CN111537647A - Ultra-high performance liquid chromatography analysis method for crimson-cleft-camaldine in flos farfarae - Google Patents

Abstract

The invention relates to an ultra-high performance liquid chromatographic analysis method for senilin in coltsfootia, comprising the following steps: taking a reference substance of senilin, adding methanol to prepare a solution containing 20 μg per 1 ml; , add methanol, sonicate, filter, wash the residue with methanol, filter, combine the filtrates, recover the solvent under reduced pressure, add 0.5% aqueous sulfuric acid to dissolve or suspend, and perform solid phase extraction; chromatography conditions: mobile phase is acetonitrile-0.1% Phosphoric acid aqueous solution=15:85; flow rate 0.3ml/min; chromatographic column: UPLC column, UV detector, detection wavelength 220nm; column temperature: 30℃; injection volume: 1μl; 1 μl was injected into the ultra-high performance liquid chromatograph. The method does not need to be used in conjunction with mass spectrometry and does not need to add ion ^- pairing reagents, and can complete the detection of keloids within 5 minutes. low cost.

Description

Ultra-high performance liquid chromatographic analysis method of senilin in coltsfootia

technical field

The invention relates to a method for detecting quality standards of coltsfoot, in particular to an ultra-high performance liquid chromatography analysis method for senilin in coltsfoot.

Background technique

Coltsfoot is the dried flower buds of the Compositae (Tussilago farfara L.), which was first recorded in the "Shen Nong's Herbal Classic" and is listed as a middle grade. , The effect of relieving cough and resolving phlegm is used for chronic cough, asthma and phlegm, labor cough and hemoptysis. Coltsfoot is a commonly used Chinese herbal medicine, which is "the most ancient and modern prescription for warming the lungs to treat cough". The 2015 edition of "Chinese Pharmacopoeia" contains Ermu Ansou Pills, Chuanbei Xue Li ointment, Cough Juhong Oral Liquid, Cough Juhong Pills, Cough Relieving and Phlegm Pills, Kechuan Shun Pills 14 kinds of Chinese patent medicines, such as bellflower winter flower tablets, lung-running and coughing pills, lung-clearing and phlegm-clearing pills, orange-red pills, orange-red tablets, orange-red capsules, orange-red granules, as well as children's lung cough granules, which are commonly used in pediatrics.

Coltsfoot is widely distributed, mainly produced in Northeast my country, North China, East China, Northwest China and Hubei, Hunan, Jiangxi, Guizhou, Yunnan, Tibet and other places, often born in valley wetlands or under forest. There are also distributions in India, Iran, Pakistan, Russia, Western Europe and North Africa. The chemical composition of coltsfoot is complex, mainly containing various compounds such as flavonoids, terpenes, phenolic acids, alkaloids, etc. effect. In addition to active ingredients, coltsfoot flowers contain high content of pyrrolizidine alkaloids (PAs), which have strong liver toxicity, but there are no such ingredients in the current quality standards. Limitation control and risk reminder, long-term use of this medicinal material, there is a greater risk of drug safety.

Pyrrolizidine alkaloids (PA) widely exist in a variety of plants. According to statistics, about 3% of the flowering plants in the world, that is, more than 6,000 species of plants contain PAs. So far, PAs have been detected in 13 families of plants, most of which belong to the comfrey family, the Asteraceae family, and the legume family. Toxicological studies have shown that PAs with double bonds at the 1 and 2 positions of the pyrrolidine ring have liver toxicity and are called hepatotoxic pyrrolizidine alkaloids (HPA). HPA can cause irreversible liver cell damage and lead to adverse consequences such as Hepatic Sinusoidal Obstruction Syndrome (HSOS), giant cell disease, liver fibrosis and cirrhosis, and even death in severe cases. HPA enters the liver and is oxidized and metabolized by cytochrome P450 to form dehydropyrrolizidine alkaloid (DHPA). -DNA complexes, the formation of DHPA-DNA complexes is cell type specific, mainly formed in hepatic sinusoidal endothelial cells rather than liver parenchyma cells, DHPA-DNA is difficult to reverse after formation, and accumulates in the body, after taking a certain amount Lead to hepatic sinusoidal obstruction syndrome (HSOS), and DHPA-DNA complex can lead to a variety of gene mutations, and then induce the formation of liver cancer.

