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Volume 8, Issue 5 (Suppl)
J Chromatogr Sep Tech, an open access journal
ISSN: 2157-7064
Chromatography 2017
August 07-09, 2017
August 07-09, 2017 | Rome, Italy
4
th
World Congress on
Chromatography
High performance and ultra-high performance liquid chromatography for determination of organic
acids - intermediates of branched-chain amino acids biosynthesis in
Escherichia coli
strains
Elizaveta Fedorova, D O Varlamova, A D Kivero, K D Guk
and
L R Ptitsyn
Ajinomoto-Genetika Research Institute, Moscow, Russia
R
ecent advances in the development of branched-chain amino acids (BCAA) production strains is mainly based on using of
system metabolic engineering strategies. Such approach needs thorough understanding of BCAA biosynthetic pathways
and its regulations. In particular, the qualitative and quantitative evaluation of organic acids (OA), which are intermediates
of BCAA biosynthesis, may help to clarify the key points of this process. Currently, different analytical methods are used for
OA determination, including gas chromatography (GC), HPLC and mass spectrometry (MS). From other hand, the UHPLC
approach is able to provide the analysis of numeral culture fluid (CF) samples with high sensitivity and high speed. The
assessment of possibility to apply UHPLC and HPLC approaches for OA determination in CF of
E. coli
strains is the goal of
this work. The following OA were analyzed: ketoisovalerate (KIV), 2-isopropylmalate (2-IPM), 3-isopropylmalate (3-IPM),
ketoisocaproate (KIC), α-keto-β-methylvalerate (KMV), ketoisobutyrate (KIB), ketoglutarate (KG). For each method, the
capacity factor (K׳), number of theoretical plates (N), asymmetry of the peak (Fasy), relative standard deviation by area
(RSDPA), relative standard deviation of migration retention time (RSDRT) were calculated. It was shown that method UHPLC
has lower qualitative detection limit, lower Fasy, RSDPA, and RSDRT. The time for analysis of one sample was much lower
in case of UHPLC (5 min) in comparison with those for HPLC (10 min). Quantitative analysis of OA was performed in CF
samples of
E. coli
strains. Two intermediates: KIV (1.53 mg/l from UHPLC and 1.87 mg/l from HPLC) and 2-IPM (2.07
mg/l from UHPLC and 2.37 mg/l from HPLC) were detected in analyzed samples. It was shown that the method UHPLC for
determination of OA in CF of
E. coli
strains has a number of advantages, higher sensitivity and less analysis time, compared
with HPLC.
Biography
Elizaveta Fedorova completed her Graduation from Russian State University named after A. N. Kosygin in 2006. She has been employed as an Analytical Chemist/
Engineer at Ajinomoto-Genetika Research Institute in 2007 and as Junior Research Associate since 2013. Her research interests include the development of
UHPLC methods for analysis of organic acids in cultural fluids. She is the author of original research papers published in international journals.
lorensdar@yandex.ruElizaveta Fedorova et al., J Chromatogr Sep Tech 2017, 8:5(Suppl)
DOI: 10.4172/2157-7064-C1-032