阅读说明：本技术 一种使用高效液相色谱测定粪便中褪黑素的方法 (Method for determining melatonin in excrement by using high performance liquid chromatography ) 是由 熊家军 李可可 何长久 齐德生 陈建国 许学林 荆焕松 孙旭阳 于 2020-03-23 设计创作，主要内容包括：本发明公开了一种使用高效液相色谱测定粪便中褪黑素的方法,包括：将待测粪便捣碎混匀,用冻干机冻干,甲醇浸提待测粪便,离心取上清液用氮吹仪吹干；用乙腈正己烷溶液溶解,离心取下层液,经微孔滤膜过滤；上机进行高效液相色谱检测,采用外标法对粪便样品中褪黑素进行定量。本发明首次实现了对粪便中褪黑素的提取和检测,检测方法的灵敏度高、专一性强、稳定性好、结果准确可靠,可以建立动物非创伤性褪黑素检测新方法,阐明动物内源褪黑素分泌昼夜节律。(The invention discloses a method for determining melatonin in excrement by using high performance liquid chromatography, which comprises the following steps: smashing and uniformly mixing the excrement to be detected, freeze-drying the excrement to be detected by using a freeze dryer, extracting the excrement to be detected by using methanol, centrifuging the obtained product, and taking supernatant liquid to blow and dry the obtained supernatant liquid by using a nitrogen blowing instrument; dissolving with acetonitrile n-hexane solution, centrifuging to obtain supernatant, and filtering with microporous membrane; and (4) performing high performance liquid chromatography detection on the excrement sample by using a machine, and quantifying melatonin in the excrement sample by using an external standard method. The invention realizes the extraction and detection of the melatonin in the excrement for the first time, the detection method has high sensitivity, strong specificity, good stability and accurate and reliable result, and a new animal non-traumatic melatonin detection method can be established to clarify the circadian rhythm of endogenous melatonin secretion of animals.)

1. A method for determining melatonin in excrement by using high performance liquid chromatography is characterized by comprising the following steps: the method comprises the following steps: smashing and uniformly mixing the excrement to be detected, freeze-drying the excrement to be detected by using a freeze dryer, extracting the excrement to be detected by using methanol, centrifuging the obtained product, and taking supernatant liquid to blow and dry the obtained supernatant liquid by using a nitrogen blowing instrument; dissolving with acetonitrile n-hexane solution, centrifuging to obtain supernatant, and filtering with microporous membrane; and (4) performing high performance liquid chromatography detection on the excrement sample by using a machine, and quantifying melatonin in the excrement sample by using an external standard method.

2. The method for determining melatonin in feces by using high performance liquid chromatography as claimed in claim 1, wherein: and (3) dissolving the mixture by using an acetonitrile normal hexane solution, centrifuging to take the supernatant, filtering by using a microporous filter membrane, and repeatedly centrifuging to take the supernatant.

3. The method for determining melatonin in feces by using high performance liquid chromatography as claimed in claim 1, wherein: the adding amount ratio of the feces to be detected to the methanol is 0.1g to 6 ml.

4. The method for determining melatonin in feces by using high performance liquid chromatography as claimed in claim 1, wherein: the centrifugation process is carried out at 12000RPM at 4 ℃ for 10 min.

5. The method for determining melatonin in feces by using high performance liquid chromatography as claimed in claim 1, wherein: the acetonitrile-n-hexane solution was a mixture of 1ml of acetonitrile water (4: 6, V/V) and 1ml of n-hexane.

6. The method for determining melatonin in feces by using high performance liquid chromatography as claimed in claim 1, wherein: the microporous filter membrane is a filter membrane with the pore diameter of 0.22 mu m.

7. The method for determining melatonin in excrement by using high performance liquid chromatography as claimed in claim 1, wherein the chromatographic conditions of the high performance liquid chromatography detection are that the chromatographic column is C18 chromatographic column 250 × 4.6.6 mm and 5um, the column temperature is 25 ℃, the detector is a fluorescence detector, the excitation wave Ex is 285nm, the emission wave Em is 345nm, the flow rate is 0.5m L/min, and the sample injection volume is 20u L.

8. The method for determining melatonin in feces by using high performance liquid chromatography as claimed in claim 1, wherein: the standard curve for quantifying melatonin by the external standard method is as follows: y is 0.0859x +0.065, wherein: y represents the peak area and x represents the melatonin concentration (ng/ml).

Technical Field

The invention relates to the technical field of melatonin detection, and particularly relates to a method for determining melatonin in excrement by using high performance liquid chromatography.

