阅读说明：本技术 一种定量核磁共振氢谱测定杜仲胶含量的方法 (Method for measuring content of gutta-percha by quantitative nuclear magnetic resonance hydrogen spectrum ) 是由 赵红波 柴晓飞 吕印美 杨丽 王岳华 董昭苹 董淑杰 崔丽华 徐晓婷 孟庆春 于 2021-07-06 设计创作，主要内容包括：本发明属于分析技术领域,具体涉及一种定量核磁共振氢谱测定杜仲胶含量的方法。通过设定特定的测定过程中参数,排除溶剂中杂质及溶剂本身氢峰的干扰,以及被测组分和杂质之间的信号干扰,通过纵向弛豫时间T1值设定核磁共振波谱仪的脉冲倾倒角和弛豫时间,再用所述核磁共振波谱仪测定待测溶液的核磁氢谱。最终使得测定积分准确,检测结果重现性好。该方法快捷并且不需要目标测试物的标准品做外标,只需要高纯度的对照品作为参比即可。本发明能够快速、准确、稳定的测定杜仲胶含量。克服了常规运用提纯后直接称量的误差。本发明具有成本低,操作简单,结果准确,在测试样品时无需杜仲胶标准品,重复性好等优点,可快速评价杜仲提取物中杜仲胶的含量。(The invention belongs to the technical field of analysis, and particularly relates to a method for determining the content of gutta-percha by quantitative nuclear magnetic resonance hydrogen spectrum. The method comprises the steps of eliminating the interference of impurities in a solvent and hydrogen peaks of the solvent and the signal interference between a detected component and the impurities by setting parameters in a specific detection process, setting a pulse flip angle and a relaxation time of a nuclear magnetic resonance spectrometer by a longitudinal relaxation time T1 value, and detecting a nuclear magnetic hydrogen spectrum of a solution to be detected by the nuclear magnetic resonance spectrometer. Finally, the determination integration is accurate, and the detection result has good reproducibility. The method is quick, and only a high-purity reference substance is needed as a reference, and a standard substance of a target test substance is not needed to be used as an external standard. The method can rapidly, accurately and stably determine the content of the gutta-percha. The error of direct weighing after conventional application and purification is overcome. The method has the advantages of low cost, simple operation, accurate result, no need of a gutta-percha standard substance during sample testing, good repeatability and the like, and can quickly evaluate the content of the gutta-percha in the eucommia ulmoides extract.)

1. A method for measuring the content of gutta-percha by quantitative nuclear magnetic resonance hydrogen spectrum is characterized by comprising the following specific steps:

(1) accurately weighing a gutta-percha sample to be measured and an internal standard substance 1,placing 3, 5-trimethoxy benzene in a clean and dry nuclear magnetic tube, adding a deuterated solvent into the nuclear magnetic tube, sealing, placing in a fume hood, completely dissolving the sample, and performing¹H NMR test; wherein, the gutta percha (measured by the estimated gutta percha amount of the gutta percha sample to be detected): 1,3, 5-trimethoxybenzene: chloroform (12 to 37.5) mg: (8-15) mg: (0.4-0.6) ml;

(2) adjusting the instrument parameters: setting parameters of a nuclear magnetic resonance spectrometer, wherein the resonance frequency is 400MHz, the pulse pouring angle is 30-90 degrees, the measuring temperature is 23 +/-2 ℃, the acquisition time is 3s, the sampling frequency NS is 16-32 times, the relaxation time D1 is 15-30 s, shimming and sampling are carried out, and the obtained map is subjected to phase adjustment and baseline correction;

(3) integrating quantitative peaks of the gutta-percha sample and the 1,3, 5-trimethoxybenzene, wherein a proton peak of delta 5.12 is selected as the quantitative peak of the gutta-percha; selecting a proton peak of delta 6.09 as the quantity peak of the 1,3, 5-trimethoxybenzene, integrating for multiple times, averaging when the relative standard deviation of the integration result is less than 1%, and calculating by adopting the following formula according to the integration result:

in the formula:

w_sis the mass fraction of the sample containing the gutta-percha;

w_iis the mass fraction of the internal standard;

A_sand n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, respectively;

M_sis the molecular mass of an individual population of samples, m_sIs the weighed mass of the sample;

A_iand n_iThe integral area of the quantitative peak of the internal standard substance and the number of protons contained in the quantitative peak are respectively;

M_iis the molecular mass of the internal standard;

m_iis the weighed mass of the internal standard substance.

