CN107505422B - Method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time - Google Patents
Method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time Download PDFInfo
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- CN107505422B CN107505422B CN201710638303.XA CN201710638303A CN107505422B CN 107505422 B CN107505422 B CN 107505422B CN 201710638303 A CN201710638303 A CN 201710638303A CN 107505422 B CN107505422 B CN 107505422B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
A method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time comprises the following steps: 1) preparing a sample to be detected; 2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows: the chromatographic column is an Inertsil ODS-3 column, the sample loading amount of a detection sample is 10 mu l, the column temperature is 26 +/-2 ℃, and the mobile phase is as follows: gradient leaching or mixed solution of 80-98% phosphate buffer solution and 2-20% methanol, wherein the flow rate is 0.6-1.41 mL/min; when gradient leaching is carried out: when the time is 0-10 min, the time is a mixed solution of 95-100% of phosphate buffer solution and 0-5% of methanol; when the time is 11-40 min, the time is 85% -95% phosphate buffer solution or mixed solution of water and 5% -15% methanol; the concentration of the phosphate buffer solution is 20-60 mmol/l, and the pH value is 6.3-6.8; compared with the prior art, the method has the characteristics of simplicity, high sensitivity and good repeatability.
Description
Technical Field
The invention belongs to the technical field of detection and analysis, and particularly relates to a method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time.
Background
Adenosine Triphosphate (ATP) is a direct source of energy required by all vital activities of tissue cells in a living body, is known as 'molecular currency' of intracellular energy, can promote the repair and regeneration of various cells of the body, enhances the metabolic activity of the cells, and has stronger pertinence to the treatment of various diseases. ATP can be decomposed into Adenosine Diphosphate (ADP) and Adenosine Monophosphate (AMP), Adenosine (Adenosine) is an important intermediate for synthesizing ATP, 2-deoxynucleotide (2-AD) is one of important elements for synthesizing DNA, and the five compounds coexist in some plants and fungi. In the analysis and detection of some crops, the separation and detection of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate and deoxynucleotide are of great significance. At present, capillary electrophoresis separation detection methods are used, such as: chinese university of pharmacy proceedings, 1993, 24 (4): 219-221, named as separation analysis of adenosine triphosphate and decomposition products thereof by micellar electrokinetic capillary chromatography; journal of analytical chemistry, 10 months 2003, vol 19, No. 5, article number: 1006-6144(2003)05-0433-02, entitled adenosine triphosphate adenosine diphosphate and adenosine monophosphate. There are also methods using High Performance Liquid Chromatography (High Performance Liquid Chromatography, which is also known as "High pressure Liquid Chromatography", "High Performance Liquid Chromatography", "High resolution Liquid Chromatography", "modern column Chromatography", etc., High Performance Liquid Chromatography is an important branch of Chromatography, and uses Liquid as a mobile phase, and a High pressure infusion system is used to pump mobile phases such as single solvents with different polarities or mixed solvents, buffers, etc. with different proportions into a chromatographic column containing a stationary phase, and after each component in the column is separated, the mobile phases enter a detector for detection, thereby realizing analysis of a sample), such as research bulletin, 1997, 17 (4): 245-247, named high performance liquid chromatography for determining ATP preparation content. However, the prior art is directed to a method for detecting and separating three compounds of adenosine triphosphate, adenosine diphosphate and adenosine monophosphate, and five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate and deoxynucleotide cannot be detected and separated at one time, and the problems of troublesome operation, low sensitivity and poor repeatability exist.
Disclosure of Invention
The invention aims to provide a method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time, which is simple, high in sensitivity and good in repeatability.
The technical scheme of the invention is as follows:
the invention provides a method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time, which is characterized by comprising the following steps: it comprises the following steps:
1) preparing a sample to be detected;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows: the chromatographic column is an Inertsil ODS-3 column, the sample loading amount of a detection sample is 10 mu l, the column temperature is 10-40 ℃, and the mobile phase is as follows: gradient leaching or mixed solution of 80-98% phosphate buffer solution and 2-20% methanol, wherein the flow rate is 0.6-1.41 mL/min;
when gradient leaching is carried out: when the time is 0-10 min, the time is a mixed solution of 95-100% of phosphate buffer solution and 0-5% of methanol; when the time is 11-40 min, the time is 85% -95% phosphate buffer solution or mixed solution of water and 5% -15% methanol;
the concentration of the phosphate buffer solution is 20-60 mmol/l, and the pH value is 6.3-6.8.
Preferably, the column temperature is 20-30 ℃.
Preferably, the flow rate is 0.8 to 1.21 mL/min.
Preferably, the mobile phase is: a mixed solution of 95% phosphate buffer solution and 5% methanol, wherein the elution time is 40 min; the concentration of the phosphate buffer solution is 30mmol/l or 40mmol/l, and the pH value is 6.7.
Preferably, the mobile phase is: taking phosphate buffer solution for 0-10 min, and taking 95% phosphate buffer solution and 5% methanol for 11-40 min; the concentration of the phosphate buffer was 30mmol pH 6.7 or 40mmol pH 6.5.
