Amino acid analysis has been used in the food and feed industries to verify and characterize materials and processes. The total amino acid content, as well as the proportions of growth-limiting amino acids, is an essential characteristic of the nutritional value of feeds. Since most of the amino acids are bound in protein, this determination requires acid hydrolysis of the sample. The total amino acids are then available for analysis.
In addition to being components of proteins, amino acids are also important intermediates in metabolic pathways. Amino acid analysis can therefore identify the genetic background, origin, and history of food products. This form of analysis must accommodate the larger number of compounds present as free amino acids that are not found in protein hydrolysates. A different separation is then required.
Figure 1 - Representative chromatograms for hydrolysate amino acids and free amino acids using the UPLC Amino Acid Analysis Solution methods, both at 10 pmol on column.
A variety of techniques have been used for the determination of amino acid content. There is a continuing need for improved accuracy, precision, and ruggedness, preferably with higher throughput. The resolving power of UltraPerformance LC® (UPLC®, Waters Corp., Milford, MA) has been combined with the well-established pre-column derivatization of AccQ∙Tag Ultra™. A robust turnkey solution, the UPLC Amino Acid Analysis Solution, has been developed to meet the needs of the food and feed industries. The improved resolution and sensitivity provide unequivocal identification and consistent quantitation of the amino acids. Standard methods are provided for the analysis of hydrolysate amino acids as well as for the separation of a larger set of free amino acids. These standard separations are shown in Figure 1.
Three applications have been chosen to show the utility of this method. The amino acid composition of a hydrolyzed poultry feed was measured to determine its nutritional content. Starting fermentation barley malts were identified and characterized through the analysis of free amino acids to characterize the raw materials used in a manufacturing process. Finally, the progress of a fermentation was monitored with amino acid analysis at different stages to illustrate the role of metabolic processes.
Materials and methods
Poultry feed samples were hydrolyzed independently and provided as part of a collaborative study. The supplied samples were stored at –80 °C in sealed glass ampules, purged with argon. On each of five days of analysis, a new ampule of hydrolyzed sample was used.
Three different starting malts (malted barley grains) were obtained (freshly milled) at a local brewing supply store: 2-row, 6-row, and pilsener. Samples were taken at the initial suspension in water to characterize the starting malts. The brewing process was monitored, with samples being taken at the beginning, apex, yeast dormancy, and end of primary fermentation.
Derivatization was performed on samples diluted with water (10× dilution for poultry diet, and 4× dilution for both starting malt and fermentation samples), as specified in the UPLC Amino Acid Analysis Solution.1 Since the derivatives are stable, samples were processed in convenient batches of 25–50. Five replicate derivatizations were performed for each day of analysis for the hydrolyzed poultry feed, with triplicate injections of each derivatized sample. All derivatized samples were subjected to analysis with the UPLC Amino Acid Analysis Solution. The system utilizes a UPLC reversed-phase separation and was monitored with UV detection. The defined Hydrolysate method was used for analysis of the feed hydrolysates, and the defined Cell Culture method was used for analysis of all other samples.
Results and discussion
Nutritional content of poultry feed
Table 1 - Poultry feed data summary, showing mol% mean values, standard deviations, and % RSD for 75 independent determinations
The robustness and accuracy of the method were tested with the analysis of the poultry feed hydrolysates. These samples are usually challenging because of the high content of minerals and other nonproteinaceous materials. Samples were derivatized and analyzed on five separate days, with freshly prepared reagents and eluents. Multiple columns were used throughout the five days of analyses. The reliability of the method is summarized in Table 1 with the results of 75 replicate injections. The mole percentage of the amino acids ranges from 2 to 16%, with reproducibility over the full sample set typically better than 1% RSD. With multiple independent preparations of the poultry feed, the same answers were obtained repeatedly, despite the many interferences present in animal feeds.
Characterization of barley malt varieties
Figure 2 - Chromatographic overlay showing the variable free amino acid amounts in different starting fermentation malts.
The three different starting malts were chosen to represent the range of barley varieties, regional origin, history, and malting process. Most amino acids are found in the range of 2–6% on a molar basis. Proline, in contrast, represents 25–30% of the total free amino acid content. Comparison of the amino acid profiles of the three different starting malts showed a significant difference in the proportions of some amino acids, as shown in Figure 2. For example, asparagine is noticeably higher for the 6-row malt as compared to the pilsener malt, while glycine is at a very similar level. This simple analysis can be used to monitor the identity and properties of the starting raw material used in a food process.
Characterization of a fermentation process
Figure 3 - Chromatographic overlay showing the changes in free amino acid levels throughout a fermentation process. Note: The derivatization peak seen in the figure is a variable by-product of the reagent that forms during derivatization and is not indicative of the properties of the sample.
The process of fermentation is a series of metabolic events that can be reflected in changes in the free amino acid profile. Figure 3 shows an overlay of analyses of samples taken at different stages of the same fermentation. While most amino acids decreased throughout the fermentation, ornithine actually increased by almost 20-fold. When the yeast activity ceased, the ornithine level declined by more than half. The profiling of such patterns indicates the progress of a particular batch and may suggest required adjustments.
Use of the UPLC Amino Acid Analysis Solution was illustrated through the examples described in this paper. Sample preparation for all of these examples was simple and required no prefractionation. Due to the sensitivity of the method, very small samples are required. In addition, because the derivatives are so stable, large numbers of samples can be derivatized and processed in a batch. The two defined chromatographic methods proved suitable for these applications.
The replicate preparation and analyses of the hydrolyzed poultry feed samples demonstrate the robustness and stability of the method, even with these complex samples. Because of the reliability of the results, confident and well-informed decisions can be made regarding the quality of animal feed blends.
Free amino acids can be monitored for both material characterization and process control. This can be used as an incoming inspection test for the identification of high value foods and for the detection of adulteration.
Analysis of free amino acids can also be used to monitor a process. In this way, the physiological processes that affect product quality can be identified. The rapid analytical method provides for recognition of important changes and adjustment of the ongoing process. The specific product characteristics that are identified may be incorporated into quality testing of the finished product.
The UPLC Amino Acid Analysis Solution is characterized by improved resolution and sensitivity in a robust method with reduced run time. The application examples shown here illustrate its utility for the food and feed industries.
- UPLC®Amino Acid Analysis Solution System Guide, 71500129702, Rev. B, Waters Corp.: Milford, MA.
Ms. Hewitson is Applications Chemist; Dr. Wheat is Principal Scientist and Life Science Laboratory Manager; and Dr. Diehl is Director, Chemistry Applied Technology, Waters Corp., 34 Maple St., Milford, MA 01757, U.S.A.; tel.: 508-482-2305; fax: 508-482-3100; e-mail: email@example.com.