2. The method for quantitative nuclear magnetic resonance hydrogen spectrometry of gutta-percha content according to claim 1, wherein the mass ratio of the gutta-percha to the internal standard substance 1,3, 5-trimethoxybenzene is 1.5-2.5: 1.

3. The method for quantitative nuclear magnetic resonance hydrogen spectrometry of gutta-percha content as in claim 1, wherein said deuterated solvent is selected from deuterated chloroform, deuterated benzene, deuterated acetone, deuterated o-dichlorobenzene.

4. The method for quantitative nuclear magnetic resonance hydrogen spectrometry of gutta-percha content as in claim 3, wherein said deuterated solvent is selected from deuterated chloroform.

5. The method for determining the content of the gutta-percha through the quantitative nuclear magnetic resonance hydrogen spectrum according to claim 1, wherein the relaxation time D1 is 15 s; the pulse flip angle is 30 °.

Technical Field

The invention belongs to the technical field of analysis, and particularly relates to a method for determining the content of gutta-percha by quantitative nuclear magnetic resonance hydrogen spectrum.

Background

Eucommia bark is a peculiar rare economic tree species in China, contains a large amount of medical components, also contains a large amount of gutta-percha in tissues such as leaves, barks, fruit peels and the like, and is an important collagen plant with the widest application range in the world. Gutta-percha belongs to hard rubber, is a high polymer of trans-isoprene, has the same composition as natural rubber and has some similar properties, and is called as sister of natural rubber. The gutta percha has good insulativity, acid and alkali resistance, friction resistance and corrosion resistance, can be vulcanized to prepare high-elasticity bodies, can also control the crosslinking degree to generate new materials, such as shape memory functional materials, and is also an important raw material for manufacturing submarine cables, airplane tires and the like. Because the eucommia ulmoides gum is hard, soft and wear-resistant, has small side effect on human bodies, and is also used for making artificial joints. In a word, through vulcanization modification or blending with other materials and deep processing, the gutta percha can develop a series of novel functional materials and industrial materials covering the fields of plastics and rubber, and has wide application. It is unique in discovery of 'rubber-plastic duality', and develops the application field of the gutta percha. The comprehensive development and utilization of the composite material have attracted wide attention of relevant departments of the country.

At present, the determination of the gutta-percha content mainly adopts a gravimetric method, namely that the gutta-percha content (%) ═ (refined gum weight/sample weight) x 100%, and the gutta-percha is obtained after a series of extraction, does not contain other substances, and has no reliable basis to prove that the extracted substances are the gutta-percha completely. At present, no method can accurately and rapidly determine the content of the gutta-percha.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for determining the content of eucommia ulmoides gum by quantitative nuclear magnetic resonance hydrogen spectrum. The method comprises the steps of eliminating the interference of impurities in a solvent and hydrogen peaks of the solvent and the signal interference between a detected component and the impurities by setting parameters in a specific detection process, setting a pulse dumping angle and a relaxation time of a nuclear magnetic resonance spectrometer by a longitudinal relaxation time T1 value, and detecting a nuclear magnetic hydrogen spectrum of a solution to be detected by the nuclear magnetic resonance spectrometer. Finally, the determination integration is accurate, and the detection result has good reproducibility. The method is quick, and only a high-purity reference substance is needed as a reference, and a standard substance of a target mapping test substance is not needed to be used as an external standard. The method can rapidly, accurately and stably determine the content of the gutta-percha. The error of direct weighing after conventional application and purification is overcome.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

(1) accurately weighing a gutta-percha sample to be measured and an internal standard substance 1,3, 5-trimethoxybenzene, placing the gutta-percha sample and the internal standard substance in a clean and dry nuclear magnetic tube, adding a deuterated solvent into the nuclear magnetic tube, placing the tube in a fume hood after sealing, and performing the steps after the sample is completely dissolved¹H NMR measurement.

