CN108828085B - Quantitative detection method for total amino acids of transfer factor capsule - Google Patents
Quantitative detection method for total amino acids of transfer factor capsule Download PDFInfo
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Abstract
The invention discloses a method for detecting the content of total amino acids in a transfer factor capsule, which solves the technical problems that only free amino acids are detected but bonded amino acids forming polypeptide substances cannot be detected in the prior art, and the separation degree of each amino acid peak and an unknown component peak is poor in the amino acid detection process because transfer factors are multi-component biochemical drugs, and meanwhile, the quantitative detection of 18 common amino acids is realized by adopting a one-test-and-multiple-evaluation method through a glutamic acid reference substance, so that the technical problems that the external standard method requires more reference substances, the detection cost is higher and the operation is complicated are solved. The invention takes glutamic acid as an internal reference substance, and determines relative correction factors and relative retention time of other 17 amino acids through systematic verification and research; realizing the quantitative detection of 18 amino acids. The detection method is accurate and reliable, has good separation effect and strong specificity, and can effectively detect and monitor the total amino acids in the transfer factor capsule.
Description
Technical Field
The invention belongs to the technical analysis field of multi-component biochemical drugs, and particularly relates to a quantitative detection method for total amino acids of a transfer factor capsule.
Background
The Transfer Factor (TF) is also called transmission factor, is a natural bidirectional immunomodulator, and is an ideal medicine for treating related diseases such as low immune function, deficiency and the like at present. The transfer factor belongs to polypeptide substance, and is a composite molecule composed of polynucleotide, polypeptide and other components. Wherein the small molecular peptide substance is a functional marker of the transfer factor and is used as a main functional component of the transfer factor. Amino acids are the basic units that make up polypeptides and are key quality indicators for transfer factors. Therefore, the accurate determination of the total amino acid content in the transfer factor has important significance for optimizing the production process of the transfer factor and controlling the product quality.
However, the prior art only detects the content of free amino acid in the transfer factor, and can not detect the bonded amino acid which forms the polypeptide substance; and because the transfer factor components are complex, the existing detection technology is adopted to detect the condition that the peaks of adjacent components are overlapped to a large extent, so that the detection result has large deviation. Meanwhile, in the prior art, an external standard method is adopted for quantitative detection, 18 amino acid reference substances are needed, the detection cost is high, and the operation is complicated.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide the method for quantitatively detecting the total amino acids in the transfer factor capsule, which is simple to operate and low in detection cost, and can realize accurate quantitative analysis of the total amino acids in the transfer factor capsule, thereby realizing effective supervision and control on the production of the transfer factor capsule.
The invention is realized by the following technical scheme:
the invention discloses a method for quantitatively detecting total amino acids of a transfer factor capsule, which comprises the following steps:
1) preparation of test solution for total amino acid determination derivation
Precisely weighing 0.9-1.2 g of transfer factor capsule content, adding 0.1mol/L hydrochloric acid solution to fully dissolve the transfer factor, shaking up, standing, taking supernate, filtering with a microporous membrane, then precisely taking 2.0mL of subsequent filtrate, placing into a reaction container, adding 6mL of concentrated hydrochloric acid solution, charging nitrogen, sealing, hydrolyzing at 110 ℃ for 24h, cooling to obtain transfer factor acid hydrolysis solution, unsealing the transfer factor acid hydrolysis solution, performing water bath evaporation to dryness, adding 10mL of 0.1mol/L hydrochloric acid solution to dissolve residues, and obtaining a sample solution for total amino acid determination derivatives;
2) preparing a test solution for measuring the content of total amino acids
Precisely measuring 4.0mL of a sample solution for determination of total amino acid, 2.0mL of a 0.1mol/L phenylisothiocyanate acetonitrile solution and 1.0mL of a 1mol/L triethylamine acetonitrile solution, mixing, shaking up, reacting at room temperature for 1h, extracting with 8.0mL of n-hexane, standing for 10min, filtering a lower layer solution with a microporous filter membrane, and taking a subsequent filtrate to obtain a sample solution for determination of total amino acid content;
3) determination of the Standard Curve
Preparing a standard curve series solution by taking glutamic acid as a reference substance, obtaining a chromatogram by high performance liquid chromatography, and performing linear regression on the peak area of the solution by the concentration of the glutamic acid according to a least square method to obtain a standard curve equation and a linear correlation coefficient;
4) precisely measuring 2 mu L of the total amino acid content determination sample solution prepared in the step 2), injecting into a high performance liquid chromatograph, recording a chromatogram, obtaining 18 amino acid chromatographic peaks which take glutamic acid as an internal reference substance and are positioned according to relative retention time in the total amino acid content determination sample solution, substituting the area of each chromatographic peak into a standard curve equation to obtain the content of each amino acid, multiplying the content by relative correction factors of each amino acid relative to glutamic acid, and summing to calculate the total amino acid content in the transfer factor capsule.
