Monitoring metabolite uptake and excretion in the culture medium is a noninvasive technique that is used for the metabolic study of cleaving embryos after in vitro fertilization. Low sample consumption, the versatility of the detection, and optimal sensitivity and selectivity are essential elements for extracellular metabolome analyses, and can be conveniently achieved by combining CE with mass spectrometric detection. This paper reports a method for amino acid determination in a limited volume sample (8 μL) of spent culture media collected after the cultivation of in vitro fertilized embryos. Special attention was focused on the sample preparation procedure. The sample was processed with acetonitrile, which facilitates online sample preconcentration via field-amplified sample stacking, and undesired sample evaporation was significantly reduced by the simultaneous addition of dimethyl sulfoxide. Key parameters that affected electrophoretic separation and mass spectrometric detection were investigated, including the type of buffers and organic solvent, optimization of their concentrations, and finally the settings for their ionization. The separation and quantification of 19 amino acids were achieved using 15% acetic acid as the background electrolyte with a sheath liquid consisting of an equimolar mixture of methanol and water. The applicability of the optimized system was demonstrated by determining the amino acid profile in 40 samples of spent cultivation medium in this pilot study. This developed method also has great potential for amino acid analyses in minute sample volumes of other biological matrices.

Department of Biochemistry Faculty of Science Masaryk University Brno Czech Republic

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Steptoe, P. C., Edwards, R. G., Lancet 1978, 2, 366.

Zaninovic, N., Irani, M., Meseguer, M., Fertil. Steril. 2017, 108, 722-729.

Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, Hum. Reprod. 2011, 26, 1270-1283.

Zheng, W., Zhang, S., Gu, Y., Gong, F., Kong, L., Lu, G., Lin, G., Liang, B., Hu, L., Front. Physiol. 2021, 12, 2073.

Seli, E., Botros, L., Sakkas, D., Burns, D. H., Fertil. Steril. 2008, 90, 2183-2189.

Marhuenda-Egea, F. C., Gonsálvez-Álvarez, R., Martínez-Sabater, E., Lledó, B., Ten, J., Bernabeu, R., Metabolomics 2011, 7, 247-256.

Celá, A., Mádr, A., Ješeta, M., Žáková, J., Crha, I., Glatz, Z., Electrophoresis 2018, 39, 3040-3048.

D'Alessandro, A., Federica, G., Palini, S., Bulletti, C., Zolla, L., Mol. Biosyst. 2012, 8, 953-958.

Norton, M. E., Jacobsson, B., Swamy, G. K., Laurent, L. C., Ranzini, A. C., Brar, H., Tomlinson, M. W., Pereira, L., Spitz, J. L., Hollemon, D., Cuckle, H., Musci, T. J., Wapner, R. J., N. Engl. J. Med. 2015, 372, 1589-1597.

Gardner, D. K., Wale, P. L., Fertil. Steril. 2013, 99, 1062-1072.

Leese, H. J., Baumann, C. G., Brison, D. R., McEvoy, T. G., Sturmey, R. G., Mol. Hum. Reprod. 2008, 14, 667-672.

Gardner, D. K., Wale, P. L., Collins, R., Lane, M., Hum. Reprod. 2011, 26, 1981-1986.

Goto, K., Kumasako, Y., Koike, M., Kanda, A., Kido, K., Nagaki, M., Otsu, E., Kawabe, F., Kai, Y., Utsunomiya, T., Reprod. Med. Biol. 2018, 17, 289-296.

Summers, M. C., Biggers, J. D., Hum. Reprod. Update 2003, 9, 557-582.

Leese, H. J., Biggers, J. D., Mroz, E. A., Lechene, C., Anal. Biochem. 1984, 140, 443-448.

Leese, H. J., Reproduction 2012, 143, 417-427.

Morbeck, D. E., Krisher, R. L., Herrick, J. R., Baumann, N. A., Matern, D., Moyer, T., Fertil. Steril. 2014, 102, 759-766. e9.

Whitten, W., Nature 1956, 177, 96.

Quinn, P., Kerin, J. F., Warnes, G. M., Fertil. Steril. 1985, 44, 493-498.

Gardner, D. K., Lane, M., Calderon, I., Leeton, J., Fertil. Steril. 1996, 65, 349-353.

Block, J., Bonilla, L., Hansen, P. J., Theriogenology 2009, 71, 1063-1071.

Li, J., Li, Y., Zhang, W., Chen, Z., Fan, G., J. Sep. Sci. 2013, 36, 1817-1825.

Wen, J., Zhang, W. T., Cao, W. Q., Li, J., Gao, F. Y., Yang, N., Fan, G. R., Molecules 2014, 19, 4907-4923.

Wuethrich, A., Haddad, P. R., Quirino, J. P., Electrophoresis 2016, 37, 1139-1142.

Zhu, G., Sun, L., Yan, X., Dovichi, N. J., Anal. Chem. 2014, 86, 6331-6336.

Mádr, A., Celá, A., Klejdus, B., Pelcová, M., Crha, I., Žáková, J., Glatz, Z., Electrophoresis 2015, 36, 1244-1250.

Motiei, M., Vaculikova, K., Cela, A., Tvrdonova, K., Khalili, R., Rumpik, D., Rumpikova, T., Glatz, Z., Saha, T., J. Clin. Med. 2020, 9, 4094.

Onjiko, R. M., Portero, E. P., Moody, S. A., Nemes, P., J. Vis. Exp. 2017, 130, 56956.

Zhang, W., Ramautar, R., Electrophoresis 2021, 42, 381-40.

van Mever, M., Hankemeier, T., Ramautar, R., Electrophoresis 2019, 40, 2349-2359.

Desiderio, C., Iavarone, F., Rossetti, D. V., Messana, I., Castagnola, M., J. Sep. Sci. 2010, 33, 2385-2393.

Hirayama, A., Tomita, M., Soga, T., Analyst 2012, 137, 5026-5033.

Drábková, P., Andrlová, L., Hampl, R., Kanďár, R., Physiol. Res. 2016, 65, 823-832.

Houghton, F. D., Hawkhead, J. A., Humpherson, P. G., Hogg, J. E., Balen, A. H., Rutherford, A. J., Leese, H. J., Hum. Reprod. 2002, 17, 999-1005.

Brison, D. R., Houghton, F. D., Falconer, D., Roberts, S. A., Hawkhead, J., Humpherson, P. G., Lieberman, B. A., Leese, H. J., Hum. Reprod. 2004, 19, 2319-2324.

Dawson, K. M., Collins, J. L., Baltz, J. M., Biol. Reprod. 1998, 59, 225-232.