In 1976, Australian researchers isolated 25 mg (yield 0.0025%) of Senkirkine from 1 kg of coltsfoot flower for the first time. The structure was identified by mass spectrometry, hydrogen NMR and comparison of reference substances. . Sennil is a macrocyclic diester type alkaloid of osso clematis, and it is the highest content of HPA in coltsfoot, and the content is generally 0.002-0.010%, that is, each gram of medicinal materials contains 20-100 μg. About one-third of the batches exceeded the limit (0.004%) of the HPA component adonifrin base (also pyrrolizidine alkaloid) under the Qianliguang item in the 2015 edition of the Chinese Pharmacopoeia. . Calculated according to the daily dosage of 5-10g stipulated in the "Chinese Pharmacopoeia", the daily intake of senilin is 100-1000μg.

Gramine is the most important hepatotoxic component in coltsfoot. Mice were injected intraperitoneally with clematine extracted from coltsfoot flower for 7 days, and the serum alanine aminotransferase (GOT), aspartate aminotransferase (GPT) and total bilirubin (TBIL) in the serum biochemical indexes were the same as those in the control group. The ratio was significantly increased by about 6 times; however, mild edema of hepatocytes was observed in pathological sections, punctate necrosis of hepatocytes, and inflammatory cell infiltration in the hepatic lobules and portal areas. Therefore, it is of great significance to establish a method for the determination of senilin in coltsfootia to control its quality and toxicity.

Due to the low content of clematis in Coltsfoot (generally less than 1/10,000), it is difficult to directly measure the content by conventional liquid chromatography. Most of the existing research reports use LC/MS to measure, Liquid chromatography-mass spectrometry (LC-MS) has extremely high sensitivity and strong structural identification ability, and is currently the main method for the determination of keloids, and there are many reports in the literature. For example: Qiao Yue et al., "Simultaneous determination of pyrrolizidine alkaloids and nitrogen oxides in coltsfoot flowers based on UPLC-MS/MS" published in the Chinese Journal of Experimental Formulary in 2019. The author established a UPLC-MS/MS method in coltsfootia medicinal materials, simultaneously determined the content of 15 pyrrolizidine alkaloids, and carried out quantitative research on 5 components including senilin from the medicinal materials. The quantitative method was ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. However, this method has the following problems: (1) The price of the LC/MS instrument is relatively high, generally requiring more than 2 million yuan. At present, many medicinal herb decoction pieces enterprises do not have the ability to purchase this instrument. Therefore, using this method for detection will greatly increase the number of enterprises. (2) High-purity liquid nitrogen and mass spectrometry-grade reagents are required for the measurement, and the test cost is much higher than other test methods such as ultra-high performance liquid chromatography and high performance liquid chromatography; (3) liquid nitrogen Mass spectrometry has higher technical requirements for experimental operators. Compared with ultra-high performance liquid chromatography, experimenters need several times more training time to master and operate the instrument proficiently; (4) For content determination In terms of accuracy and test precision, the accuracy and test precision of LC-MS assay are not as good as that of ultra-high performance liquid chromatography.