Preferably, in the step 3) and the step 4), the detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;
the mobile phases used for gradient elution were:
mobile phase A: according to the proportion of methanol: acetonitrile: 20 parts of water: 60:20 in volume ratio;
mobile phase B: according to the sodium acetate buffer solution: solution prepared by the volume ratio of the mobile phase A to 97: 3;
the concentration of the sodium acetate buffer solution is 0.1mol/L, and the pH value is 6.0-6.5;
detection wavelength: 210-320 nm;
the sample injection amount is 1-10 mu L;
the flow rate is 0.5-2 mL/min;
the column temperature is 25-50 ℃.
Still more preferably, the ratio of change of mobile phase a to mobile phase B upon gradient elution is as follows:
0min, mobile phase B: 100 percent;
15min, mobile phase A: 14%, mobile phase B: 86 percent;
20min, mobile phase A: 17%, mobile phase B: 83 percent;
50min, mobile phase A: 44%, mobile phase B: 56 percent;
50.1min, mobile phase A: 100 percent;
55min, mobile phase B: 100 percent;
65min, mobile phase B: 100 percent.
More preferably, the particle size of the octadecylsilane chemically bonded silica is 5-10 μm; the inner diameter of the chromatographic column is 2-5 mm, and the length is 10-30 cm.
Preferably, in the step 1), the transfer factor acid hydrolysis solution contains 1.6-2.4 mg/mL of total amino acids; in the step 2), the content of the total amino acid is determined, and the total amino acid contained in the test solution is 0.4-0.8 mg/mL; filtering the supernatant with 0.45 μm microporous membrane; in the step 2), the lower layer solution is filtered by a 0.22-0.45 mu m microporous filter membrane.
Preferably, the prepared transfer factor acid hydrolysis liquid contains 1.6-2.4 mg/mL of total amino acids; the content of the total amino acid in the prepared test solution is measured, and the total amino acid content in the test solution is 0.4-0.8 mg/mL.
Preferably, in step 3), the specific method for determining the standard curve is as follows:
(1) preparing reference substance stock solution
Precisely weighing 15.0mg of glutamic acid reference substance, placing the glutamic acid reference substance in a 100mL measuring flask, adding 0.1mol/L hydrochloric acid solution for dissolving, diluting to scale, and shaking up to obtain reference substance stock solution;
(2) preparing standard curve series solution
Precisely measuring 1mL, 2mL, 4mL and 10mL of reference substance stock solutions respectively, placing in a 20mL measuring flask, adding water to dilute to scale, shaking up, and using as reference solutions 1-4, and using reference solution 5 and reference substance stock solution;
(3) derivatization of solutions of the Standard Curve series
Respectively precisely measuring 1-5 control solutions with the volume of 4.0mL, respectively placing the control solutions into 20mL test tubes with plugs, adding 2.0mL of 0.1mol/L phenyl isothiocyanate acetonitrile solution and 1.0mL of 1mol/L triethylamine acetonitrile solution, shaking uniformly, reacting at room temperature for 1h, adding 8mL of n-hexane for extraction, standing for 10min, filtering the lower layer solution with an organic filter membrane to obtain 5 derived standard curve series solutions;
(4) measuring and calculating a standard curve
Respectively and precisely measuring 2 mu L of each of the 5 derived standard curve series solutions, respectively injecting into a high performance liquid chromatograph, recording a chromatogram, and performing linear regression on the peak area of the standard curve series solution according to a least square method by using the concentration of glutamic acid to obtain a standard curve equation and a linear correlation coefficient.
Preferably, in step 4), the relative calibration factor of each amino acid with respect to glutamic acid is determined by using glutamic acid as an internal reference and using a standard curve method, and the detection results of the relative calibration factors of 18 amino acids are as follows:
still further preferably, the method further comprises examining an indicator of the relative correction factor's durability, i.e. examining the relative correction factor's durability under different chromatographic conditions.