Wherein, the gutta percha (measured by the estimated gutta percha amount of the gutta percha sample to be detected): 1,3, 5-trimethoxybenzene: chloroform (12 to 37.5) mg: (8-15) mg: (0.4-0.6) ml to ensure that the quantitative peak difference between the object to be detected and the internal standard substance is not large, the solution can be completely dissolved in the solvent, the concentration peak is moderate, and the determination error is reduced

Preferably, the mass ratio of the gutta-percha (measured by the estimated gutta-percha amount in the gutta-percha sample to be detected) to the internal standard substance 1,3, 5-trimethoxybenzene is 1.5-2.5: 1, and the result obtained in the ratio range has a good linear relation;

the gutta-percha is a polymer, the chemical structural formula of which is shown in the formula I, the gutta-percha is not very good in solubility or even insoluble in a common solvent, so that a specific solvent is required to be dissolved when a quantitative nuclear magnetic hydrogen spectrum technology is adopted, and the weighed internal standard substance and a sample are ensured to be completely dissolved in a deuterated reagent. The inventor finds that the deuterated chloroform, the deuterated benzene, the deuterated acetone and the deuterated o-dichlorobenzene can well dissolve the gutta-percha within a certain period of time and have no interference on a target quantitative signal peak of a sample. In view of better solubility, signal peak interference and cost issues, deuterated chloroform is preferably used in the present invention.

(2) Adjusting the instrument parameters:

setting parameters of a nuclear magnetic resonance spectrometer, wherein the resonance frequency is 400MHz, the pulse pouring angle is 30-90 degrees, the measuring temperature is 23 +/-2 ℃, the acquisition time is 3s, the sampling frequency NS is 16-32 times, the relaxation time D1 is 15-30 s, shimming and sampling are carried out, and the obtained map is subjected to phase adjustment and baseline correction;

through T1 experiments, the T1 value of the obtained substance is 6S, D1 is more than or equal to 2.5T1, D1 is 15-30S, and the relaxation time (D1) is long enough to enable the atomic nucleus to be completely relaxed, so that the integrated signal intensity is in direct proportion to the number of the atomic nucleus, but the larger the D1 value is, the longer the time for collecting the sample is, and the preferable D1 is 15S; the pulse dumping angle is 30-90 degrees, the sensitivity is good when a 90-degree pulse is adopted, the analysis time is long, the advantage of rapid nuclear magnetism quantification cannot be reflected, when a 30-degree pulse small dumping angle is adopted, an accurate analysis result can be obtained according to a T1 value, the analysis time can be shortened, the analysis efficiency is improved, and therefore the pulse dumping angle is preferably 30 degrees.

(3) Integrating quantitative peaks of the gutta-percha sample and the 1,3, 5-trimethoxybenzene, wherein a proton peak of delta 5.12 is selected as the quantitative peak of the gutta-percha; selecting a proton peak of delta 6.09 as the quantity peak of the 1,3, 5-trimethoxybenzene, integrating for multiple times, averaging when the relative standard deviation of the integration result is less than 1%, and calculating by adopting the following formula according to the integration result:

in the formula:

w_sis the mass fraction of the sample containing the gutta-percha;

w_iis the mass fraction of the internal standard;

A_sand n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, respectively;

M_sis the molecular mass of an individual population of samples, m_sIs the weighed mass of the sample;

A_iand n_iThe integral area of the quantitative peak of the internal standard substance and the number of protons contained in the quantitative peak are respectively;

M_iis the molecular mass of the internal standard;

m_iis the weighed mass of the internal standard substance.

In order to avoid the interference of new impurities added into the sample on signal analysis, the purity of the internal standard substance is higher and better, and 1,3, 5-trimethoxybenzene with the purity of 99.7 percent is selected in the invention.

Based on variability and artificial differences in phase adjustment and baseline correction, the quantitative peak integration area is preferably the average of 5 integrations per signal, with the number increasing or decreasing depending on the stability of the integration.

Compared with the prior art, the invention has the following beneficial effects:

the invention firstly uses nuclear magnetic resonance hydrogen spectrum to calculate the content of the gutta-percha (eucommia ulmoides extract). The error of direct weighing after conventional application and purification is overcome.

