阅读说明：本技术 检测藏红花素的试剂盒及其方法 (Kit for detecting crocin and method thereof ) 是由 王秀娟 陈凤明 袁真 冯峰 于 2021-09-23 设计创作，主要内容包括：本发明公开了检测藏红花素的方法和试剂盒。该方法包括：将待测样本进行提取处理,以便得到提取液；以及利用色谱-串联质谱对所述提取液进行检测,以便获得所述藏红花素的含量。该方法利用色谱-串联质谱进行检测,能对藏红花素进行定量检测,检测的准确度和灵敏度高。(The invention discloses a method and a kit for detecting crocin. The method comprises the following steps: extracting a sample to be detected to obtain an extracting solution; and detecting the extracting solution by using a chromatography-tandem mass spectrum so as to obtain the content of the crocin. The method can be used for quantitative detection of crocin by using chromatography-tandem mass spectrometry, and has high detection accuracy and sensitivity.)

1. A method for detecting crocin, comprising:

extracting a sample to be detected to obtain an extracting solution; and

detecting the extracting solution by using a chromatography-tandem mass spectrum so as to obtain the content of the crocin.

2. The method of claim 1, wherein the extraction process comprises:

mixing the sample to be tested with an extraction solvent to obtain a mixture;

carrying out ultrasonic extraction on the mixture so as to obtain an ultrasonic extracting solution; and

and centrifuging the ultrasonic extracting solution, and extracting a supernatant, wherein the supernatant is the extracting solution.

3. The method according to claim 2, characterized in that the extraction solvent is an aqueous ethanol solution, preferably a 50 vol% aqueous ethanol solution.

4. The method according to claim 2, wherein the temperature condition of the ultrasonic extraction is 35-50 ℃ and the time condition is 30-50 minutes.

5. The method of claim 1, wherein the chromatography in the chromatography-tandem mass spectrometry is high performance liquid chromatography.

6. The method of claim 5, wherein the high performance liquid chromatography column is an ACQUITY UPLC HSS T3 column.

7. The method of claim 5, wherein the mobile phase A of the high performance liquid chromatography is an aqueous solution containing 5mM ammonium acetate, and the mobile phase B is methanol.

8. The method of claim 7, wherein the elution by high performance liquid chromatography is a gradient elution,

optionally, the gradient elution conditions are: 0-0.2 min, and 20% of mobile phase B: 0.2-2 min, and the mobile phase B is 50%: 2-4 min, wherein the mobile phase B is 100%; 4-6 min, wherein the mobile phase B is 20%; 6-6.2 min, and 20% of mobile phase B.

9. The method of claim 8, wherein the chromatographic conditions of the chromatograph-tandem mass spectrometer are:

column temperature: 34-35 ℃;

sample introduction amount: 3 mu L of the solution;

flow rate: 0.3 mL/min.

Optionally, the mass spectrometry conditions of the chromatography-tandem mass spectrometry are:

ionization mode: ESI⁺；

Electrospray voltage: 4500V;

detection mode: a FULL scan/data dependency (FULL MASS/dd-MS2) mode is adopted;

the scanning range is 200-1200 m/z;

collision energy: 35V.

10. A kit for detecting crocin, comprising a reagent, standard, auxiliary material or a combination of at least one thereof used in the method for detecting crocin of claims 1-9.

Technical Field

The invention relates to the field of analytical chemistry, in particular to a method for detecting crocin and a kit for detecting crocin.

Background

Saffron, also known as saffron and Crocus, is the dry stigma of Crocus sativus L.of Iridaceae and has the effects of promoting blood circulation, removing blood stasis, cooling blood, removing toxic substances, resolving stagnation and tranquilizing. Crocin, a main active substance of saffron, is a series of ester glycosides formed by combining crocetin and different amounts of glycosides, and has the effects of centrally lowering blood pressure, preventing atherosclerosis and preventing cervical cancer. Because of its powerful pharmacological action, the market demand of saffron is higher and higher, and the quality of saffron on the market is good and uneven, so that the phenomenon of secondary good is frequent.

The method for detecting crocin in the prior art comprises Japanese pharmacopoeia-JP 16, British pharmacopoeia-BP 2013 and ISO-3632, and the content of crocin in saffron is quantified by ultraviolet-visible spectrophotometry. However, it is difficult to perform accurate quantitative detection of crocin by the above method.

Therefore, a method for quantitatively detecting crocin is yet to be studied.

However, in most cases, the qualitative and quantitative determination of the sample by mass spectrometry and chromatography is not found, and a liquid chromatography-mass spectrometry method for crocin should be developed because crocin is a substance with a large polarity, and the detection signal in the mass spectrometry is low and the retention in the chromatography is difficult.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a method for detecting crocin, which uses chromatography-tandem mass spectrometry to perform detection, can perform quantitative detection on crocin, and has high detection accuracy and sensitivity.