Mroczek et al., Journal of Chromatography A, 2002 (Simultaneous determination of N-oxides and free bases of pyrrolizidinealkaloids by cation-exchange solid-phase extraction and ion-pair high-performance liquid chromatography), the authors established cation exchange Solid-phase extraction was used to enrich the pyrrolizidine alkaloids in the water extract of Coltsfoot, using n-hexane sulfonic acid as the ion-pair reagent to establish an ion-pair high performance liquid chromatography (High Performance Ion Pair Chromatography, HPIPC), using C8 chromatography Column, the mobile phase is acetonitrile-1% phosphoric acid aqueous solution (containing 5mM sodium n-hexane sulfonate) gradient elution, at 220nm wavelength to detect kelvin base, the detection time is about 20 minutes, the limit of quantification is about 0.2μg·ml -1. However, this method still has the following problems: (1) due to the use of ion-pair chromatography, sodium n-hexane sulfonate is used as an ion-pair reagent to be added to the mobile phase, and under chromatographic conditions, sodium n-hexane sulfonate and Kelvin A chromatographic method in which the base forms a hydrophobic ion-pair compound, thereby controlling the retention behavior of kelvin base. The principle of this method is different from the general reversed-phase HPLC method. The salt ion-pairing reagents can easily damage the column life. (2) The added salt ion-pair reagent not only forms detectable substances with the compound to be tested, but also forms detectable substances with many other components. What is detected on the chromatogram is the addition of chemical components and n-hexanesulfonic acid. When other chromatographic conditions remain unchanged, the chromatogram obtained by ion-pair chromatography is often more complex than that obtained by reversed-phase HPLC. As for the kelvin base peak, it is suspected that other chromatograms are embedded in the chromatographic peak. chromatographic peaks. (3) Gradient elution is used in this document, the elution process is more complicated, the chromatographic baseline is more unstable, and the chromatogram is not good.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the object of the present invention is to provide an ultra-high performance liquid chromatographic analysis method of senilin in Coltsfoot. The method is based on a reversed-phase ultra-high performance liquid chromatography-ultraviolet detector, which does not need to be used in conjunction with mass spectrometry, and does not need to add ion-pairing reagents. ·ml ^-1 ), good accuracy and reproducibility, and low detection cost.

To achieve the above object, the present invention specifically adopts the following technical solutions to realize:

An ultra-high performance liquid chromatographic analysis method for senilin in coltsfoot, comprising the following steps:

(1) Preparation of reference solution

Take an appropriate amount of kelvin base reference substance, accurately weigh it, add methanol to make a solution containing 20μg per 1ml, for use;

(2) Preparation of the test solution

Take the coltsfoot flower powder that has passed through the No. 4 sieve, accurately weigh it, put it in a stoppered conical flask, add methanol, ultrasonically treat, filter, wash the residue with methanol, filter, combine the filtrates, recover the solvent under reduced pressure, add 0.5% sulfuric acid The aqueous solution is dissolved or suspended, and solid-phase extraction is performed;

The specific process of the solid-phase extraction is as follows: after first activating the cationic solid-phase extraction column with methanol, equilibrating the solid-phase extraction column with 0.5% sulfuric acid aqueous solution, then adding the sample solution of sulfuric acid water to the solid-phase extraction column, and rinsing with methanol to remove Mixed, the concentration is 8% ammonia water-methanol=1:3 solution elution, collect the eluate, recover the solvent under reduced pressure, add formic acid, make up to volume with water, filter with organic membrane, and set aside;

(3) Chromatographic conditions

Mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution=15:85; flow rate 0.3ml/min; chromatographic column: UPLC column, UV detector, detection wavelength 220nm; column temperature: 30℃; injection volume: 1μl;

(4) Determination: Precisely draw 1 μl each of the reference solution and the test solution, inject it into an ultra-high performance liquid chromatograph, and measure.

As a preference of the present invention, when the test solution is prepared, the power of ultrasonic treatment is 300W and the frequency is 40kHz.

As a preference of the present invention, the filler of the solid phase extraction column is a cation exchange resin, and the capacity is 500 mg/6 mL.

As a preference of the present invention, the column length of the UPLC column is 50 mm, the inner diameter is 2.1 mm, and the particle size is 1.7 μm.