The principle of quantitative nuclear magnetic resonance hydrogen spectrum is that the NMR spectrum signals of organic compounds containing H are in direct proportion to the number of atoms, so that the resonance peaks of a measured object and an internal standard substance are required to be close to the corresponding structural forms when experimental parameters are set. The selection of hydrogen needs to eliminate the interference of impurities in the solvent and the hydrogen peak of the solvent, and the signal interference between the detected component and the impurities, otherwise, the integration is inaccurate, and the reproducibility of the detection result is poor and inaccurate. Meanwhile, in the quantitative nuclear magnetic resonance hydrogen spectrum, peaks at different positions correspond to different relaxation times and pulse flip angles, so that the proper relaxation time and pulse flip angle are required to be selected for the quantitative peaks. According to the method, the longitudinal relaxation time T1 value of the gutta percha target peak in the deuterated reagent is measured, the influence of parameters such as the pulse dumping angle and the relaxation time on the measurement result is determined, the pulse dumping angle is finally adopted to be 30 degrees, the relaxation time is finally adopted to be 15s, the calculation result is high in accuracy, the time is saved, and the high efficiency is guaranteed.

The invention adopts the delta 5.12 proton peak (which is the proton chemical shift of the double bond position of gutta percha) and the delta 6.09 (the double bond proton chemical shift of the internal standard substance) which participate in the calculation of the compound, avoids the interference of a deuterogen reagent and other impurity peaks, and ensures the accuracy of the result.

The invention preferably adopts deuterated chloroform as a solvent, thereby not only ensuring the solubility of the sample, but also avoiding the interference peak of the deuterated reagent at the position of the calculated peak, improving the spectrogram resolution and saving the cost.

The invention adopts the internal standard substance as 1,3, 5-trimethoxybenzene with the purity of 99.7 percent, and controls the sample weighing mass ratio (calculated by the content of gutta percha) to the internal standard substance within the limited range of the invention, thereby ensuring the good separation degree of the two in the nuclear magnetic hydrogen spectrum and reducing the interference of the calculation peak, and further improving the accuracy of the detection result.

Drawings

FIG. 1 is a chemical structural formula of 1,3, 5-trimethoxybenzene;

FIG. 2 shows the hydrogen nuclear magnetic resonance spectrum of example 1;

FIG. 3 is a hydrogen nuclear magnetic resonance spectrum of example 2;

FIG. 4 is a hydrogen nuclear magnetic resonance spectrum of example 3;

FIG. 5 shows the hydrogen nuclear magnetic resonance spectrum of example 4;

FIG. 6 shows the hydrogen nuclear magnetic resonance spectrum of example 5;

FIG. 7 shows the hydrogen nuclear magnetic resonance spectrum of example 6;

FIG. 8 is a hydrogen nuclear magnetic resonance spectrum of example 7;

FIG. 9 shows the hydrogen spectrum for NMR in example 8;

FIG. 10 shows the hydrogen nuclear magnetic resonance spectrum of example 9.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the scope of the present invention is not limited thereto:

the instrument used in the present invention was a bruker avance 400MHz nuclear magnetic resonance spectrometer.

Example 1

Estimating a sample with the gutta-percha content of 93% by a weight method, accurately weighing 18.2mg of a crude eucommia ulmoides extract product and 8.0mg of an internal standard substance 1,3, 5-trimethoxybenzene, placing the crude eucommia ulmoides extract product and the 8.0mg of the internal standard substance 1,3, 5-trimethoxybenzene into a clean and dry nuclear magnetic tube, adding 0.5mL of deuterated chloroform solvent into the nuclear magnetic tube, placing the nuclear magnetic tube in a fume hood after sealing for 2h, shaking the nuclear magnetic tube to enable the internal solution to be uniform after the sample is completely dissolved, and performing¹H NMR measurement. Setting instrument parameters, homogenizing and sampling under the experimental conditions, performing phase adjustment and baseline correction after obtaining a map, integrating quantitative peaks of an eucommia ulmoides gum sample and 1,3, 5-trimethoxy benzene for 5 times respectively, taking an average value when the relative standard deviation of an integration result is less than 1%, and calculating according to the integration result by using the following formula:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 30 degrees, D₁And (4) acquiring the sample for 15s, wherein the accumulation times are 32 times, the testing temperature is 23 +/-2 ℃, and the NMR spectrum of the sample is determined.