Preferably, in step 4), the results of measuring the relative retention time of 18 amino acids in the test solution are as follows:
| serial number | Name of amino acid | Relative retention time | Serial number | Name of amino acid | Relative retention time |
| 1 | Aspartic acid | 0.83 | 10 | Tyrosine | 3.57 |
| 2 | Glutamic acid | 1.00 | 11 | Valine | 3.85 |
| 3 | Serine | 1.69 | 12 | Methionine | 4.09 |
| 4 | Glycine | 1.80 | 13 | Cystine | 4.52 |
| 5 | Histidine | 1.98 | 14 | Isoleucine | 4.66 |
| 6 | Arginine | 2.19 | 15 | Leucine | 4.74 |
| 7 | Threonine | 2.27 | 16 | Phenylalanine | 5.16 |
| 8 | Alanine | 2.37 | 17 | Tryptophan | 5.30 |
| 9 | Proline | 2.44 | 18 | Lysine hydrochloride | 5.56 |
。
Compared with the prior art, the invention has the following beneficial technical effects:
firstly, the invention adopts an acid hydrolysis method to hydrolyze polypeptide substances in the transfer factor into basic constituent unit amino acid, thereby realizing the simultaneous determination of the content of free amino acid in the transfer factor capsule and the content of amino acid composing the polypeptide.
Secondly, the optimal detection chromatographic condition is determined by the measures of screening a chromatographic column, mobile phase composition, mobile phase pH value, chromatographic column temperature, optimizing elution time gradient and the like; the liquid chromatography result proves that the condition of the invention is adopted for detection, the peak shape of each amino acid peak in the map is good, the separation degree is high, the overlapping proportion between adjacent component peaks is greatly reduced, and the detection accuracy is improved.
Thirdly, the chromatographic conditions are determined through systematic verification and research, and the relative correction factors and the relative retention time of the 18 common amino acids are measured, which shows that the glutamic acid can be used as an internal reference to realize the quantitative detection of the 18 common amino acids by adopting the one-measurement-multiple-evaluation method, thereby solving the technical problems of more reference substances, higher detection cost and fussy operation of an external standard method used in the prior art in the field.
In conclusion, the method can realize accurate quantitative analysis of the total amino acids in the transfer factor capsule, thereby realizing effective supervision and control on the production of the transfer factor capsule.
Drawings
FIG. 1 is a liquid chromatogram of a reference solution of 18 amino acids.
FIG. 2 is a liquid chromatogram of a test solution of a transfer factor capsule in an embodiment of the invention.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.
The invention discloses a method for quantitatively detecting total amino acids of a transfer factor capsule, which comprises the following steps:
1) preparing a test solution for determining and deriving total amino acid;
2) preparing a test solution for measuring the content of total amino acid;
3) the total amino acid content in the transfer factor capsule is determined by adopting a one-test-multiple-evaluation method.
In the step 1), the preparation method of the test solution for measuring the total amino acid content of the transfer factor capsule specifically comprises the following steps:
a. precisely weighing about 1.0g of transfer factor capsule content, placing into a 20ml test tube with a plug, adding 0.1mol/L hydrochloric acid solution, placing on an oscillator to oscillate so as to dissolve the transfer factor, shaking up, and standing; filtering the supernatant with 0.45 μm microporous membrane, precisely measuring 2.0mL of the subsequent filtrate, placing in a digestion tube, adding 6mL of concentrated hydrochloric acid, charging nitrogen, sealing, hydrolyzing at 110 deg.C for 24 hr, and cooling to obtain the transfer factor acid hydrolysate.
b. Unsealing the transfer factor acid hydrolysis solution, placing the transfer factor acid hydrolysis solution in an evaporation dish, and evaporating the transfer factor acid hydrolysis solution in a water bath to dryness; the residue was dissolved in 10mL of 0.1mol/L hydrochloric acid solution to prepare a sample solution for deriving the total amino acid assay.
Preferably, in step a, the oscillation time is not less than 2 minutes, and the nitrogen charging time is not less than 30 seconds.
Preferably, in the step a, the concentration of the sample in the transfer factor acid hydrolysis solution has a large influence on the determination result of the total amino acid content, the concentration of the hydrolyzed sample is too high, the amino acid in the sample is not completely hydrolyzed, and the detection result of the total amino acid content is lower; the concentration of the hydrolyzed sample is too low, the damage degree of amino acid in the sample is larger, and the detection result of the total amino acid content is also lower. The concentration of the hydrolyzed sample contains 1.6-2.4 mg/mL of total amino acids, preferably 2.0 mg/mL.
In the step 2), 4.0mL of the sample solution for determination and derivation of the total amino acid prepared in the step 1) is precisely measured and placed in a 20mL test tube with a plug; adding 2.0mL of 0.1mol/L phenyl isothiocyanate acetonitrile solution and 1mL of 1mol/L triethylamine acetonitrile solution, shaking up, reacting for 1 hour at room temperature, adding 8mL of n-hexane for extraction, standing for 10 minutes, taking the lower layer solution, filtering with a microporous filter membrane (the pore diameter is 0.22-0.45 mu m), and taking the subsequent filtrate to obtain the sample solution for measuring the total amino acid content.