Advantages and positive effects of the present invention:

(1) The method provided by the present invention does not require the use of a liquid chromatography-mass spectrometer, and only uses an ultra-high performance liquid chromatograph to achieve high sensitivity, the limit of quantification is 0.1 μg·ml ^-1 , and the repeatability, test precision and The accuracy is better than that of LC/MS, and the operation is relatively simple, and the cost of an ultra-high performance liquid chromatograph is about 500,000 yuan, which is far lower than that of LC/MS. The reagents can be selected at the chromatographic grade during detection. , the detection cost is also significantly lower than that of LC/MS.

(2) The method provided by the present invention does not use ion pair reagents, which greatly reduces the damage to the chromatographic column and the detection cost.

(3) The present invention has constant current elution, good chromatographic peak shape, short retention time, stable chromatographic baseline, and the quality of the spectrum is much better than that of the ion-pair chromatogram, and has the advantages of good precision, convenient and fast detection and low cost.

Description of drawings

Fig. 1 The chemical structural formula of kelvin base;

Fig. 2 UPLC chromatogram of senilic acid reference substance;

Fig. 3 No. 1 UPLC chromatogram of coltsfoot flower sample;

Fig. 4 The ultraviolet spectrum of kelvin base;

Figure 5. The standard curve of senilin.

Detailed ways

The present invention is further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention, but the present invention is not limited by this.

Instruments and Reagents

Instrument: Waters H-CLASS ultra-high performance liquid chromatograph, diode array detector; chromatographic column: ACQUITYBEH Shield RP18, 2.1×50mm, 1.7um; electronic balance Mittler XS105DU 1/100,000 electronic balance.

Reagent: Keshi senilic acid alkali reference substance (product of German PhytoLab company); coltsfoot medicinal material is collected from the place of origin.

Example 1

The ultra-high performance liquid chromatographic analysis method of clematis in coltsfoot, including the following steps:

(1) Preparation of reference solution

Take an appropriate amount of kelvin base reference substance, accurately weigh it, add methanol to make a solution containing 20μg per 1ml, for use;

(2) Preparation of test solution for content determination

Withdraw 2.00 g of winter flower powder (passed through a No. 4 sieve), accurately weigh it, put it in a stoppered conical flask, add 40 ml of methanol, ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 min, filter, and wash the residue twice with 5 ml of methanol, Filter, combine the filtrates, recover the solvent under reduced pressure at 50°C to about 1ml, and then add 10ml of 0.5% sulfuric acid aqueous solution to dissolve or suspend, and perform solid phase extraction;

The specific process of the solid phase extraction is as follows: first activate a cationic solid phase extraction column (cation exchange resin, capacity 500 mg/6 mL) with 5 ml of methanol, equilibrate the solid phase extraction column with a 0.5% aqueous sulfuric acid solution, and then add the above-mentioned sample solution of sulfuric acid water to the solid phase extraction column. Add it to the solid phase extraction column, rinse with 20 ml of methanol to remove impurities, and elute with 10 ml of 8% ammonia water-methanol=1:3 solution, collect the eluate, recover the solvent under reduced pressure at 50 °C to about 2 ml, add 1 ml of formic acid, Dilute to 5ml with water, filter with 0.22μm organic filter, set aside;

(3) Chromatographic conditions

Mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution (15:85); flow rate 0.3ml/min; chromatographic column: UPLC column (column length is 50mm, inner diameter is 2.1mm, particle size is 1.7μm), detection wavelength: 220nm; Temperature: 30℃; Injection volume: 1μl;

(4) Determination: Precisely draw 1 μl each of the reference solution and the test solution, inject it into an ultra-high performance liquid chromatograph, and measure.

Methodological investigation:

Linear relationship: Precisely draw the reference substance stock solution, add methanol to prepare reference substance series solutions of 2.048, 5.12, 10.24, 20.48, 40.96 ^, 81.82 μg·ml ^{-1 1} ) is the abscissa, and the peak area y is the ordinate to draw a standard curve (see Figure 5), and perform linear regression to obtain the regression equation:

y=34745x–573; r=1.0000

The results showed that the concentration of reference substance had a good linear relationship with the peak area in the range of 2.048～81.82μg·ml ^-1 .

