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Ultra-performance liquid chromatography (UPLC) for the determination of organic acids – The intermediates of branched-chain amino acid biosynthesis in Escherichia coli strains
Journal of Liquid Chromatography & Related Technologies ( IF 1.0 ) Pub Date : 2020-10-18 , DOI: 10.1080/10826076.2020.1832894
Elizaveta N. Fedorova 1 , Darya O. Varlamova 1 , Alexandr D. Kivero 1 , Kristina D. Guk 1 , Leonid R. Ptitsyn 1
Affiliation  

Abstract Simple and rapid reverse-phase ultra-performance liquid chromatography with UV-detection (RP-UPLC-UV) was developed and validated for the simultaneous analysis of 2-isopropylmalate (2-IPM), 3-isopropylmalate (3-IPM), ketoisovalerate (KIV), ketoisocaproate (KIC), and α-keto-β-methylvalerate (KMV) in the culture fluid (CF) of branched-chain amino acid (BCAA) Escherichia coli strain producers. The RP-UPLC-UV method was developed using a Waters Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of phosphate monobasic salt adjusted to a pH of 3.5 and methanol solution. The flow rate of the mobile phase was 0.5 mL min−1 with gradient elution. The RP-UPLC-UV method was compared with reverse-phase high-performance liquid chromatography with UV-detection (RP-HPLC-UV), ion-exchange high-performance liquid chromatography with UV-detection (IE-HPLC-UV), and ion-exchange high-performance liquid chromatography with electrochemical detection (IE-HPLC-ECD) and a postcolumn buffer for organic acid analysis. The proposed RP-UPLC-UV method was highly sensitive (LOD for KIV was 2.5 × 10−3 mg mL−1, for 2-IPM – 4.5 × 10−3 mg mL−1, for 3-IPM – 4.4 × 10−3 mg mL−1, for KIC – 3.4 × 10−3 mg mL−1, and for KMV – 2.8 × 10−3 mg mL−1) and successfully applied for the analysis of organic acids in the CF of E. coli strain producers. Graphical abstract

中文翻译:

用于测定有机酸的超高效液相色谱 (UPLC) – 大肠杆菌菌株中支链氨基酸生物合成的中间体

摘要 开发并验证了简单快速的反相超高效紫外检测反相液相色谱 (RP-UPLC-UV) 可同时分析苹果酸 2-异丙酯 (2-IPM)、苹果酸 3-异丙酯 (3-IPM)、支链氨基酸 (BCAA) 大肠杆菌菌株生产者的培养液 (CF) 中的酮异戊酸 (KIV)、酮异己酸 (KIC) 和 α-酮-β-甲基戊酸 (KMV)。RP-UPLC-UV 方法是使用 Waters Acquity BEH C18 色谱柱(100 mm × 2.1 mm,1.7 μm)开发的,流动相由调节至 pH 为 3.5 的磷酸二氢盐和甲醇溶液组成。流动相的流速为 0.5 mL min-1,梯度洗脱。RP-UPLC-UV 方法与带紫外检测的反相高效液相色谱法 (RP-HPLC-UV) 进行了比较,带紫外检测的离子交换高效液相色谱 (IE-HPLC-UV) 和带电化学检测的离子交换高效液相色谱 (IE-HPLC-ECD) 和柱后缓冲液用于有机酸分析。提出的 RP-UPLC-UV 方法高度灵敏(KIV 的 LOD 为 2.5 × 10−3 mg mL−1,2-IPM 的 LOD 为 4.5 × 10−3 mg mL−1,3-IPM 的 LOD 为 4.4 × 10− 3 mg mL−1,对于 KIC – 3.4 × 10−3 mg mL−1,对于 KMV – 2.8 × 10−3 mg mL−1) 并成功应用于分析大肠杆菌菌株 CF 中的有机酸生产者。图形概要 5 × 10−3 mg mL−1,对于 3-IPM – 4.4 × 10−3 mg mL−1,对于 KIC – 3.4 × 10−3 mg mL−1,以及对于 KMV – 2.8 × 10−3 mg mL− 1) 并成功应用于大肠杆菌菌株生产者 CF 中有机酸的分析。图形概要 5 × 10−3 mg mL−1,对于 3-IPM – 4.4 × 10−3 mg mL−1,对于 KIC – 3.4 × 10−3 mg mL−1,以及对于 KMV – 2.8 × 10−3 mg mL− 1) 并成功应用于大肠杆菌菌株生产者 CF 中有机酸的分析。图形概要
更新日期:2020-10-18
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