Preferably, the concentration of the total amino acid content determination test sample solution has a large influence on the total amino acid content determination result, the concentration of the detection test sample solution is too high, the amino acid peak area and the concentration are not linear, and the total amino acid content determination result is lower; the concentration of the solution of the test sample is too low, the detection sensitivity is reduced, and the detection result is inaccurate. The concentration of the test sample preferably contains 0.4-0.8 mg/mL of total amino acids, and most preferably 0.6 mg/mL.
Step 3), adopting a 'one-test-multiple-evaluation' method to determine the content of the total amino acids in the transfer factor capsule, wherein the specific method comprises the following steps:
a. the relative correction factor is determined by taking glutamic acid which is the amino acid with the highest content and relatively stable property in the transfer factor capsule as an internal reference and determining the relative correction factors of the rest amino acids.
b. Relative to the correction factor durability examination, the examination contents include: the relative correction factors are robust to various chromatographic conditions (minor changes in the composition ratio of the mobile phase, pH, column temperature, flow rate, and detection wavelength, etc., and the use of different instruments, different brands or different lot numbers of the same type of chromatographic column, different operators, etc.).
c. The 18 kinds of amino acids are located in chromatographic peak under the condition of drawing up chromatogram, and when the durability of the relative correction factor is examined, the relative retention time of the peaks of the rest amino acids is calculated by taking glutamic acid as an internal reference, and the durability is also examined.
d. Preparing linear series solution by taking glutamic acid as a reference substance according to the measured relative correction factor and relative retention time, and measuring a standard curve; and positioning chromatographic peaks of the rest 17 amino acids according to the relative retention time, calculating the content of each amino acid peak from the standard curve, multiplying by corresponding relative correction factors, and adding to obtain the total amino acid content.
The 18 amino acids include: aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cystine, isoleucine, leucine, phenylalanine, tryptophan and lysine hydrochloride.
In the step d, when a glutamic acid reference substance is used for determining a standard curve, the linear concentration range has a large influence on the determination result of the total amino acid content of the transfer factor capsule, the concentration of the glutamic acid reference substance is too high, the peak area and the concentration of the glutamic acid are not linear, and the detection result of the total amino acid content is lower; the concentration of the glutamic acid reference substance is too low, the detection sensitivity is reduced, and the detection result is inaccurate. The concentration range of the glutamic acid reference substance is preferably 7.5-150 mu g/mL.
The total amino acid content of the transfer factor capsule adopts a high performance liquid chromatography, and detection chromatographic conditions are established according to the properties of 18 common amino acids and the condition of free amino acids contained in the transfer factor capsule; and carrying out systematic methodology verification research on the established chromatographic conditions to ensure the durability, reproducibility and accuracy of the established chromatographic conditions.
The detection chromatographic conditions comprise: octadecylsilane chemically bonded silica is used as a filler chromatographic column; gradient elution was carried out using methanol-acetonitrile-water (20:60:20) as mobile phase A and 0.1mol/L sodium acetate buffer (pH adjusted to 6.3 with glacial acetic acid) -mobile phase A (97:3) as mobile phase B.
The granularity of the octadecyl bonded silica gel chromatographic column filler is 5-10 mu m, preferably 5 mu m; the inner diameter is 2-5 mm, and preferably 4.6 mm; the length is 10cm to 30cm, preferably 25 cm.
The pH value of the sodium acetate buffer solution in the mobile phase B is 6.0-6.5, preferably 6.3.
The volume ratio of the mobile phase A to the mobile phase B gradient elution is 0:100(0min), 14:86(15min), 17:83(20min), 44:56(50min), 100:0(50.1min), 100:0(55min), 0:100(56min), 0:100(65 min).
In the detection method, the detection wavelength is 210-320 nm, preferably 254 nm.
The sample injection amount in the detection method is 1-10 mu L, and preferably 2 mu L.
The flow rate in the detection method is 0.5-2 mL/min, preferably 1 mL/min.
In the detection method, the column temperature is 20-50 ℃, and preferably 30 ℃.
Examples of screening of the assay conditions used in the course of the method for quantitative determination of total amino acids according to the present invention are specifically described below.
EXAMPLE 1 screening of transfer factor Capsule hydrolysis conditions
Screening hydrolysis conditions of the transfer factor capsules by using a 3-factor 3 horizontal orthogonal test, wherein 3 factors considered by the hydrolysis conditions are hydrolysis sample concentration, hydrolysis time and hydrolysis acid volume respectively; the 3-factor 3 horizontal orthogonal test designed according to the above-mentioned optimal level values of 3 factors is shown in table 1, and the analysis of the orthogonal test results is shown in table 2:
table 1 transfer factor capsule hydrolysis condition screening orthogonal test table
| Level of | Concentration of hydrolyzed sample A | Time of hydrolysis B | Volume of hydrolyzed acid C |
| 1 | 2.0mg/ml | 22h | 3mL |
| 2 | 2.4mg/ml | 24h | 6mL |
| 3 | 2.8mg/ml | 26h | 9mL |
TABLE 2 transfer factor Capsule hydrolysis condition screening orthogonal test result analysis table
As can be seen from table 2, the optimal combination of hydrolysis conditions was determined by a 3-factor 3 horizontal orthogonal test as: hydrolysis sample concentration 2.4mg/mL, hydrolysis time 24 hours, hydrolysis acid volume 6 mL. And the most significant factor influencing the detection result of the sample is the concentration of the hydrolyzed sample through the worst analysis.
