CN108931592A - A kind of quantitative detecting method of oral liquid capable of transferring factors nucleotide substance - Google Patents
A kind of quantitative detecting method of oral liquid capable of transferring factors nucleotide substance Download PDFInfo
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Abstract
The invention discloses a kind of quantitative detecting methods of oral liquid capable of transferring factors nucleotide substance, belong to multicomponent biochemical drug quality research field.Method includes：1) nucleotide analog matter reference substance is utilized, is detected with high performance liquid chromatography and obtains chromatogram, constructs calibration curve equation；2) test solution is made in oral liquid capable of transferring factors to be detected, the corresponding chromatographic peak area of each nucleotide is extracted from chromatogram；3) area for the chromatographic peak that the calibration curve equation and step 2) obtained according to step 1) obtains, calculates the content of transfer factor oral solution nucleotide substance.It can effectively solve the problem that using method of the invention and be only capable of general one substance of measurement in the prior art, and detection interfering substance is more, the technical problem of testing result poor reproducibility.Checking research through the system detection method is interfered without other substances, specificity is strong, accurate and reliable, can be effectively to free nucleotide detection and monitoring in transfer factor oral solution.
Description
Technical field
The invention belongs to multicomponent Biochemical Drugs quality analysis studying technological domains, and in particular to a kind of oral liquid capable of transferring factors
The quantitative detecting method of nucleotide substance.
Background technique
Transfer factor (transter factor, abbreviation TF) is also known as transmission factor, by the leaching with cellular immunity function
Bar cell generates, and the immune response of body is participated in as a kind of specific cell factor, can improve animal body cellular immunity function
Energy.Transfer factor oral solution is to take orally the immunopotentiator for form of medication.Transfer factor belongs to polypeptides matter, be by
The compound molecule of polynucleotides, polypeptide and other compositions composition.Wherein small-molecular peptides substance and ucleotides substance are transfers
The function marker of the factor, and as its main composition.Therefore, the nucleotide content in Accurate Determining transfer factor for
Optimization process for producing transfer factors and control product quality have great importance.
So far, the relevant report about transfer factor nucleotide content assaying method is using orcin method
Chromogenic reaction is detected by reference substance of D-ribose.The principle of this method is the ribose that orcinol reagent and nucleolytic generate
Reaction generates furfural colour developing, calculates Ribose concentration by D-ribose regression curve, but pentose has this reaction, specificity is not strong；This
The outer method is only to can detect ribonucleic acid (RNA) according to the chromogenic reaction of the ribose moieties in conjunction with purine bases in RNA and contain
Amount, undetectable DNA (DNA) content.It is not single-minded to be directed to ribose due to the extracting method of transfer factor solution
Ucleotides substance, during the extraction process may there is also a large amount of deoxyribonucleotide substances, but the above method is not right
Substance of this kind is measured and controls.
Thus, it will be seen that the prior art is only capable of general one substance of measurement, and detection interfering substance is more, detection knot
Fruit poor reproducibility.
Summary of the invention
In order to overcome the problems of the above-mentioned prior art, the purpose of the present invention is to provide a kind of oral liquid capable of transferring factors
The quantitative detecting method of nucleotide substance, this method have accuracy height, favorable reproducibility, advantage easy to operate.
The present invention is to be achieved through the following technical solutions：
The quantitative detecting method of oral liquid capable of transferring factors nucleotide substance disclosed by the invention, includes the following steps：
1) calibration curve equation of ucleotides substance is constructed
Precision measures each reference substance ucleotides substance and stock solution is made, and building standard curve institute then is made in stock solution
The reference substance serial solution needed；It is detected using high performance liquid chromatography and obtains chromatogram, with each nucleotide in ucleotides substance
Concentration carries out linear regression to nucleotide peak area each in chromatogram, according to least square method, finds out calibration curve equation；Its
In, chromatographic test strip part is：
Using octadecylsilane chemically bonded silica as filler chromatographic column；
Gradient elution is carried out with mobile phase A and Mobile phase B, wherein：Mobile phase A is methanol；Mobile phase B is that pH value is 3.5
~3.6 phosphate buffer solution；
In detection process：Wavelength is 210~320nm, and sample volume is 1~50 μ L, and flow velocity is 0.6~1.0mL/min, column temperature
It is 33~37 DEG C；
2) detection of oral liquid capable of transferring factors nucleotide substance
Precision measures oral liquid capable of transferring factors to be detected and test solution is made, using high performance liquid chromatography with step
Chromatogram is obtained under rapid 1) identical chromatographic test strip part, extracts each nucleotide in oral liquid capable of transferring factors nucleotide substance
Corresponding chromatographic peak area；
3) content of oral liquid capable of transferring factors nucleotide substance calculates
According to step 1) obtain calibration curve equation and step 2) obtain chromatographic peak area, be calculated transfer because
The content of sub- oral administration solution nucleotide substance.
