Abstract:
The present study was undertaken to develop a simple yet effective HPLC/UV method based on a reversed-phase separation and quantification of the 20 essential amino acids with dual wavelengths. The hydrolysis of the proteins was done by inorganic acid. The derivatised amino acids are identified with UV detector at 262nm and 338nm. The proposed method for amino acid analysis (AAA) offers excellent accuracy and a precision of about 5% relative standard deviation. The method was validated with certified reference materials (CRM) of amino acids and. AAA can be used for the quantification of amino acids, in complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. Amino acid testing also enables the analysis of protein complexes or mixtures of proteins such as protein powder supplements.
1. Objective and Introduction:
Amino acid analysis (AAA) seems to lose some importance over the recent years. One of the reasons might be the fact that AAA needs sophisticated analytical devices and has remained a complex, relatively expensive, and nontrivial task. Conventional AAA can be performed based on pre-column derivatisation, for example, with phenylisothiocyanate, FMOC (fluorenylmethyloxycarbonyl chloride), OPA (o-phthaldialdehyde), or 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate. An important advantage of this approach is the use of reversed-phase (RP) chromatography. However, a very good separation performance is required to avoid overlapping peaks. Another traditional option is post-column derivatisation, which needs ion-exchange columns, run with highly optimized buffers and dedicated post-column derivatisation devices. The most important chemistry is based on ninhydrin, leading to highly colored products, which can be detected photometrically in the visible range. In addition, gas-chromatographic (GC) techniques have been applied for amino acid analysis for a long time. This approach always requires one or several derivatisation steps to make the analytes sufficiently volatile. Recently, GC was applied for chiral amino acid analysis on spacecrafts, such as the robotic lander Philae. More recently, several mass-spectrometric methods for amino acid analysis have emerged. LC-MS/MS-based methods often do not need any derivatisation; however, they do not achieve complete chromatographic separation in many cases; they heavily rely on the mass-spectrometric selectivity. Due to complexity, duration, cost, and effort, amino acid analysis is not used in many cases, in which it would be helpful. The present study was undertaken in order to develop a relative cost efficient yet simple method for analysis of amino acids form a wide variety of matrices.
2. Methods & Materials:
2.1 Reagents & standards:
Amino acids standards were procured from Merck India. Hydrochloric acid, Acetonitrile, Methanol, FMOC, Borate buffer were also procured from Merck India and were of GR grade.
2.2. Hydrolysis of Protein Samples:
Approx 0.5g of sample was hydrolyzed with 25mL 6N hydrochloric acid at 110ºC for 24hr. The hydrolyzed samples were neutralized with 6N sodium hydroxide solution to pH 7.0. The sample was filtered through Whatmann No 1 filter paper in 250mL volumetric flask and volume was made up with distilled water.
2.3. Derivatisation of amino acid:
Derivatisation of amino acids to their respective fluorescent derivatives was achieved using OPA and FMOC reagents at pH 10.2 and 60∞C.
3. Result and discussion:
3A. Preparation of standard curve of standard amino acids by HPLC:
Calibration curve was plotted, in the range of 0.5 mg/kg to 2.5 mg/kg (Figure-1-20) and correlation co-efficient r > 0.98 by applying weighing factor 1/X (Table-1).
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| Fig-1: Aspartic acid | Fig-2: Glutamic acid | Fig-3: Serine |
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| Fig-4: Asparagine | Fig-5: Glutamine | Fig-6: Histidine |
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| Fig-7: Threonine | Fig-8: Arginine | Fig-9: Alanine |
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| Fig-10: Tyrosine | Fig-11: Glycine | Fig-12: Valine |
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| Fig-13: Methionine | Fig-14: Tryptophan | Fig-15: Phenyl alanine |
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| Fig-16: Isoleucine | Fig-17: Leucine | Fig-18:Lycine |
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| Fig-19: Hydroxy-proline | Fig-20: Proline |
3B. QUANTIFICATION OF AMINO ACIDS IN DIFFERENT FOOD COMODITIES:
The different food commodities. Milk and chocolate were taken for the quantification of amino acids. The analyses were done in triplicates. The chromatogram and results are depicted in table 2-5.