As shown in FIG. 4, in¹In H NMR spectrum, A_s＝1.00,A_iThe gutta-percha content in the gutta-percha sample was calculated to be 87.56% according to the formula, and repeated 5 times with a standard error of 0.17% and a relative standard deviation of 0.18%.

Example 2

Estimating a sample with the gutta-percha content of 93% by a weight method, accurately weighing 17.9mg of a crude eucommia ulmoides extract product and 10.1mg of an internal standard substance 1,3, 5-trimethoxybenzene, placing the crude eucommia ulmoides extract product and the 10.1mg of the internal standard substance 1,3, 5-trimethoxybenzene into a clean and dry nuclear magnetic tube, adding 0.5mL of deuterated chloroform solvent into the nuclear magnetic tube, placing the nuclear magnetic tube in a fume hood after sealing for 2 hours, shaking the nuclear magnetic tube to enable the internal solution to be uniform after the sample is completely dissolved, and performing¹H NMR measurement. Setting instrument parameters under the experimental conditions, shimming and sampling, performing phase adjustment and baseline correction after obtaining a map, integrating quantitative peaks of an eucommia ulmoides gum sample and 1,3, 5-trimetoxybenzene for 5 times respectively, taking an average value when a relative standard deviation of an integration result is less than 1%, and calculating according to the integration result by using the following formula:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 30 degrees, D₁The cumulative number of times of collection was 28 times for 20s, the test temperature was 23 + -2 deg.C, and the NMR spectrum of the sample was determined.

As shown in fig. 5, in¹In H NMR spectrum, A_s＝1.00,A_iCalculating the content of gutta-percha in the gutta-percha sample to be 87.89% according to the formula, and repeating the steps5 times, the standard error is 0.17%, and the relative standard deviation is 0.20%.

Example 3

Estimating a sample with the gutta-percha content of 93% by a gravimetric method, accurately weighing 12.4mg of a crude eucommia ulmoides extract product and 15.0mg of an internal standard substance 1,3, 5-trimethoxybenzene, placing the crude eucommia ulmoides extract product and the 15.0mg of the internal standard substance 1,3, 5-trimethoxybenzene into a clean and dry nuclear magnetic tube, adding 0.5mL of deuterated chloroform solvent into the nuclear magnetic tube, placing the nuclear magnetic tube in a fume hood after sealing for 2 hours, shaking the nuclear magnetic tube to enable the internal solution to be uniform after the sample is completely dissolved, and performing¹H NMR measurement. Setting instrument parameters under the experimental conditions, shimming and sampling, performing phase adjustment and baseline correction after obtaining a map, integrating quantitative peaks of an eucommia ulmoides gum sample and 1,3, 5-trimetoxybenzene for 5 times respectively, taking an average value when a relative standard deviation of an integration result is less than 1%, and calculating according to the integration result by using the following formula:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 30 degrees, D₁The NMR spectrum of the sample was determined at 30s, 16 times for the cumulative number of acquisitions, and a test temperature of 23. + -. 2 ℃.

As shown in FIG. 6, in¹In H NMR spectrum, A_s＝1.00,A_iThe gutta-percha content in the gutta-percha sample was calculated to be 87.48% according to the formula, and the repetition was performed 5 times with a standard error of 0.19% and a relative standard deviation of 0.20%.

Example 4

Estimating a sample with the gutta-percha content of 93% by a weight method, accurately weighing 37.1mg of a crude eucommia ulmoides extract product and 15.0mg of an internal standard substance 1,3, 5-trimethoxybenzene, placing the crude eucommia ulmoides extract product and the 15.0mg of the internal standard substance 1,3, 5-trimethoxybenzene into a clean and dry nuclear magnetic tube, adding 0.5mL of deuterated chloroform solvent into the nuclear magnetic tube, placing the nuclear magnetic tube in a fume hood after sealing for 2 hours, shaking the nuclear magnetic tube to enable the internal solution to be uniform after the sample is completely dissolved, and performing¹H NMR measurement. Setting instrument parameters under the experimental conditions, shimming and sampling, performing phase adjustment and baseline correction after obtaining a map, integrating quantitative peaks of an eucommia ulmoides gum sample and 1,3, 5-trimetoxybenzene for 5 times respectively, taking an average value when a relative standard deviation of an integration result is less than 1%, and calculating according to the integration result by using the following formula:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 60 degrees and D₁And (4) acquiring the sample for 15s, wherein the accumulation times are 32 times, the testing temperature is 23 +/-2 ℃, and the NMR spectrum of the sample is determined.