Background

Melatonin (Melatonin, MT) is an indole hormone with the chemical name N-acetyl-5-methoxytryptamine, and is a broad-spectrum physiological regulator. Melatonin is mainly derived from the pineal gland of mammals, and is proved to be the endogenous leucocyte scavenger with the strongest antioxidant effect, which is known at present and has important effects on sleep, gonadal development promotion and circadian rhythm. Animals find the existence of melatonin which has the effects of regulating circadian rhythm, photoperiod and growth development, the functions of the melatonin in the animals comprise the effects of scavenging free radicals, enhancing immunity, inhibiting aging and the like, the diversity of physiological and pharmacological functions of the melatonin is generally regarded by scholars at home and abroad, the food and drug administration in the United states approves the melatonin as a common dietary supplement, and the Ministry of health in China successively approves more than 20 products containing the melatonin as health-care food for improving sleep, and millions of people take the melatonin for a long time all over the world. By extracting and detecting the MT content in the plant material, the reasonable drinking amount of related food can be evaluated, and scientific basis is provided for development and utilization of healthy diet and functional health food of people.

At present, methods for detecting melatonin in animal tissues, blood plasma and blood serum exist in China, for example, in the 10 th phase 2010 of Hubei agricultural science, the content of the melatonin in the plasma of the cashmere goat is measured by taking the cashmere goat as a test material and adopting a radioactive immunoassay method to measure the level of the melatonin in the plasma of the cashmere goat at night within a one-year period. When animal tissues, blood plasma and blood serum are obtained, certain damage can be caused to the animal body, the production performance of the animal body is influenced, and meanwhile, a large amount of manpower and material resources are required to be used to cause economic loss. The method for detecting melatonin by collecting animal excrement is convenient to sample, can avoid the trauma to animals and reduce economic loss, has complex animal excrement components, and has difficulty in excrement pretreatment when melatonin is detected by the animal excrement, but no reports of animal excrement pretreatment and melatonin detection methods in the animal excrement exist at present. Therefore, it is necessary to study a method for detecting melatonin using animal excrements and an excrement pretreatment process.

Disclosure of Invention

The invention aims to provide a method for determining melatonin in excrement by using high performance liquid chromatography, which has the advantages of high sensitivity, strong specificity, good stability and accurate and reliable result.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for determining melatonin in stool using high performance liquid chromatography, comprising: smashing and uniformly mixing the excrement to be detected, freeze-drying the excrement to be detected by using a freeze dryer, extracting the excrement to be detected by using methanol, centrifuging the obtained product, and taking supernatant liquid to blow and dry the obtained supernatant liquid by using a nitrogen blowing instrument; dissolving with acetonitrile n-hexane solution, centrifuging to obtain supernatant, and filtering with microporous membrane; and (4) performing high performance liquid chromatography detection on the excrement sample by using a machine, and quantifying melatonin in the excrement sample by using an external standard method.

Preferably, the steps of repeated centrifugation and supernatant taking are added between the steps of dissolving by using acetonitrile-n-hexane solution, centrifuging and taking the supernatant and filtering by using a microporous filter membrane.

Preferably, the ratio of stool to be tested to methanol addition is 0.1g to 6 ml.

Preferably, the centrifugation is performed at 12000RPM at 4 deg.C for 10 min.

Preferably, the acetonitrile-n-hexane solution is a solution prepared by mixing 1ml of acetonitrile water (4: 6, V/V) and 1ml of n-hexane.

Preferably, the chromatographic conditions of the high performance liquid chromatography detection comprise a chromatographic column of C18, a chromatographic column of 250 × 4.6.6 mm and 5um, a column temperature of 25 ℃, a detector of fluorescence, an excitation wave Ex of 285nm, an emission wave Em of 345nm, a flow rate of 0.5m L/min and a sample injection volume of 20u L.

Preferably, the standard curve for quantifying melatonin by the external standard method is as follows: y is 0.0859x +0.065, wherein: y represents the peak area and x represents the melatonin concentration (ng/ml).

The invention has the beneficial effects that: the method pretreats animal feces, realizes extraction and detection of melatonin in the feces for the first time, has high sensitivity, strong specificity, good stability and accurate and reliable result, can establish a new method for detecting animal non-traumatic melatonin, and clarifies the circadian rhythm of endogenous melatonin secretion of animals.

Drawings

Fig. 1 is a melatonin standard curve of a control of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific embodiments. This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the invention to those skilled in the art, and the present invention will only be defined by the appended claims.

1. Chromatographic conditions

The chromatographic column comprises a C18 chromatographic column 250 × 4.6.6 mm and 5um, the column temperature is 25 ℃, the detector is a fluorescence detector, the excitation wave Ex is 285nm, the emission wave Em is 345nm, the flow rate is 0.5m L/min, and the sample injection volume is 20u L.