Preferably, the mobile phase is: phosphate buffer solution is used for 0-10 min, and 95% water and 5% methanol are used for 11-40 min; the concentration of the phosphate buffer solution is 30mmol, and the pH value is 6.7.
Preferably, the mobile phase is: phosphate buffer solution is used for 0-10 min, and 95% water and 15% methanol are used for 11-30 min; the concentration of the phosphate buffer solution is 30mmol, and the pH value is 6.7; the column temperature was 26 ℃.
Preferably, the chromatographic column is an Inertsil ODS-3C18 column, with dimensions of 250mm × 4.6mm, i.d.5 μm.
After the method is adopted, the beneficial effects of the invention are as follows:
1. the five compounds can be separated and detected at one time by adopting an HPLC method, and can be completed within 40min, so that the method is quick and simple, good in repeatability and high in sensitivity, and a method is provided for agricultural product detection in agriculture.
2. The concentration of the phosphate buffer solution adopted by the mobile phase is low, the damage to the column can be reduced, the use amount of phosphate is reduced by the gradient leaching method, and the service life of the column is effectively prolonged.
3. And the adopted mobile phase also avoids the use of various organic matters, and has less dosage and pollution reduction.
Drawings
FIG. 1 is a detection map according to a first embodiment of the present invention.
FIG. 2 is a detection map of example two of the present invention.
FIG. 3 is a detection map of the third embodiment of the present invention.
FIG. 4 is a detection map of the fourth embodiment of the present invention.
FIG. 5 is a detection map of example five of the present invention.
FIG. 6 is a detection map of example six of the present invention.
In the drawings: the appearance sequence of the five peaks is Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), Adenosine Monophosphate (AMP), adenosine and deoxynucleotide.
Detailed Description
1. Reagents and instrumentation:
the potassium dihydrogen phosphate, the sodium hydroxide and the methanol are all domestic analytical purifications; the phosphate buffer solution is prepared from potassium dihydrogen phosphate and sodium hydroxide.
Agilent 1250 high performance liquid chromatograph (Agilent corporation, usa), G1322A vacuum degasser, G1367B autosampler, G1316A column oven; the chromatographic column is an Inertsil ODS-3C18 column with the size of 250mm multiplied by 4.6mm, i.d.5 μm; an electronic balance.
2. Liquid condition:
mobile phase: a is phosphate buffer solution, B is methanol, chromatographic column is Inertsil ODS-3C18 column, its size is 250mm x 4.6mm, i.d.5 μm, sample injection volume is 10 μ L, column temperature is 25-30 deg.C, flow rate is 1 ml/min; the detection wavelength of the detector is 254-260 nm.
The samples to be tested were: 1.497ml of Adenosine (133.62mg/l), 1.486ml of 2-AD (134.6mg/l), 1.825ml of AMP (109.581mg/l), 0.907ml of ATP (220.456mg/l) and 1.170ml of ADP (170.88mg/l) are taken and are made into a volume of 10ml by using ultrapure water, and the concentration of the mixed standard sample is respectively 20 mg/l.
A method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time is characterized in that: it comprises the following steps:
1) preparing a sample to be detected;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows: the chromatographic column is an Inertsil ODS-3 column, the sample loading amount of a detection sample is 10 mu l, the column temperature is 10-40 ℃, and the mobile phase is as follows: gradient leaching or mixed solution of 80-98% phosphate buffer solution and 2-20% methanol, wherein the flow rate is 0.6-1.41 mL/min;
when gradient leaching is carried out: when the time is 0-10 min, the time is a mixed solution of 95-100% of phosphate buffer solution and 0-5% of methanol; when the time is 11-40 min, the time is 85% -95% phosphate buffer solution or mixed solution of water and 5% -15% methanol;
the concentration of the phosphate buffer solution is 20-60 mmol/l, and the pH value is 6.3-6.8.
Preferably, the column temperature is 20-30 ℃.
Preferably, the flow rate is 0.8 to 1.21 mL/min.
Preferably, the mobile phase is: a mixed solution of 95% phosphate buffer solution and 5% methanol, wherein the elution time is 40 min; the concentration of the phosphate buffer solution is 30mmol/l or 40mmol/l, and the pH value is 6.7.
Preferably, the mobile phase is: taking phosphate buffer solution for 0-10 min, and taking 95% phosphate buffer solution and 5% methanol for 11-40 min; the concentration of the phosphate buffer was 30mmol pH 6.7 or 40mmol pH 6.5.
Preferably, the mobile phase is: phosphate buffer solution is used for 0-10 min, and 95% water and 5% methanol are used for 11-40 min; the concentration of the phosphate buffer solution is 30mmol, and the pH value is 6.7.
Preferably, the mobile phase is: phosphate buffer solution is used for 0-10 min, and 95% water and 15% methanol are used for 11-30 min; the concentration of the phosphate buffer solution is 30mmol, and the pH value is 6.7; the column temperature was 26 ℃.