EXAMPLE 2 screening of chromatographic conditions for determination of Total amino acid content in transfer factor capsules
Screening transfer factor capsule total amino acid content determination chromatographic conditions by using a 3-factor 3 horizontal orthogonal test, wherein 3 factors of chromatographic condition investigation are mobile phase B composition proportion, chromatographic column temperature and mobile phase flow rate respectively; the 3-factor 3 horizontal orthogonal test designed based on the above-mentioned optimal level values of the 3 factors is shown in table 3, and the analysis of the orthogonal test results is shown in table 4.
TABLE 3 transfer factor Capsule Total amino acid content determination chromatographic Condition screening orthogonal test Table
| Level of | Composition ratio A of mobile phase B | Column temperature B of chromatographic column | Flow rate of mobile phase C |
| 1 | 98:2 | 25℃ | 0.8mL/min |
| 2 | 97:3 | 30℃ | 1.0mL/min |
| 3 | 96:4 | 35℃ | 1.2mL/min |
TABLE 4 transfer factor Capsule Total amino acid content determination chromatogram condition screening orthogonal test result analysis table
As can be seen from table 4, the optimal combination of hydrolysis conditions was determined by a 3-factor 3 horizontal orthogonal test as: the composition ratio of the mobile phase B is 0.1mol/L sodium acetate buffer solution (pH value is adjusted to be 6.3 by glacial acetic acid) -the mobile phase A (97:3), the temperature of a chromatographic column is 30 ℃ and the flow rate of the mobile phase is 1.0 mL/min; and the most significant factor influencing the detection result of the sample is the composition proportion of the mobile phase B through range analysis.
Example 3 Single control method for determining the content of total amino acids in transfer factor capsules
1. Determination of relative correction factor
The relative correction factors of the rest amino acids are determined by taking glutamic acid which is the amino acid with the highest content and relatively stable property in the transfer factor capsule as an internal reference. The specific calculation method of the relative correction factor can be calculated according to the detection principle of the lambertian law, that is, according to the lambertian law, the amount (mass or concentration) of the component is proportional to the response of the detector in a certain linear range. In the quality evaluation of multiple indexes (s, a, b, …, i, …), a typical effective component in the object is taken as an internal reference substance(s), and relative correction factors (RCF, fsa, fsb, fsc, …) between the internal reference substance and other objects (a, b, …, i, …) are established, and f is calculated according to the following formulasi＝fs/fi＝(As/Cs)/(Ai/Ci) (ii) a Measured according to the method described aboveAnd (3) obtaining a correction factor by dividing the slope of the standard curve of the internal reference glutamic acid by the slope of the standard curve of other amino acids to be detected according to the standard curve equation of 18 amino acids.
Through calculation, the detection results of the 18 amino acids relative correction factors related by the invention are shown in the following table 5:
TABLE 5 relative correction factor test results
2. Relative correction factor durability review
The content of the investigation includes: the relative correction factors are robust to various chromatographic conditions (minor changes in the composition ratio of the mobile phase, pH, column temperature, flow rate, and detection wavelength, etc., and the use of different instruments, different brands or different lot numbers of the same type of chromatographic column, different operators, etc.). The durability test conditions are shown in the following table 6, and it can be seen that the test results are all satisfactory.
TABLE 6 relative correction factor durability examination conditions
| Condition | Value to be determined | Investigation of value 1 | Investigation value 2 |
| Composition ratio of mobile phase B | 97：3 | 98：2 | 96：4 |
| pH value of mobile phase | 6.3 | 6.1 | 6.5 |
| Column temperature | 30 |
25℃ | 35℃ |
| Flow rate of flow | 1.0mL/min | 0.8mL/min | 1.2mL/min |
| Detection wavelength | 254nm | 252nm | 256nm |
| Different chromatographic columns | Kromasil | Shimadzu | Feilou door |
| Different instruments | Agilent | Daian (UV) | Daian (DAD) |
| Different persons | Inspector A | Inspector B | Inspector C |
3. Relative retention time
The 18 kinds of amino acids are located in chromatographic peak under the condition of drawing up chromatogram, and when the durability of the relative correction factor is examined, the relative retention time of the peaks of the rest amino acids is calculated by taking glutamic acid as an internal reference, and the durability is also examined. The results of the 18 amino acids relative retention time measurements are shown in table 7 below:
TABLE 7 results of relative retention time measurements
4. Chromatographic conditions
An Agilent high performance liquid chromatograph is adopted, and octadecylsilane chemically bonded silica is used as a filler chromatographic column; gradient elution is carried out by taking methanol-acetonitrile-water (20:60:20) as a mobile phase A and taking 0.1mol/L sodium acetate buffer solution (pH value is adjusted to be 6.3 by glacial acetic acid) -the mobile phase A (97:3) as a mobile phase B; the flow rate is 1.0mL per minute, the detection wavelength is 254nm, the column temperature is 30 ℃, and the injection volume is 2 muL.