| Table 2:HPLC chromatogram of Amino acids in Milk |
Experiment has been carried out in triplicates. Results are expressed in Mean ± Standard Deviation
| Table 3: Percentage calculation of amino acids in Milk | ||||
| Milk | Retention time | Conc (mg/kg) | Final conc (mg/kg) | % |
| Glutamic Acid | 3.524+ 0.007 | 3.31+ 0.03 | 31797.12+32.82 | 3.17+ 0.03 |
| Serine | 6.451+ 0.009 | 0.56+ 0.02 | 5353.45+5.92 | 0.53+ 0.01 |
| Threonine | 7.743+ 0.005 | 1.51+ 0.02 | 14450.96+6.29 | 1.44+ 0.02 |
| Arginine | 8.28+ 0.008 | 0.38+ 0.01 | 3665.73+9.18 | 0.36+ 0.02 |
| Alanine | 8.886+ 0.006 | 0.37+ 0.01 | 3558.54+6.82 | 0.35+ 0.02 |
| Tyrosine | 10.515+ 0.100 | 0.20+ 0.02 | 1956.81+6.86 | 0.19+ 0.02 |
| Glycine | 11.187+ 0.085 | 0.89+ 0.03 | 8595.48+14.81 | 0.85+ 0.01 |
| Valine | 11.779+ 0.057 | 0.11+ 0.02 | 1080.42+4.11 | 0.11+ 0.02 |
| Tryptophan | 12.591+ 0.029 | 0.39+ 0.02 | 3775.53+6.62 | 0.37+ 0.02 |
| Isoleucine | 13.589+ 0.020 | 0.15+ 0.01 | 1477.54+7.28 | 0.14+ 0.02 |
| LycinemonoHCL | 15.466+ 0.015 | 0.20+ 0.01 | 1958.73+3.73 | 0.19+ 0.02 |
| Hydroxyproline | 14.851+ 0.040 | 0.79+ 0.02 | 7598.84+2.98 | 0.75+ 0.02 |
| Proline | 17.155+ 0.067 | 1.09+ 0.01 | 10515.26+6.40 | 1.05+ 0.02 |
| 9.5784 | ||||
| Table 4: HPLC chromatogram of Amino acids in Chocolate |
Experiment has been carried out in triplicates. Results are expressed in Mean± Standard Deviation
| Table5: Percentage calculationof Amino acids in Chocolate | ||||
| Chocolate | Retention time | Conc (mg/kg) | Final conc (mg/kg) | % |
| Alanine | 8.863±0.006 | 0.225±0.001 | 2327.03±8.85 | 0.23±0.01 |
| Tyrosine | 10.073±0.006 | 0.175±0.004 | 1823.20±4.17 | 0.18±0.015 |
| Valine | 11.759± 0.043 | 0.231± 0.003 | 2375.18±6.80 | 0.24± 0.01 |
| Tryptophan | 12.453± 0.013 | 0.485±0.003 | 5026.56±6.72 | 0.50±0.015 |
| Phenyl Alanine | 13.145± 0.005 | 0.527± 0.002 | 5441.93±4.88 | 0.54±0.015 |
| Leucine | 13.919± 0.005 | 0.296±0.003 | 3053.52±6.18 | 0.31± 0.015 |
| LycinemonoHCL | 15.166± 0.006 | 1.082±0.002 | 11176.69±266.01 | 1.12± 0.025 |
| Proline | 17.14± 0.039 | 0.553± 0.001 | 5696.32±12.2 | 0.57± 0.025 |
| 3.692 | ||||
The method was demonstrated to provide accurate results for all 20 amino acids in the various substrates and can be used for routine analysis of samples in a short period of time. The present study was carried out to analyze the amino acid contents in three different matrices viz. milk and chocolate. In milk total amino acid percentage was found to be 9.5%, however in Chocolate it was found to be comparatively less (3.6%).
In milk concentration of glutamic acid was found to be highest (3 mg/kg) and in case of chocolate the highest concentration was found for Lycine (1mg/kg).
4.Conclusion:
The present study was successfully carried out in milk and chocolate for the profiling and quantification of 20 amino acids in a single run and can also be used for determining the total protein content of the matrix. The method was found to be precise and accurate. This method has a good prospect for routine analysis of amino acid in these two food commodities. This method may also be used for the estimation of amino acids in other food commodities.