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Evaluation of a High Performance Liquid Chromolography (hplc) Mehtod for Amino Acid Analysis in Feed with Precolumn Derivatization and Fluorescence Detection

A. B. M. Khaleduzzaman
Ph. D student, Department of Animal Nutrition, Bangladesh Agricultural University,Mymensingh, 2202, Bangladesh.

Z. H. Khandaker
Department of Animal Nutrition, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh

M. S. H. Khan
Pacific Pharmaceuticals Ltd. Kanchpur, Narayangonj, Bangladesh

L. A. Banu
Animal Nutrition Section, Department of Livestock Services, Farmgate, Dhaka-1215, Bangladesh

M. K. A. Khan
Animal Nutrition Section, Department of Livestock Services, Farmgate, Dhaka-1215, Bangladesh

Abstract/ Executive Summary:

An experiment was conducted to determine the amino acids content in wheat sample which is often used as internal check in animal feed analysis by pre-column derivatization method using ortho-phthaldialdehyde (OPA) and betamercaptoethanol followed by high-performance liquid chromatography (HPLC). Fluorescence detection was used for the assay of OPA derivatives of amino acids with the detection wavelength set at Ex 340 nm and Em 455 nm. Ortho-phthaldialdehyde reagent itself does not fluorescence and hence produces no peak on the chromatogram and also produces a very low level of background noise. From standard amino acid mixture fourteen amino acids (Asp, Glu, Ser, Gly, Thr, Arg, Ala, Tyr, Met, Val, Phe, Ile, Leu, and Lys) were separated in 55 min with fine resolution. Good reproducibility and accuracy of the method were demonstrated by the determination of amino acids in wheat sample. The precision for the retention time of amino acids (n = 10 injections over 3 days) between the days showed average standard deviation (SD) of 0.43 and coefficient of variation (CV) of 3.32%. The average SD and CV of peak area repeatability over n = 10 injections were 0.72 and 14.93% also indicated the higher sensitivity of the method. The results of the amino acids in wheat samples suggested that the method can be potentially applied for the determination of amino acids in wheat as well as other cereal feeds used in animal diets.

Keywords: Amino acids, Ortho-phthaldialdehyde, Wheat, Pre-column derivatization, HPLC, Fluorescence detection

Location: Department of Animal Nutrition, Bangladesh Agricultural University

Start Date: 00-00-2008

End Date: 00-00-2008

Program Area: Animal Health and Management

Commodity: Wheat

Objectives:

1. To determine the amino acid contents in wheat sample (check sample) by pre-column derivatization with ortho-phthaldialdehyde (OPA) followed by fluorescence detection in high-performance liquid chromatography (HPLC).

Methodology:

Chromatography system - The amino acid analysis was performed in a KNAUER high pressure binary gradient system, consisting of 2 HPLC pumps (Type 64) equipped with micro-pump head, Rheodyne manual injector, system controller (Software: KNAUER, Eurochrom 2000, Version 1.57 for system control, data acquisition and analysis), dynamic mixing chamber, a high temperature oven with temperature control unit, and coupling with fluorescence detector fixed for OPA. A Vertex Column (Lichrospher 100 RP-18 endcapped 5 μm) 250 x 4 mm ID from KNAUER was used. A guard column cartridge was used to protect the column. Chemicals and reagents - Ortho-phthaldialdehyde and all other reagents were of analytical grade from Merck, Germany. Methanol and acetonitrile were HPLC grade from Merck, Germany. Deionized water was generated using an ultra pure water purification system. Hydrochloric acid, phenol, mono and disodium hydrogen phosphate were extra pure from Merck (Germany). Amino acids standard solution was from Sigma, USA. Borate buffer was supplied from Chrom, Germany. 12N-phenol HCl solution - One or two crystals of phenol were added to 12N HCl solution. Mobile phase - Eluent-A : 93% 12.5 mM phosphate buffer and 7% acetonitrile (pH 6.5). Eluent-B : 100% methanol. Prepared freshly and was filtered through 0.22 μm membrane filter (Millipore). Derivatizing reagents - Reaction mixture-1 : 10 ml of 100 mM boric acid buffer solution (pH 9.1) containing 10 μl of β-mercaptoethanol. Reaction mixture was prepared freshly everyday. Reaction mixture-2 : 10 ml of 100 mM boric acid buffer solution (pH 9.1), add 3 ml of OPA (10mg/3 ml ethanol). Reaction mixture was prepared freshly everyday. Preparation of calibration standard and internal standard - Calibration standard was 2.5 mM mixture of 17 amino acids with the exception in case of cystine (1.25 mM). The amino acids in the standard mixture were Asp, Glu, Ser, Thr, Tyr, Met, Val, Ala, Phe, Pro, Leu, Arg, Lys, Ile, Gly, His and Try. In order to prepare 2.5 mM internal standard stock solution, 6.45 mg of alpha amino butyric acid (AABA) was added to 25 ml of 0.1M HCl solutions. Internal standard stock solution was stored at -200C. Five hundred micro liters (500 μl) of amino acid standard mixture (2.5 mM) was added to 500 μl of internal standard stock solution (2.5 mM) to prepare calibration standard with internal standard. Sample preparation and hydrolysis - The well mixed wheat sample was finely ground to pass through 0.25 mm sieve and divided into 10 equal portions. Seven hundred milligrams of powdered samples (equivalent to around 10 mg nitrogen content) were taken in each digestion tubes separately and were hydrolysed using liquid-phase hydrolysis procedure suggested by AOAC International (2000). Methionine and cystine might undergo oxidation during acid hydrolysis. To prevent that oxidation, the wheat sample was subjected to oxidation with performic acid. The excess reagent was removed by using vacuum evaporator. Equal volumes of 12N-phenol HCl solution and 2.5 mM internal standard stock solution mixed together where resultant strength of HCl solution became 6N and the concentration of internal standard was 1.25 mM. Twenty micro liters of 6N-phenol HCl acid solution containing internal standard of 1.25 mM AABA was added to performic acid treated wheat sample and hydrolysed for 24 h at 1150 C. After cooling, 500 μl of protein hydrolysate was taken in an eppendorf tube and centrifuged at 3000 rpm for 10 minutes. The clear supernatant was collected and filtered through a 0.45 µm filter (Millipore) which was considered as hydrolysate. Derivatization with OPA - Ten micro liters of calibration standard solution was added to 175 µL of reaction mixture-1, votexed and then added to 175 µL of reaction mixture-2. Twenty micro liters of derivatized standard sample was injected within 2 minutes of derivatization in the HPLC system. In case of sample 10 µL of hydrolysate was dried with nitrogen flushing and was added 185 µL of reaction mixture-1 and vortexed. Then 175 µL of reaction mixture-2 was added and vortexed again. Twenty micro liters of derivatized sample was injected in the HPLC system.

Source of Information: Bang. J. Anim. Sci. 2008, 37(2) : 66 - 73; ISSN 0003-3588

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Ortho-phthaldialdehyde reagent itself does not fluorescence and hence produces no peak on the chromatogram and also produces a very low level of background noise. From standard amino acid mixture fourteen amino acids (Asp, Glu, Ser, Gly, Thr, Arg, Ala, Tyr, Met, Val, Phe, Ile, Leu, and Lys) were separated in 55 min with fine resolution. Good reproducibility and accuracy of the method were demonstrated by the determination of amino acids in wheat sample. The precision for the retention time of amino acids (n = 10 injections over 3 days) between the days showed average standard deviation (SD) of 0.43 and coefficient of variation (CV) of 3.32%. The average SD and CV of peak area repeatability over n = 10 injections were 0.72 and 14.93% also indicated the higher sensitivity of the method. The results of the amino acids in wheat samples suggested that the method can be potentially applied for the determination of amino acids in wheat as well as other cereal feeds used in animal diets.

Knowledge Management: Journal