As shown in FIG. 7, in¹In H NMR spectrum, A_s＝1.00,A_iThe gutta-percha content in the gutta-percha sample was calculated to be 87.72% according to the formula, and repeated 5 times with a standard error of 0.19% and a relative standard deviation of 0.20%.

Example 5

Estimating a sample with the gutta-percha content of 93 percent by weight,accurately weighing 12.1mg of eucommia ulmoides extract crude product and 8.0mg of internal standard substance 1,3, 5-trimethoxybenzene, placing in a clean and dry nuclear magnetic tube, adding 0.5mL of deuterated chloroform solvent into the nuclear magnetic tube, sealing, placing in a fume hood for 2h, shaking the nuclear magnetic tube to make the internal solution uniform after the sample is completely dissolved, and performing¹H NMR measurement. Setting instrument parameters, homogenizing and sampling under the experimental conditions, performing phase adjustment and baseline correction after obtaining a map, integrating quantitative peaks of an eucommia ulmoides gum sample and 1,3, 5-trimethoxy benzene for 5 times respectively, taking an average value when the relative standard deviation of an integration result is less than 1%, and calculating according to the integration result by using the following formula:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 60 degrees and D₁The cumulative number of times of collection was 28 times for 20s, the test temperature was 23 + -2 deg.C, and the NMR spectrum of the sample was determined.

As shown in FIG. 8, in¹In H NMR spectrum, A_s＝1.00,A_iThe gutta-percha content in the gutta-percha sample was calculated to be 87.31% according to the formula, and repeated 5 times with a standard error of 20% and a relative standard deviation of 0.21%.

Example 6

Estimating a sample with the gutta-percha content of 93 percent by a weight method, accurately weighing 37.5mg of a crude eucommia ulmoides extract product and 8.1mg of an internal standard substance 1,3, 5-trimethoxybenzene, and placing the crude eucommia ulmoides extract product and the internal standard substance in clean and dry nuclear magnetismAdding 0.5mL of deuterated chloroform solvent into the nuclear magnetic tube, sealing, placing in a fume hood for 2h, dissolving the sample completely, shaking the nuclear magnetic tube to make its internal solution uniform, and performing¹H NMR measurement. Setting instrument parameters, homogenizing and sampling under the experimental conditions, performing phase adjustment and baseline correction after obtaining a map, integrating quantitative peaks of an eucommia ulmoides gum sample and 1,3, 5-trimethoxy benzene for 5 times respectively, taking an average value when the relative standard deviation of an integration result is less than 1%, and calculating according to the integration result by using the following formula:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 60 degrees and D₁The NMR spectrum of the sample was determined at 30s, 16 times for the cumulative number of acquisitions, and a test temperature of 23. + -. 2 ℃.

As shown in FIG. 9, in¹In H NMR spectrum, A_s＝1.00,A_iThe gutta-percha content in the gutta-percha sample was calculated to be 87.48% according to the formula, and repeated 5 times with a standard error of 0.18% and a relative standard deviation of 0.21%.

Example 7

Estimating a sample with the gutta-percha content of 93% by a weight method, accurately weighing 13.9mg of a crude eucommia ulmoides extract product and 12.3mg of an internal standard substance 1,3, 5-trimethoxybenzene, placing the crude eucommia ulmoides extract product and the internal standard substance 1,3, 5-trimethoxybenzene into a clean and dry nuclear magnetic tube, adding 0.5mL of a deuterated chloroform solvent into the nuclear magnetic tube, placing the nuclear magnetic tube in a fume hood for 2 hours after sealing, and finishing samplingAfter full dissolution, the nuclear magnetic tube is shaken to make the solution in the nuclear magnetic tube uniform¹H NMR measurement. Setting instrument parameters under the experimental conditions, shimming and sampling, performing phase adjustment and baseline correction after obtaining a map, integrating quantitative peaks of an eucommia ulmoides gum sample and 1,3, 5-trimetoxybenzene for 5 times respectively, taking an average value when a relative standard deviation of an integration result is less than 1%, and calculating according to the integration result by using the following formula:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 90 degrees, D₁And (4) acquiring the sample for 15s, wherein the accumulation times are 32 times, the testing temperature is 23 +/-2 ℃, and the NMR spectrum of the sample is determined.