The ucleotides substance includes 4 kinds of ribonucleotides, 4 kinds of deoxyribonucleotides and 4 kinds of nucleosides, specially：
Adenylic acid, cytidylic acid, uridylate, guanylic acid, adenyl-deoxyribonucleotide, cytimidine are de-
Oxygen nucleotide, thymidylic acid, guanine deoxyribonucleoside acid, adenosine, cytidine, uridine
And guanosine.
Preferably, when gradient elution, the variation ratio of mobile phase A and Mobile phase B is as follows：
0min, mobile phase A：3%, Mobile phase B：97%；
10min, mobile phase A：3%, Mobile phase B：97%；
10.1min mobile phase A：10%, Mobile phase B：90%；
20min, mobile phase A：10%, Mobile phase B：90%；
20.1min mobile phase A：3%, Mobile phase B：97%；
30min, mobile phase A：3%, Mobile phase B：97%.
Preferably, the phosphate buffer solution is the mixed solution of disodium hydrogen phosphate and sodium dihydrogen phosphate, and the mixing is molten
Disodium hydrogen phosphate concentration is 0.05mol/L in liquid, and the concentration of sodium dihydrogen phosphate is 0.05mol/L.
Preferably, in step 1), precision measures each reference substance ucleotides substance 20.0mg, is placed in 100mL measuring bottle, adds
Water dissolves and is diluted to scale, shakes up, as reference substance stock solution；Then, respectively it is accurate measure reference substance stock solution 0.1mL,
0.5mL, 1mL, 2.5mL and 5mL are respectively placed in 25mL measuring bottle, are diluted with water to scale, are shaken up, and it is molten that reference substance series is made
Liquid.
Preferably, in step 2), precision measures transfer factor oral solution 5mL to be detected, is placed in 10mL measuring bottle, adds
Water is diluted to scale, shakes up and test solution is made.
Preferably, the filler particle size of octadecyl silane chromatographic column is 5~10 μm, and internal diameter is 2~5mm, and length is
10~30cm.
Preferably, the pH value of Mobile phase B is 3.55；Detection wavelength is 260nm；Sample volume is 2 μ L, flow velocity 0.8mL/
Min, column temperature are 35 DEG C.
Compared with prior art, the invention has the following beneficial technical effects：
First, the present invention measures transfer factor oral solution center compared with the higher HPLC method of strong, accuracy using specificity
Thuja acid content, can effectively exclude interference of the substances such as pentose to testing result, and testing result is more acurrate compared with the prior art, can
It leans on.
Second, pass through screening chromatographic column, flowing phase composition, flowing phase pH value, chromatographic column column temperature and optimization elution time ladder
The measures such as degree, it is determined that detection chromatographic condition；It is detected using the condition, in liquid chromatogram, each nucleotide peak peak shape is good, separation
Degree is high, and testing result is accurate and reliable.
Third, the present invention may be implemented to measure while ribonucleic acid (RNA) and DNA (DNA) content, because
This is highly suitable for the detection and monitoring of the nucleotide in transfer factor oral solution.
4th, the prior art is using orcin method due to test operation complexity, and link is more, and disturbing factor is more, and measurement side
Method is the poor ultraviolet spectrophotometry of specificity, therefore testing result reproducibility is poor.And the method for the present invention test operation letter
Just, process control is tested, and measuring method is specificity and the high high performance liquid chromatography of accuracy；Therefore testing result is reappeared
Property is good.
Detailed description of the invention
Fig. 1 is 12 kinds of nucleotide reference substance solution chromatograms.
Fig. 2 is transfer factor oral solution test solution chromatogram.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
Transfer factor oral solution used in the embodiment of the present invention is " golden flower transfer factor oral solution ", national drug standard
H20013288。
The screening of 1 transfer factor oral solution nucleotide assay chromatographic condition of embodiment
With 3 factor, 3 horizontal quadrature experiment sieving transfer factor oral solution nucleotide assay chromatographic condition, chromatography
3 factors that condition is investigated are respectively Mobile phase B phosphate buffer pH value, chromatographic column column temperature and flow rate of mobile phase.According to upper
3 factor, the 3 horizontal quadrature test table for stating the optimum level value design of 3 factors is shown in Table 1, and orthogonal experiments analysis is shown in Table 2, table
3。
1 transfer factor oral solution nucleotide assay chromatographic condition of table screens orthogonal test table
2 transfer factor oral solution nucleotide assay chromatographic condition of table screens orthogonal experiments analytical table 1
3 transfer factor oral solution nucleotide assay chromatographic condition of table screens orthogonal experiments analytical table 2
Determine that the optimum level value of 3 factors is respectively Mobile phase B as can be seen from the above results investigating by single factor experiment
Phosphate buffer pH value 3.55,35 DEG C of chromatographic column column temperature, flow rate of mobile phase 0.8ml/min.Accordingly, it is determined that optimal chromatostrip
Part group is combined into：Mobile phase B phosphate buffer pH value 3.55,35 DEG C of chromatographic column column temperature, flow rate of mobile phase 0.8ml/min；And it passes through
Variance analysis finds that 3 factors are not significant.
