Determination of amino acids in fodders and raw materials using capillary electrophoresis WWW.LUMEX.RU

Methods for analysis of AA contents in fodders Ion-exchange chromatography (in the amino acids analyzer) RP-HPLC with gradient elution Calculation method ________________________________________________________________ CE - only for free form of AAs (juice, beer) WWW.LUMEX.RU

2 schemes of AAs analysis in fodders by CE Complete analysis of proteinogenous amino acids (20 AAs) Express analysis of technologically important amino acids – Lys, Met, Thr, Cys-Cys, Trp. Pre-capillary derivatization Direct detection (without derivatization) WWW.LUMEX.RU

Direct determination of technologically important AAs Detection Optimization of separation, including: on 190 nm (Lys, Met, Thr, Cys-Cys) acid hydrolysis on 219 nm (Trp) alkaline hydrolysis pH and composition of BGE additives to the BGE (organic solvents, surfactants, macrocycles) WWW.LUMEX.RU

CZE separation of the 19 underivatized amino acids Buffer: 10 mM borate with the addition 10 mM -CD (pH 9.18) Voltage: +20 kV; Detection:190 nm. WWW.LUMEX.RU

Assay characteristics for technologically important amino acids Parameters LOQ LOD mg l-1 Range b r % R.S.D. % (n=9) Lys 40 – 200 16.30 0.9982 40 0.40 9 20 Met 10 – 100 2.95 0.9998 10 0.10 4 5 Thr 20 – 100 6.65 0.9997 0.20 Cys-Cys 8.81 0.9988 7 Trp 0.1 – 50 1.45 0.1 0.02 0.05 WWW.LUMEX.RU

Percentage of the amino acids in samples, (%SD) The data of the underivatized amino acids determination in the real samples using HPLC, CZE and IEC Percentage of the amino acids in samples, (%SD) CZE RP-HPLC IEC (arbitral method) Fish flour Lys 0.85  0.11 0.94  0.16 0.84  0.09 Met 1.42  0.17 1.65  0.20 1.35  0.12 Thr 1.05  0.13 1.10  0.16 1.02  0.11 Cys-Cys 0.56  0.08 0.65  0.13 0.69  0.12 Mill cake 1.15  0.16 1.18  0.19 1.09  0.14 0.75  0.10 0.69  0.10 0.69  0.09 0.72  0.12 0.80  0.14 0.75  0.16 0.24  0.04 0.36  0.07 0.23  0.04 Mixed fodder 1.52  0.18 1.49  0.13 1.27  0.15 0.45  0.06 0.53  0.06 0.52  0.08 1.11  0.14 0.98  0.15 1.00  0.15 0.33  0.04 0.29  0.04 0.41  0.05 WWW.LUMEX.RU

Direct determination of Trp Alkaline hydrolysis Temperature 40°C Detection on 219 nm: increase of detection sensitivity decrease of interference of concomitant AAs WWW.LUMEX.RU

The complete analysis of proteinogenous amino acids Only for derivative forms PITC advantages: simplicity of use effective separation of the derivatives sufficient reproducibility of the reaction condition good solubility of the derivatives in water WWW.LUMEX.RU

Derivatization of AAs with PITC CH NH 2 COOH AAs pH 10.5 NH C S CH R COOH PTC - AAs N C S PITC + O PTH - AAs R S NH N WWW.LUMEX.RU

CE analysis of PTC-AAs Voltage: 25 kV; BGE: phosphate with ß-CD Temperature: 20°C No pressure was applied WWW.LUMEX.RU

CE analysis of PTC-AAs Controlled pressure during analysis Highly precise changeable temperature of a capillary cooling Voltage: 25 kV; BGE: phosphate with ß-CD Temperature: 30°C Pressure 30 mbar was applied after 17 min WWW.LUMEX.RU

b-CD as a component of BGE Chiral activity for Trp, Tyr, Phe ! Protein molecules consist of L-AAs, but mixed fodders’ preparation is followed by insertion of synthetic AAs. Technology nonobservance – use racemates or D-AAs. Fodders quality control and falsifications reveal. WWW.LUMEX.RU