermann, R.; Zrostlikovss, J.; Spranger, J.; Ristow, M. J. Chromatogr., A 2005, 1086 (1-2), 83–90. (6) Strelkov, S.; Elstermann, M. v.; Schomburg, D. Biol. Chem. 2004, 385 (9), 853–861. (7) Villas-Bo ˆas, J. M.; Kesson, M.; Smedsgaard, J.; Nielsen, J. Yeast 2005, 22 (14), 1155–1169. (8) Koek, M. M.; Muilwijk, B.; vanderWerf, M. J.; Hankemeier, T. Anal. Chem. 2006, 78 (4), 1272–1281. (9) Koek, M. M.; Muilwijk, B.; van Stee, L. L. P.; Hankemeier, T. J. Chromatogr., A 2008, 1186 (1-2), 420–429. (10) Woodruff-Pak, D. S. J. Alzheimer’s Dis. 2008, 15 (4), 507–521. (11) Harvey, B. K.; Wang, Y.; Hoffer, B. J. Acta Neurochir. Suppl. 2008, 101, 89–92. (12) van de Ven, R. C.; Kaja, S.; Plomp, J. J.; Frants, R. R.; van den Maagdenberg, A. M.; Ferrari, M. D. Arch. Neurol. 2007, 64 (5), 643–646. Anal. Chem. 2010, 82, 156–162 10.1021/ac9015787  2010 American Chemical Society 156 Analytical Chemistry, Vol. 82, No. 1, January 1, 2010 Published on Web 11/30/2009