2 high effective liquid chromatography for measuring transfer factor oral solution nucleotide content of embodiment
1, chromatographic condition：It is filler color with octadecylsilane chemically bonded silica using Agilent high performance liquid chromatograph
Compose column；Using methanol as mobile phase A, with 0.05mol/L disodium hydrogen phosphate and 0.05mol/L sodium dihydrogen phosphate (with phosphoric acid tune
3.55) saving pH value to be is that Mobile phase B carries out gradient elution；Flow velocity is 0.8ml per minute, Detection wavelength 260nm, column temperature 35
DEG C, sampling volume is 2 μ l.The gradient elution time is shown in Table 4:
4 gradient elution timetable of table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 3 | 97 |
| 10 | 3 | 97 |
| 10.1 | 10 | 90 |
| 20 | 10 | 90 |
| 20.1 | 3 | 97 |
| 30 | 3 | 97 |
2, the measurement of standard curve：
1) preparation of reference substance stock solution：Precision weighs 4 kinds of nucleotide, and (adenylic acid, cytidylic acid, urine are phonetic
Pyridine nucleotide, guanylic acid), (adenyl-deoxyribonucleotide, deoxycytidylic acid, thymus gland are phonetic for 4 kinds of deoxynucleotides
Pyridine deoxynucleotide, guanine deoxyribonucleoside acid) and 4 kinds of nucleosides (adenosine, cytidine, uridine, bird are fast
Purine nucleosides) each 20.0mg of reference substance, it is placed in 100mL measuring bottle, is dissolved in water and is diluted to scale, shake up, store as reference substance
Standby liquid.
2) preparation of standard curve serial solution：Respectively it is accurate measure reference substance stock solution 0.1mL, 0.5mL, 1mL,
2.5mL, 5mL are placed in 25ml measuring bottle, are diluted with water to scale, shake up, as reference substance serial solution 1~5.
3) it measures：It is accurate respectively to measure above-mentioned reference substance serial solution 1~5 each 2 μ L, it is injected separately into liquid chromatograph,
Chromatogram is recorded, as a result as shown in Figure 1.Each nucleotide peak area is linearly returned by least square method with each nucleotide concentration
Return, finds out standard curve regression equation and linearly dependent coefficient (r>0.999).
3, the measurement of sample
1) preparation of test solution：Precision measures transfer factor oral solution 5mL, is placed in 10mL measuring bottle, adds water dilute
It releases to scale, shakes up to obtain the final product.
2) it measures, precision measures above-mentioned 2 μ L of test solution, injects liquid chromatograph, chromatogram is recorded, as a result such as Fig. 2
It is shown, nucleotide content is found out from regression equation.
The comparison of 3 experimental result of embodiment
It is surveyed using the orcin method (determined by ultraviolet spectrophotometry) of high performance liquid chromatography and the prior art of the invention
Determine transfer factor oral solution nucleotide content results to compare
High performance liquid chromatography is respectively adopted and orcin method measures and contains with 10 batches of transfer factor oral solution nucleotides
Amount, testing result see the table below 5：
5 two methods of table measure transfer factor oral solution nucleotide content results comparison sheet
The results show that due to survey while ribonucleic acid and deoxyribonucleic acid electrochemical biosensors may be implemented in high performance liquid chromatography
It is fixed, and orcin method is only capable of measurement rna content；Therefore high performance liquid chromatography testing result is significantly higher than orcin method.
Therefore, high effective liquid chromatography for measuring transfer factor oral solution nucleotide content strong, accuracy compared with orcin method specificity
High, favorable reproducibility.