TABLE 8 elution gradient time Table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 0 | 100 |
| 15 | 14 | 86 |
| 20 | 17 | 83 |
| 50 | 44 | 56 |
| 50.1 | 100 | 0 |
| 55 | 100 | 0 |
| 56 | 0 | 100 |
| 65 | 0 | 100 |
5. Determination of the Standard Curve
1) Preparation of reference stock solution
Precisely weighing 15.0mg of glutamic acid reference substance, placing the glutamic acid reference substance in a 100mL measuring flask, adding 0.1mol/L hydrochloric acid solution for dissolving and diluting to scale, and shaking up to be used as reference substance stock solution.
2) Preparation of Standard Curve series solutions
Precisely measuring 1mL, 2mL, 4mL and 10mL of reference stock solutions respectively, placing in a 20mL measuring flask, adding water to dilute to scale, and shaking up to obtain reference solutions 1-4; control solution 5 was the same as the control stock solution.
3) Derivatization of solutions of the Standard Curve series
Respectively precisely measuring 4.0mL of the standard curve series solution, and dividing the standard curve series solution into 20mL test tubes with plugs; adding 2.0mL of 0.1mol/L phenyl isothiocyanate acetonitrile solution and 1.0mL of 1mol/L triethylamine acetonitrile solution, shaking up, reacting for 1 hour at room temperature, adding 8mL of n-hexane for extraction, standing for 10 minutes, taking the lower layer solution, filtering with 0.45um organic filter membrane, and taking the subsequent filtrate to obtain the final product.
4) Measurement of
And precisely measuring 2 mu L of the derivatized standard curve series solutions respectively, injecting the solutions into a liquid chromatograph, and recording the chromatogram. And performing linear regression on the peak area of the glutamic acid according to a least square method by using the concentration of the glutamic acid to obtain a standard curve equation and a linear correlation coefficient (r is more than 0.999). Table 9 below is the standard curve equation for glutamic acid measured for 3 consecutive days:
TABLE 9 glutamic acid standard curve equation
| Serial number | Standard curve equation of glutamic acid |
| 1 | Y(Peak area)＝5.68*C(glutamic acid concentration)-1.59 |
| 2 | Y(Peak area)＝5.49*C(glutamic acid concentration)-2.58 |
| 3 | Y(Peak area)＝5.58*C(glutamic acid concentration)-3.24 |
6. Determination of samples
1) Preparation of test solution
Precisely weighing about 1.5g of transfer factor capsule content, placing the content in a 25mL measuring flask, adding a proper amount of 0.1mol/L hydrochloric acid solution, oscillating for 2min to dissolve the transfer factor, adding 0.1mol/L hydrochloric acid solution to dilute to scale, shaking up, filtering, precisely weighing 2mL of subsequent filtrate, placing the subsequent filtrate in a digestion tube, adding 6mL of hydrochloric acid, filling nitrogen, sealing, and hydrolyzing at 110 ℃ for 24 hours. Hydrolyzing the solution for 24 hours; cooling, unsealing, putting in an evaporating dish, and evaporating in water bath to dryness; the residue was dissolved in 10mL of 0.1mol/L hydrochloric acid solution to prepare a sample solution for deriving the total amino acid assay.
2) Derivatization of test solutions
Precisely measuring 4.0mL of the test solution, and placing the test solution into a 20mL test tube with a plug; adding 2.0mL of 0.1mol/L phenyl isothiocyanate acetonitrile solution and 1.0mL of 1mol/L triethylamine acetonitrile solution, shaking up, reacting for 1 hour at room temperature, adding 8mL of n-hexane for extraction, standing for 10 minutes, taking the lower layer solution, filtering with 0.45um organic filter membrane, and taking the subsequent filtrate to obtain the final product.