As shown in FIG. 10, in¹In H NMR spectrum, A_s＝1.00,A_iThe gutta-percha content in the gutta-percha sample was calculated to be 88.13% according to the formula, and repeated 5 times with a standard error of 0.18% and a relative standard deviation of 0.19%.

Example 8

Estimating a sample with the gutta-percha content of 93% by a weight method, accurately weighing 15.9mg of a crude eucommia ulmoides extract product and 13.1mg of an internal standard substance 1,3, 5-trimethoxybenzene, placing the crude eucommia ulmoides extract product and the 13.1mg of the internal standard substance 1,3, 5-trimethoxybenzene into a clean and dry nuclear magnetic tube, adding 0.5mL of deuterated chloroform solvent into the nuclear magnetic tube, placing the nuclear magnetic tube in a fume hood after sealing for 2 hours, shaking the nuclear magnetic tube to enable the internal solution to be uniform after the sample is completely dissolved, and performing¹H NMR measurement. In the aboveSetting instrument parameters under experimental conditions, shimming and sampling, performing phase adjustment and baseline correction after obtaining a map, integrating quantitative peaks of an gutta percha sample and 1,3, 5-trimethoxybenzene for 5 times respectively, taking an average value when a relative standard deviation of an integration result is less than 1%, and calculating by using the following formula according to the integration result:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 90 degrees, D₁The cumulative number of times of collection was 28 times for 20s, the test temperature was 23 + -2 deg.C, and the NMR spectrum of the sample was determined.

As shown in FIG. 9, in¹In H NMR spectrum, A_s＝1.00,A_iThe gutta-percha content in the gutta-percha sample was calculated to be 87.81% according to the formula, and repeated 5 times with a standard error of 0.19% and a relative standard deviation of 0.19%.

Example 9

Estimating a sample with the gutta-percha content of 93% by a gravimetric method, accurately weighing 12.9mg of a crude eucommia ulmoides extract product and 13.6mg of an internal standard substance 1,3, 5-trimethoxybenzene, placing the crude eucommia ulmoides extract product and the 13.6mg of the internal standard substance 1,3, 5-trimethoxybenzene into a clean and dry nuclear magnetic tube, adding 0.5mL of deuterated chloroform solvent into the nuclear magnetic tube, placing the nuclear magnetic tube in a fume hood after sealing for 2 hours, shaking the nuclear magnetic tube to enable the internal solution to be uniform after the sample is completely dissolved, and performing¹H NMR measurement. Setting instrument parameters under the above experimental conditions, shimming, sampling, and performing phase adjustment and baseline correction after obtaining the atlasPositive, the quantitative peaks of the gutta percha sample and 1,3, 5-trimethoxybenzene were integrated 5 times, respectively, and the average value was taken when the relative standard deviation of the integration results was less than 1%, and calculated according to the integration results by the following formula:

in the formula: w is a_sIs the mass fraction of the sample containing gutta-percha, w_iIs the mass fraction of the internal standard, A_sAnd n_sThe integral area of the quantitative peak of the sample and the number of protons contained in the quantitative peak, M_sIs the molecular mass, m, of a single population of samples_sIs the weighed mass of the sample, A_iAnd n_iThe integral area of the quantification peak of the internal standard substance and the number of protons contained in the quantification peak, M_iIs the molecular mass of the internal standard, m_iIs the weighed mass of the internal standard substance.

Nuclear magnetic resonance acquisition conditions: the resonance frequency of the nuclear magnetic resonance spectrometer is 400MHz, the pulse is 90 degrees, D₁The NMR spectrum of the sample was determined at 30s, 16 times for the cumulative number of acquisitions, and a test temperature of 23. + -. 2 ℃.

As shown in FIG. 10, in¹In H NMR spectrum, A_s＝1.00,A_iThe gutta-percha content in the gutta-percha sample was calculated to be 87.14% according to the formula, and repeated 5 times with a standard error of 0.20% and a relative standard deviation of 0.19%.

Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive effort by those skilled in the art.