Claims (8)
1. a kind of quantitative detecting method of oral liquid capable of transferring factors nucleotide substance, which is characterized in that include the following steps：
1) calibration curve equation of ucleotides substance is constructed
Precision measures each reference substance ucleotides substance and stock solution is made, and then stock solution is made needed for building standard curve
Reference substance serial solution；It is detected using high performance liquid chromatography and obtains chromatogram, with each nucleotide concentration in ucleotides substance
To nucleotide peak area each in chromatogram, linear regression is carried out according to least square method, finds out calibration curve equation and linear phase
Relationship number；Wherein, chromatographic test strip part is：
Using octadecylsilane chemically bonded silica as filler chromatographic column；
Gradient elution is carried out with mobile phase A and Mobile phase B, wherein：Mobile phase A is methanol；Mobile phase B is that pH value is 3.5~3.6
Phosphate buffer solution；
In detection process：Wavelength is 210~320nm, and sample volume is 1~50 μ L, and flow velocity is 0.6~1.0mL/min, column temperature 33
~37 DEG C；
2) detection of oral liquid capable of transferring factors nucleotide substance
Precision measures oral liquid capable of transferring factors to be detected and test solution is made, using high performance liquid chromatography with step 1)
Chromatogram is obtained under identical chromatographic test strip part, is extracted each nucleotide pair in oral liquid capable of transferring factors nucleotide substance and is answered
Chromatographic peak area；
3) content of oral liquid capable of transferring factors nucleotide substance calculates
The area for the chromatographic peak that the calibration curve equation and step 2) obtained according to step 1) obtains, is calculated transfer factor mouth
Take the content of solution nucleotide substance.
2. the quantitative detecting method of oral liquid capable of transferring factors nucleotide substance according to claim 1, feature exist
It is that adenylic acid, cytidylic acid, uridylate, guanylic acid, gland are fast in, the ucleotides substance
Purine deoxynucleotide, deoxycytidylic acid, thymidylic acid, guanine deoxyribonucleoside acid, adenosine,
Cytidine, uridine and guanosine.
3. the quantitative detecting method of oral liquid capable of transferring factors nucleotide substance according to claim 1, feature exist
When, gradient elution, the variation ratio of mobile phase A and Mobile phase B is as follows：
0min, mobile phase A：3%, Mobile phase B：97%；
10min, mobile phase A：3%, Mobile phase B：97%；
10.1min mobile phase A：10%, Mobile phase B：90%；
20min, mobile phase A：10%, Mobile phase B：90%；
20.1min mobile phase A：3%, Mobile phase B：97%；
30min, mobile phase A：3%, Mobile phase B：97%.
4. the quantitative detecting method of oral liquid capable of transferring factors nucleotide substance according to claim 1, feature exist
In the phosphate buffer solution is the mixed solution of disodium hydrogen phosphate and sodium dihydrogen phosphate, phosphoric acid hydrogen two in the mixed solution
Na concn is 0.05mol/L, and the concentration of sodium dihydrogen phosphate is 0.05mol/L.
5. the quantitative detecting method of oral liquid capable of transferring factors nucleotide substance according to claim 1, feature exist
In in step 1), precision measures each reference substance ucleotides substance 20.0mg, is placed in 100mL measuring bottle, is dissolved in water and dilutes
It to scale, shakes up, as reference substance stock solution；Then, respectively it is accurate measure reference substance stock solution 0.1mL, 0.5mL, 1mL,
2.5mL and 5mL is respectively placed in 25mL measuring bottle, is diluted with water to scale, is shaken up, and reference substance serial solution is made.
6. the quantitative detecting method of oral liquid capable of transferring factors nucleotide substance according to claim 1, feature exist
In in step 2), precision measures transfer factor oral solution 5mL to be detected, is placed in 10mL measuring bottle, is diluted with water to quarter
Degree, shakes up and test solution is made.
7. the quantitative detecting method of oral liquid capable of transferring factors nucleotide substance according to claim 1, feature exist
In the filler particle size of octadecyl silane chromatographic column is 5~10 μm, and internal diameter is 2~5mm, and length is 10~30cm.
8. the quantitative detecting method of oral liquid capable of transferring factors nucleotide substance according to claim 1, feature exist
In the pH value of Mobile phase B is 3.55；Detection wavelength is 260nm；Sample volume is 2 μ L, flow velocity 0.8mL/min, column temperature 35
℃。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109917058A (en) * | 2019-03-14 | 2019-06-21 | 南京农业大学 | The high-efficient liquid phase determining method of intracellular four kinds of free deoxynucleotides |
| CN110243964A (en) * | 2019-06-25 | 2019-09-17 | 西安建筑科技大学 | In a kind of water treatment technology in microbial body guanosine tetraphosphate HPLC detection method |
| CN113376287A (en) * | 2021-06-15 | 2021-09-10 | 劲牌有限公司 | Method for detecting activity of cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109917058A (en) * | 2019-03-14 | 2019-06-21 | 南京农业大学 | The high-efficient liquid phase determining method of intracellular four kinds of free deoxynucleotides |
| CN110243964A (en) * | 2019-06-25 | 2019-09-17 | 西安建筑科技大学 | In a kind of water treatment technology in microbial body guanosine tetraphosphate HPLC detection method |
| CN113376287A (en) * | 2021-06-15 | 2021-09-10 | 劲牌有限公司 | Method for detecting activity of cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography |
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