2. Preparation of reference substance solution and drawing of standard curve

Diluting a certain amount of melatonin standard substance with mobile phase to obtain melatonin concentrations of 10, 25, 50, 100, 250, 500, 1000ng/ml, and measuring with 20 μ l sample. And (4) taking the concentration of each standard solution as an abscissa and taking the peak area as an ordinate to draw a standard curve.

3. Preparation of fecal samples to be tested

3.1 sample Collection and processing

Collecting sika deer feces, taking 0.1 g/sample, and freeze-drying with a freeze dryer.

3.2 methanol extraction of melatonin

Taking 3ml of methanol to extract excrement in a freeze-drying bottle, shaking, transferring all the excrement into a 15ml centrifuge tube, adding 3ml of methanol for repeated extraction once, combining the two extracting solutions, centrifuging at 12000RPM for 10min at 4 ℃, taking supernatant, repeatedly centrifuging once, taking the supernatant, and drying by using a nitrogen blowing instrument.

3.3 degreasing and processing of n-hexane

Dissolving 1ml of acetonitrile (4: 6) and 1ml of n-hexane, centrifuging at 4 ℃ and 12000RPM for 10min, taking the supernatant, repeatedly centrifuging, taking the supernatant, filtering through a 0.22vm filter membrane, and loading on a machine for testing.

4. Determination of recovery

12 feces samples, No. 1, No. 2 and No. 3 are only added; 4. adding 100ng melatonin into No. 5 and No. 6 manure samples; 7. adding 250ng melatonin into No. 8 and No. 9 feces samples; 10. feces samples of nos. 11 and 12 were treated with 500ng melatonin in the same manner.

5. On-machine measurement

And (4) carrying out high performance liquid chromatography determination on the machine sample and the standard sample simultaneously. The chromatographic conditions were as described above.

6. Results

6.1 melatonin Linear relationship analysis

The prepared melatonin standard series solution is detected on a machine, and the concentration and the peak area of the standard solution are shown in table 1. A standard curve was prepared with the concentration (ng/ml) as abscissa and the peak area as ordinate, and the results are shown in FIG. 1. Linear equation is 0.0859x +0.065, correlation coefficient R²Is 0.9999. The linear relation between the concentration of melatonin in the range of 10-1000ng/ml and the peak area is good, and the melatonin can be used for quantitative analysis.

TABLE 1 melatonin Linear relationship analysis

6.2 precision analysis

The precision is another index for judging the quality of the method. Precision usually consists of parallelism and reproducibility. According to the regulation in the quality control standard of the national standard GB/T27404-2008 laboratory, the content of the measured components is lower than 100 mu g/kg, and the Coefficient of Variation (CV) in the laboratory is within 15 percent; the content of the tested components is lower than 10 mug/kg, and CV is within 21 percent; the content of the tested components is lower than 1 mu g/kg, and CV is within 30 percent. The test is repeated for six times by using a standard substance with the concentration of 50ng/ml, and the results are as follows: the coefficient of variation for retention time was 0.5876%, the coefficient of variation for peak height was 4.7619%, and the coefficient of variation for peak area was 5.3055%. Therefore, the parallelism and the reproducibility meet the requirements of GB/T27404-.

TABLE 2 melatonin precision test results

6.3 sample recovery

The sample standard adding recovery rate can reflect the accuracy of the detection method. According to the technical requirements of the detection method in the national standard GB/T27417-; when the content of the detected component is lower than 0.1ng/kg, the recovery rate ranges from 60% to 120%. The stool of the test is subjected to a standard adding experiment, and the result shows that when the standard is added to 1000ng/g, the standard adding average recovery rate of various samples is 83-105%; when the standard addition amount is 2500ng/g, the average recovery rate of various samples is 94-97%; when the standard adding amount is 5000ng/g, the average recovery rate of the standard adding of various samples is 106-109%. The result of the recovery rate in the feces meets the requirement of GB/T27417-2017, and the detection method established by the test has good accuracy.

TABLE 3 stool recovery with standard addition

7. Detecting the suitability of the method of the invention

The accuracy of the method of the invention was also compared with that of the E L ISA (enzyme-linked immunosorbent assay).

5 feces samples, only feces sample is added in No. 1; adding 25ng melatonin into No. 2 feces sample; adding 50ng melatonin into No. 3 feces sample; adding 250ng melatonin into No. 4 feces sample; 250ng of melatonin is added into the feces sample of No. 5, and then the melatonin in the feces sample of the panda is extracted by the method.

The detection result of the liquid chromatogram is obviously better than that of the E L ISA by comparing the detection of the melatonin in the panda feces by the liquid chromatogram with the detection of the melatonin in the panda feces by the E L ISA.

TABLE 4 stool spiked concentrations compared to E L ISA concentrations

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.