Precision test: Accurately absorb 1 μl of kelvin base reference solution, inject 6 times continuously, measure the peak area, the RSD is 0.63% (n=6) respectively, the results are shown in Table 1, and the results show that the instrument has good precision.

Table 1 Precision test results

Stability test: Precisely draw the same test solution (No. 1), and at 0, 2, 4, 8, 12 and 24 hours after preparation, measure the peak area according to the aforementioned chromatographic conditions. 769844, the RSD is 0.76%, the test solution is stable within 24h, and the results are shown in Table 2.

Table 2 Stability test results

Repeatability test: Take this product powder (No. 1), 2.00g, a total of 6 parts, and measure it according to the aforementioned method. The result shows that the average content of senilin is 0.00534%, and the RSD is 2.44%, indicating that the test has good repeatability. See Table 3.

Table 3 Repeatability test results

Recovery test: Take the same batch of samples (No. 1) with known content, 6 parts, 1.00g each, put them in conical flasks with stoppers respectively, and then accurately add Kjeldahl with a concentration of 40.96μg·mL ^-1 in turn. 1.0 mL of the reference solution of sensible alkali, and then 40 mL of methanol was added precisely, and the remaining steps were prepared according to the preparation method of the test solution, measured, and the recovery rate was calculated. The results are shown in Table 4.

Table 4 Recovery test results

The above results show that the total recovery rate of 6 parts of phyllostatin is 89.46%, and the RSD value is 2.80%, and the method has high accuracy.

Determination of sample content: The collected samples were determined according to the above method, and the content was calculated by the external standard method, as shown in Table 5.

Table 5 Sample Content Determination Results

Claims (4)

1. An ultra-high performance liquid chromatography analysis method of crimson-sodium in flos farfarae is characterized by comprising the following steps:

(1) preparation of control solutions

Taking a proper amount of a crimson-gomphrena base reference substance, precisely weighing, and adding methanol to prepare a solution containing 20 mu g of crimson-gomphrena base per 1ml for later use;

(2) preparation of test solution

Precisely weighing flos Farfarae powder passing through No. four sieves, placing into a conical flask with a plug, adding methanol, ultrasonic treating, filtering, washing residue with methanol, filtering, mixing filtrates, recovering solvent under reduced pressure, adding 0.5% sulfuric acid water solution for dissolving or suspending, and performing solid phase extraction;

the solid phase extraction comprises the following specific processes: activating a cation solid-phase extraction column by using methanol, balancing the solid-phase extraction column by using 0.5% sulfuric acid aqueous solution, adding a sample solution of sulfuric acid water onto the solid-phase extraction column, leaching and removing impurities by using methanol, eluting by using 8% ammonia water-methanol (1: 3) solution, collecting eluent, recovering a solvent under reduced pressure, adding formic acid, fixing the volume by using water, and filtering by using an organic filter membrane for later use;

(3) chromatographic conditions

The mobile phase is acetonitrile-0.1% phosphoric acid water solution 15: 85; the flow rate is 0.3 ml/min; a chromatographic column: a UPLC column, an ultraviolet detector, a detection wavelength of 220 nm; column temperature: 30 ℃; sample introduction amount: 1 mul;

(4) and (3) determination: precisely sucking 1 μ l of each of the reference solution and the sample solution, injecting into an ultra-high performance liquid chromatograph, and measuring.

2. The ultra-high performance liquid chromatography analysis method of crimson-alkali in flos farfarae of claim 1, wherein the power of ultrasonic treatment is 300W and the frequency is 40kHz when the sample solution is prepared.

3. The method for ultra high performance liquid chromatography analysis of crimson-cri.

4. The ultra performance liquid chromatography analysis method for crimson excelsior base in coltsfoot flower according to claim 1, wherein the UPLC column has a column length of 50mm, an inner diameter of 2.1mm, and a particle diameter of 1.7 μm.

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