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Determination of amino acids in Determination of amino acids in fodders and raw materials using fodders and raw materials using

capillary electrophoresiscapillary electrophoresis

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Methods for analysis of AA contents Methods for analysis of AA contents in fodderin fodderss

Ion-exchange chromatography (in the amino acids analyzer)

RP-HPLC with gradient elution Calculation method________________________________________________________________

CE - only for free form of AAs (juice, beer)

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2 schemes of AAs analysis in 2 schemes of AAs analysis in foddersfodders by CE by CE

Express analysis of technologically important amino acids – Lys, Met, Thr, Cys-Cys, Trp.

Complete analysis of proteinogenous amino acids (20 AAs)

Direct detection (without derivatization)

Pre-capillary derivatization

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Direct determination of technologically Direct determination of technologically important AAs 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)

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CZE separation of the 19 underivatized CZE separation of the 19 underivatized amino acidsamino acids

4 5 6 7 8 9 10 11 12 13 min

11 mAU

L

ys

Arg

Pro

Ala

Val

Gly

+L

eu+

Ile

Ph

e+H

is T

yr

Met

Gln Ser

T

hr

Asn

Cys-

Cys

Glu

Asp

a

20С

4 5 6 7 8 9 10 11 12 13 min

22 mAU

Ly

s A

rg

Pro

Ala

Va

l Gly

+L

eu+

Ile

Ty

r+P

he+

His

Met

Gln Ser

T

hr

Asn

Cy

s-C

ys

Glu

Asp

b

40С

Buffer: 10 mM borate with the addition 10 mM -CD (pH 9.18)

Voltage: +20 kV;

Detection:190 nm.

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Assay characteristics for technologically Assay characteristics for technologically

important amino acidsimportant amino acids Amino

acidParameters LOQ LOD

mg l-1

Rangemg l-1

b r mg l-1 % 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 20 0.20 5 10

Cys-Cys 10 – 100 8.81 0.9988 10 0.10 7 5

Trp 0.1 – 50 1.45 0.9997 0.1 0.02 5 0.05

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The data of the underivatized amino acids determination The data of the underivatized amino acids determination in the real samples using HPLC, CZE and IECin 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

Lys 1.15 0.16 1.18 0.19 1.09 0.14

Met 0.75 0.10 0.69 0.10 0.69 0.09

Thr 0.72 0.12 0.80 0.14 0.75 0.16

Cys-Cys 0.24 0.04 0.36 0.07 0.23 0.04

Mixed fodder

Lys 1.52 0.18 1.49 0.13 1.27 0.15

Met 0.45 0.06 0.53 0.06 0.52 0.08

Thr 1.11 0.14 0.98 0.15 1.00 0.15

Cys-Cys 0.33 0.04 0.29 0.04 0.41 0.05

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1 2 3 4 5 6 7 min

3.87 mAU

Capel

Trp

Met

+H

is+

Tyr

Direct determination of TrpDirect determination of Trp

Alkaline hydrolysis Temperature 40°C Detection on 219 nm:

increase of detection sensitivity

decrease of interference of concomitant AAs

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The complete analysis of The complete analysis of proteinogenous amino acidsproteinogenous 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

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Derivatization of AAs with PITC

N C S

PITC

NH C S

NH

CH R

COOH

PTC - AAs

R CH

NH2

COOH

AAs

pH 10.5

+

O

PTH - AAsR

S

NH

N

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CE analysis of PTC-AAs

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 min

6.14 mAU

capel

arg

lys

tyr

ph

eh

is

leu

,ile

met

va

lp

roth

rse

ra

la

gly

cys-

cys

glu asp

Voltage: 25 kV; BGE: phosphate with ß-CDTemperature: 20°CNo pressure was applied

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CE analysis of PTC-AAs

7 8 9 10 11 12 13 14 15 16 17 18 19 20 min

4.64 mAU

Capel

arg

lys

tyr

ph

eh

is

leu

,ile

met

va

lp

roth

rse

ra

la

gly

cys-

cys

glu

asp

cyst

eic

aci

d

Voltage: 25 kV; BGE: phosphate with ß-CDTemperature: 30°CPressure 30 mbar was applied after 17 min

Controlled pressure during analysis

Highly precise changeable temperature of a capillary cooling

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-CD as a component of BGE-CD as a component of BGE

CChiral activity for Trp, Tyr, Phe !hiral 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.