3) Measurement of
Precisely measuring 2 μ L of the derivatized sample solution, injecting into a liquid chromatograph, and recording chromatogram. And (3) positioning chromatographic peaks of 18 amino acids according to the relative retention time of each amino acid in the table 7, substituting the area of each chromatographic peak into a standard curve equation to calculate the content of each amino acid, multiplying the content by the corresponding relative correction factor of each amino acid in the table 5, and adding to obtain the total content of the amino acids.
Example 4 comparison of results of determination of total amino acid content of transfer factor capsules by single-control one-test-multiple-evaluation method and multiple-control external standard method
Respectively adopting a single-control one-test-multiple-evaluation method and a multiple-control external standard method to determine the total amino acid content in the same 10 batches of transfer factor capsules, wherein the results show that the determination results of the single-control one-test-multiple-evaluation method and the multiple-control external standard method are basically consistent; the results are shown in Table 10.
TABLE 10 comparison table of results of measuring total amino acid content of transfer factor capsule by two methods
It can be seen that the single-contrast total amino acid quantitative detection method of the invention has high detection efficiency compared with the multi-contrast external standard method, needs few contrast products and obviously reduces the detection cost.
In conclusion, according to the method disclosed by the invention, the free amino acid is subjected to pre-column derivatization by adopting a Phenyl Isothiocyanate (PITC) derivatization method; the detection chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler chromatographic column; performing gradient elution by using methanol-acetonitrile-water (20:60:20) as a mobile phase A and 0.1mol/L sodium acetate buffer solution (pH value is adjusted to be 6.3 by glacial acetic acid) as a mobile phase B, wherein the volume ratio of the gradient elution is 0:100(0min), 14:86(15min), 17:83(20min), 44:56(50min), 100:0(50.1min), 100:0(55min), 0:100(56min) and 0:100(65 min); using a UV detector, the detection wavelength is 254 nm; the column temperature is 30 ℃; the injection volume was 2. mu.l. The technical problems that only free amino acid can be detected but bonded amino acid which forms polypeptide substances can not be detected in the prior art and the separation degree of each amino acid peak and an unknown component peak is poor due to transfer factors which are multi-component biochemical drugs in the amino acid detection process are solved; meanwhile, the invention adopts a 'one-test-multiple-evaluation' method to realize the quantitative detection of 18 common amino acids by one glutamic acid reference substance, thereby solving the technical problems of more reference substances, higher detection cost and fussy operation in an external standard method. Glutamic acid is taken as an internal reference, and relative correction factors and relative retention time of other 17 amino acids are determined through systematic verification research; realizing the quantitative detection of 18 amino acids. The detection method is accurate and reliable, has good separation effect and strong specificity, and can effectively detect and monitor the total amino acids in the transfer factor capsule.
Fig. 1 and fig. 2 are representative chromatograms of the transfer factor capsule in the process of measuring the total amino acid content, fig. 1 is a chromatogram of a mixed control solution of 18 kinds of amino acids, and fig. 2 is a chromatogram of a test solution for measuring the total amino acid content. The atlas reflects that the peak shape of each amino acid peak in the total amino acid in the transfer factor capsule determined by the method is good, the separation degree is high, the overlapping proportion between adjacent component peaks is greatly reduced, and the detection accuracy is improved.
Claims (5)
1. A method for quantitatively detecting total amino acids of a transfer factor capsule is characterized by comprising the following steps:
1) preparation of test solution for total amino acid determination derivation
Precisely weighing 0.9-1.2 g of transfer factor capsule content, adding 0.1mol/L hydrochloric acid solution to fully dissolve the transfer factor, shaking up, standing, taking supernate, filtering with a microporous membrane, then precisely taking 2.0mL of subsequent filtrate, placing into a reaction container, adding 6mL of concentrated hydrochloric acid solution, charging nitrogen, sealing, hydrolyzing at 110 ℃ for 24h, cooling to obtain transfer factor acid hydrolysis solution, unsealing the transfer factor acid hydrolysis solution, performing water bath evaporation to dryness, adding 10mL of 0.1mol/L hydrochloric acid solution to dissolve residues, and obtaining a sample solution for total amino acid determination derivatives;
2) preparing a test solution for measuring the content of total amino acids
Precisely measuring 4.0mL of a sample solution for determination of total amino acid, 2.0mL of a 0.1mol/L phenylisothiocyanate acetonitrile solution and 1.0mL of a 1mol/L triethylamine acetonitrile solution, mixing, shaking up, reacting at room temperature for 1h, extracting with 8.0mL of n-hexane, standing for 10min, filtering a lower layer solution with a microporous filter membrane, and taking a subsequent filtrate to obtain a sample solution for determination of total amino acid content;
3) determination of the Standard Curve