Preferably, the chromatographic column is an Inertsil ODS-3C18 column, with dimensions of 250mm × 4.6mm, i.d.5 μm.
Example one
1) Preparing a sample to be detected;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows:
sample introduction amount: 10 μ l
Detection wavelength: 254nm
Column temperature: 26 deg.C
Flow rate: 1.0ml/min
Mobile phase: phosphate buffer (40mmol/l, pH 6.5) + methanol 95% + 5%
As a result, five mixed labels are completely and successfully separated and detected within 40min, and the map is shown in figure 1.
Example two
1) Preparing a sample to be detected;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows:
the column was equilibrated with 40mmol/l of phosphate buffer pH 6.5, with the following parameters:
sample introduction amount: 10 μ l
Wavelength: 254nm
Column temperature: 26 deg.C
Flow rate: 1.0ml/min
Mobile phase: gradient elution
As a result, three adenosine phosphate peaks are relatively early and dense, five mixed standards are completely and successfully separated and detected within 40min, and the map is shown in figure 2.
EXAMPLE III
1) Preparing a sample to be detected;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows:
the column was equilibrated with 30mmol/l of phosphate buffer pH 6.7, with the following parameters:
wavelength: 254nm
Column temperature: 26 deg.C
Flow rate: 1.0ml/min
Mobile phase: gradient elution
Five mixed labels are completely and successfully separated and detected within 40min, and the map is shown in figure 3.
Example four
1) Preparing a sample to be detected;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows:
the column was equilibrated with 30mmol/l of phosphate buffer pH 6.7, with the following parameters:
wavelength: 254nm
Column temperature: 26 deg.C
Flow rate: 1.0ml/min
Mobile phase: gradient elution
Five mixed labels are completely and successfully separated and detected within 45min, and the map is shown in figure 4.
EXAMPLE five
1) Preparing a sample to be detected;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows:
wavelength: 254nm
Column temperature: 26 deg.C
Flow rate: 1.0ml/min
Mobile phase: gradient elution
Five mixed labels are completely and successfully separated and detected within 40min, and the map is shown in figure 5.
EXAMPLE six
1) Preparing a sample to be detected;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows:
wavelength: 254nm
Column temperature: 26 deg.C
Flow rate: 1.0ml/min
Mobile phase: gradient elution
Five mixed labels are completely and successfully separated and detected within 20min, and the map is shown in figure 6.
The above examples are only preferred embodiments of the present invention, and the present invention is not limited to the above embodiments but also allows other structural changes and parameter changes, all of which are within the scope of the independent claims of the present invention.
Claims (7)
1. A method for separating and detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide at one time is characterized in that: it comprises the following steps:
1) preparing a sample to be detected; the sample to be detected is a mixed standard sample of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide;
2) carrying out HPLC detection on the sample in the step 1), wherein the detection conditions are as follows: the chromatographic column is an Inertsil ODS-3 column, the sample loading amount of a detection sample is 10 mu l, the column temperature is 26 +/-2 ℃, and the mobile phase is as follows: the flow rate of the mixed solution of 95% phosphate buffer solution and 5% methanol is 0.6-1.41 mL/min;
the concentration of the phosphate buffer solution is 20-60 mmol/l, and the pH value is 6.3-6.8.
2. The method for detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide by one-time separation according to claim 1, which comprises the following steps: the flow rate is 0.8-1.21 mL/min.
3. The method for detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide by one-time separation according to claim 1, which comprises the following steps: the elution time of the mobile phase is 40 min; the concentration of the phosphate buffer solution is 30mmol/l or 40 mmol/l; the pH was 6.7.
4. The method for detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide by one-time separation according to claim 1, which comprises the following steps: the concentration of the phosphate buffer is 30mmol/l pH 6.7 or the concentration of the phosphate buffer is 40mmol/l pH 6.5.
5. The method for detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide by one-time separation according to claim 1, which comprises the following steps: the concentration of the phosphate buffer solution is 30mmol/l, and the pH value is 6.7.
6. The method for detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide by one-time separation according to claim 1, which comprises the following steps: the concentration of the phosphate buffer solution is 30mmol/l, and the pH value is 6.7; the column temperature was 26 ℃.
7. The method for detecting five compounds of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine and deoxynucleotide by one-time separation according to claim 1, which comprises the following steps: the chromatographic column is an Inertsil ODS-3C18 column, and the size of the chromatographic column is 250mm multiplied by 4.6mm, i.d.5 mu m.
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| CN115060823B (en) * | 2022-06-13 | 2023-06-02 | 生工生物工程（上海）股份有限公司 | Method for separating nucleoside triphosphates and/or deoxynucleoside triphosphates by HPLC |
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| CN102854257A (en) * | 2011-07-01 | 2013-01-02 | 谈善军 | Method for quantitatively detecting ATP, ADP, and AMP in tissue |
| CN104237412B (en) * | 2014-09-18 | 2016-11-30 | 上海海洋大学 | A kind of high performance liquid chromatography-diode array measures the method that in aquatic products, multiple ATP closes co-product simultaneously |
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