Validation of an HPLC method for the determination of amino acids in feed (original) (raw)
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Bangladesh Journal of Animal Science, 2012
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...
The Analysis Techniques Of Amino Acid And Protein In Food And Agricultural Products
International Journal of Scientific & Technology Research, 2020
The protein content in food and agricultural products affects the physicochemical and nutritional properties of these products. This review aims to discuss the analysis techniques of protein and amino acid in food and agricultural products. The qualitative analysis can be conducted using the Hopkins-Cole, Xanthoproteic, Millon, Nitroprusside, and Sakaguchi test. In contrast, the quantitative analysis of proteins can use the Kjehldahl, Biuret, Lowry, UV Spectrophotometry, and Turbidimetry. It also discussed the immunohistochemical techniques to identify cellular or tissue constituents (antigens) by staining techniques, while Formol titration measures the hydrolysis of proteins and N-amino quickly. The amino acids can be analyzed by microbiological methods, colorimetric, high-performance liquid chromatography (HPLC), and gravimetric techniques. These methods/techniques can be chosen according to the type of sample and the purpose of the analysis so that the results can be obtained acc...
Amino Acid Analysis of Feeds in The Netherlands: Four-Year Proficiency Study
Journal of AOAC INTERNATIONAL, 1990
To improve accuracy and precision of amino acid analysis In 12 Dutch feed laboratories, a proficiency study was organized twice annually over a 4-year period. The method used included reflux acid hydrolysis for 22 h followed by evaporation, separation on a cation-exchange resin In an amino acid analyzer, and photometric detection after post-column derlvatlzatlon with ninhydrln. For the determination of sulfurcontaining amino acids, samples were oxidized prior to hydrolysis. For the determination of tryptophan, samples underwent an alkaline hydrolysis excluding oxygen, were separated by liquid chromatography on a Hypersil ODS analytical column, and were assayed by UV or fluorescence detection. The average relative standard deviations within (CVr) and between (CVR) laboratories were 3 and 7%, respectively. For some mixed feed samples, the results from the proficiency study were compared with those obtained by the International Analytical Group. For those samples, the relative standard...
Methods for Amino Acids Determination in Different Biological Extracts
2016
Sensitive, precise and accurate analytical methods for free amino acids determination in biological samples were developed. Purification by ion exchange technique was followed by two steps derivatization method to obtain trifluoroacetyl ester derivatives. GC/MS analysis was performed by using scan or SIM mode. Known amounts of internal standard, the isotopic labelled analogue of glycine, methionine or isoleucine were added to the sample, before extraction, for the quantitative analysis, followed by matrix and regression curves calculation. The methods were validated using amino acid standard samples. Analyses of dairy, corn grain, fish plasma and meat are presented. Also a trace level (picogram) analysis method of blood spots, for diagnosis of inborn errors of metabolism, is described.
Gas-liquid chromatographic analysis of amino acids in food samples
Journal of Agricultural and Food Chemistry, 1977
The gas chromatographic method of amino acid analysis requires that the sample is hydrolyzed to release the amino acids, which are then cleaned in an ion-exchange column, converted to volatile derivatives, and then separated on a polyester as well as on a silicone column. These different steps contribute to certain amounts of variation in the recovery of the individual amino acids. In addition, the various components of the food samples, especially carbohydrates, could affect the recovery of some amino acids.
Foods
Determination of the L- and D-amino acid composition in proteins is important for monitoring process-induced racemization, and thereby protein quality loss, in food and feed. Such analysis has so far been challenging due to the need for sample hydrolysis, which generates racemization, thereby leading to an overestimation of D-amino acids. Here, validation of an LC–MS/MS-based method for the simultaneous determination of L- and D-amino acids in complex biological matrixes, like food and feed, was performed in combination with deuterated HCl hydrolysis. This approach eliminated a racemization-induced bias in the L- and D-amino acid ratios. The LC–MS/MS method was applied for the analysis of 18 free amino acids, with a quantification limit of either 12.5 or 62 ng/mL, except for D-phenylalanine, for which quantification was impaired by background interference from the derivatization agent. For hydrolyzed samples, the composition of 10 L- and D-amino acids pairs could be determined in pr...
Simplified in-line sample preparation for amino acid analysis in carbohydrate containing samples
Journal of Chromatography B: Biomedical Sciences and Applications, 2001
This report describes a new, automated chromatographic procedure eliminating carbohydrates from amino acid samples prior to their analysis by anion-exchange chromatography and integrated amperometric detection. In the first step, a sample is brought onto a short cation-exchange column (trap column) in hydrogen form. Carbohydrates are passing through this column, while only amino acids are retained. Subsequently, the cation-exchange column, holding the amino acid fraction, is switched in-line with the gradient pump and separator column. The mobile phase used at the beginning of the separation (NaOH; pH 12.7) transfers amino acids from the trap column onto the anion-exchange column and the amino acid separation is completed without any interference by carbohydrates. All common amino acids are recovered following the carbohydrate removal step. The average value of their recovery is 88.1%. The calibration plots were tested between 12.5 and 500 pmol (amounts injected). The mean value of correlation coefficients of calibration plots was calculated as 0.99. The mean value of relative standard deviations from five replicates was 3.9%. The usefulness of the method is illustrated with two chromatograms of a carrot juice sample obtained before and after the in-line removal of carbohydrates.
A Rapid and Accurate Extraction Procedure for Analysing Free Amino Acids in Meat Samples by GC-MS
This study evaluated the use of a mixer mill as the homogenization tool for the extraction of free amino acids in meat samples, with the main goal of analyzing a large number of samples in the shortest time and minimizing sample amount and solvent volume. Ground samples (0.2 g) were mixed with 1.5 mL HCl 0.1 M and homogenized in the mixer mill. The final biphasic system was separated by centrifugation. The supernatant was deproteinized, derivatized and analyzed by gas chromatography. This procedure showed a high extracting ability, especially in samples with high free amino acid content (recovery = 88.73-104.94%). It also showed a low limit of detection and quantification (3.8 ⋅ 10 −4-6.6 ⋅ 10 −4 g L −1 and 1.3 ⋅ 10 −3-2.2 ⋅ 10 −2 g L −1 , resp.) for most amino acids, an adequate precision (2.15-20.15% for run-to-run), and a linear response for all amino acids (R 2 = 0.741-0.998) in the range of 1-100 g mL −1. Moreover, it takes less time and requires lower amount of sample and solvent than conventional techniques. Thus, this is a cost and time efficient tool for homogenizing in the extraction procedure of free amino acids from meat samples, being an adequate option for routine analysis.
Technical note: Rapid method for determination of amino acids in milk
Journal of Dairy Science, 2010
A rapid method for measurement of amino acids in milk was developed and validated. The method included a first step of milk protein hydrolysis, followed by the derivatization and separation of amino acids by HPLC. Six combinations of hydrolysis agent and temperature-time conditions were compared with a reference method; derivatization procedures as well as HPLC separation were improved. Hydrolysis of milk samples with 6 N HCl at 160°C for 60 min resulted in no significantly differences compared with the reference method but allowed the analysis of a greater number of milk samples in a short time. In addition, this method was characterized by high precision, low repeatability uncertainty, and high accuracy for all amino acids evaluated; the recovery mean value of the single amino acids was 98.38%. The proposed method is, therefore, accurate, simple, rapid, and suitable for large numbers of milk samples.