Preparing a standard curve series solution by taking glutamic acid as a reference substance, obtaining a chromatogram by high performance liquid chromatography, and performing linear regression on the peak area of the solution by the concentration of the glutamic acid according to a least square method to obtain a standard curve equation and a linear correlation coefficient;
4) precisely measuring 2 mu L of the total amino acid content determination test sample solution prepared in the step 2), injecting into a high performance liquid chromatograph, recording a chromatogram, obtaining 18 amino acid chromatographic peaks which take glutamic acid as an internal reference substance and are positioned according to relative retention time in the total amino acid content determination test sample solution, substituting the area of each chromatographic peak into a standard curve equation to obtain the content of each amino acid, multiplying the content by relative correction factors of each amino acid relative to the glutamic acid, and summing to calculate the total amino acid content in the transfer factor capsule;
in the step 1), the transfer factor acid hydrolysis solution contains 1.6-2.4 mg/mL of total amino acids; in the step 2), the content of the total amino acid is determined, and the total amino acid contained in the test solution is 0.4-0.8 mg/mL; in the step 2), filtering the lower-layer solution by using a 0.22-0.45 mu m microporous filter membrane;
in the step 4), the relative correction factor of each amino acid relative to glutamic acid is measured by using glutamic acid as an internal reference material and adopting a standard curve method;
determination of total amino acid content the results of the relative retention time measurements of 18 amino acids in the test solution were as follows:
in the step 3) and the step 4), the detection conditions of the high performance liquid chromatography are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;
the mobile phases used for gradient elution were:
mobile phase A: according to the proportion of methanol: acetonitrile: water = 20:60:20 in volume ratio;
mobile phase B: according to the sodium acetate buffer solution: a solution prepared by the volume ratio of the mobile phase A =97: 3;
the concentration of the sodium acetate buffer solution is 0.1mol/L, and the pH value is 6.0-6.5;
detection wavelength: 210-320 nm;
the sample injection amount is 1-10 mu L;
the flow rate is 0.5-2 mL/min;
the column temperature is 25-50 ℃;
the ratio of change of mobile phase a to mobile phase B during gradient elution was as follows:
0min, mobile phase B: 100 percent;
15min, mobile phase A: 14%, mobile phase B: 86 percent;
20min, mobile phase A: 17%, mobile phase B: 83 percent;
50min, mobile phase A: 44%, mobile phase B: 56 percent;
50.1min, mobile phase A: 100 percent;
55min, mobile phase B: 100 percent;
65min, mobile phase B: 100 percent.
2. The method for quantitatively detecting the total amino acids in the transfer factor capsule according to claim 1, wherein the particle size of the octadecylsilane chemically bonded silica is 5 to 10 μm; the inner diameter of the chromatographic column is 2-5 mm, and the length of the chromatographic column is 10-30 cm.
3. The method for quantitatively detecting the total amino acids in the transfer factor capsule according to claim 1, wherein the specific method for determining the standard curve in step 3) is as follows:
(1) preparing reference substance stock solution
Precisely weighing 15.0mg of glutamic acid reference substance, placing the glutamic acid reference substance in a 100mL measuring flask, adding 0.1mol/L hydrochloric acid solution for dissolving, diluting to scale, and shaking up to obtain reference substance stock solution;
(2) preparing standard curve series solution
Precisely measuring 1mL, 2mL, 4mL and 10mL of reference substance stock solutions respectively, placing in a 20mL measuring flask, adding water to dilute to scale, shaking up, and using as reference solutions 1-4, and using reference solution 5 and reference substance stock solution;
(3) derivatization of solutions of the Standard Curve series
Respectively precisely measuring 1-5 control solutions with the volume of 4.0mL, respectively placing the control solutions into 20mL test tubes with plugs, adding 2.0mL of 0.1mol/L phenyl isothiocyanate acetonitrile solution and 1.0mL of 1mol/L triethylamine acetonitrile solution, shaking uniformly, reacting at room temperature for 1h, adding 8mL of n-hexane for extraction, standing for 10min, filtering the lower layer solution with an organic filter membrane to obtain 5 derived standard curve series solutions;
(4) measuring and calculating a standard curve
Respectively and precisely measuring 2 mu L of each of the 5 derived standard curve series solutions, respectively injecting into a high performance liquid chromatograph, recording a chromatogram, and performing linear regression on the peak area of the standard curve series solution according to a least square method by using the concentration of glutamic acid to obtain a standard curve equation and a linear correlation coefficient.
4. The method for quantitatively detecting the total amino acids in the transfer factor capsule according to claim 1, wherein the detection results of the 18 amino acids relative to the correction factor are as follows:
5. the method for quantitatively detecting the total amino acids in the transfer factor capsules according to claim 4, further comprising examining the relative correction factor durability index, i.e., examining the relative correction factor durability under different chromatographic conditions.
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