It should be noted that the present invention is completed based on the following work of the inventors:

since crocin is a substance with larger polarity, the detection signal in the mass spectrum is lower, and the retention in the chromatogram is more difficult, and the ultraviolet-visible spectrophotometry is generally adopted, so that the crocin is difficult to detect by the chromatogram-tandem mass spectrum. The Tibetan red pigment is represented as Na in a mass spectrum⁺According to the symmetry and polarity of the ion peak, the detection sensitivity cannot be improved under the condition of adding acid or alkali, and the problem of poor mass spectrum response can be solved only by increasing the amount of the sample or the standard; for chromatography, the commonly used C18 column does not retain polar substances well, and a corresponding polar column is used, and the column used in the present invention is a UPLC HSS T3 column.

Thus, according to one aspect of the present invention, the present invention provides a method for detecting crocin. According to an embodiment of the invention, the method comprises: extracting a sample to be detected to obtain an extracting solution; and detecting the extracting solution by using a chromatography-tandem mass spectrum so as to obtain the content of the crocin.

According to the method for detecting the crocin, provided by the embodiment of the invention, the crocin can be quantitatively detected by utilizing a chromatography-tandem mass spectrum, and the detection accuracy and sensitivity are high.

In addition, the method for detecting crocin according to the above embodiment of the present invention may further have the following additional technical features:

according to an embodiment of the present invention, the extraction process includes: mixing the sample to be tested with an extraction solvent to obtain a mixture; carrying out ultrasonic extraction on the mixture so as to obtain an ultrasonic extracting solution; and centrifuging the ultrasonic product to extract a supernatant, wherein the supernatant is the extracting solution.

According to an embodiment of the invention, the extraction solvent is an aqueous ethanol solution, preferably a 50 vol% aqueous ethanol solution.

According to the embodiment of the invention, the temperature condition of ultrasonic extraction is 35-50 ℃, and the time condition is 30-50 minutes.

According to an embodiment of the invention, the chromatography in the chromatography-tandem mass spectrometry is high performance liquid chromatography.

According to the embodiment of the invention, the chromatographic column of the high performance liquid chromatography is an ACQUITY UPLC HSS T3 chromatographic column.

According to the embodiment of the invention, the mobile phase A of the high performance liquid chromatography is an aqueous solution containing 5mM ammonium acetate, and the mobile phase B is methanol.

According to an embodiment of the invention, the elution of the high performance liquid chromatography is a gradient elution,

according to an embodiment of the invention, the gradient elution conditions are: 0-0.2 min, and 20% of mobile phase B: 0.2-2 min, and the mobile phase B is 50%: 2-4 min, wherein the mobile phase B is 100%; 4-6 min, wherein the mobile phase B is 20%; 6-6.2 min, and 20% of mobile phase B.

According to an embodiment of the present invention, the chromatographic conditions of the chromatography-tandem mass spectrometry are: column temperature: 34-35 ℃; sample introduction amount: 3 mu L of the solution; flow rate: 0.3 ml/min.

According to an embodiment of the present invention, the mass spectrometry conditions of the chromatography-tandem mass spectrometry are: ESI⁺(ii) a FULL MASS ddMS2 mode; the scanning range is 200-1200 m/z; the collision energy is 35V; the electrospray voltage was 4500V.

According to another aspect of the present invention, the present invention provides a kit for detecting crocin. According to an embodiment of the present invention, the kit comprises reagents, standards, auxiliary materials or a combination of at least one of them used in the aforementioned method for detecting crocin. Therefore, the kit provided by the embodiment of the invention is used for detecting crocin, and the crocin can be quantitatively detected by using chromatography-tandem mass spectrometry, so that the detection accuracy and sensitivity are high. Here, it should be noted that the kit has all the technical features and technical effects of the foregoing method for detecting crocin, and details are not repeated herein.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

fig. 1 shows a crocin standard chromatography schematic of a C18 chromatography column according to one embodiment of the invention;

FIG. 2 shows a sample water extract chromatography schematic according to one embodiment of the invention;

FIG. 3 shows a schematic diagram of a second mass spectrum of a control according to one embodiment of the present invention;

FIG. 4 shows a schematic diagram of a secondary mass spectrum of a sample to be measured according to an embodiment of the present invention;

FIG. 5 shows a schematic chromatogram of a control according to an embodiment of the invention;

FIG. 6 shows a schematic chromatogram of a sample to be tested according to an embodiment of the invention;

FIG. 7 shows a schematic diagram of a calibration curve according to an embodiment of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

According to one aspect of the present invention, the present invention provides a method for detecting crocin.

According to the method for detecting the crocin, provided by the embodiment of the invention, the crocin can be quantitatively detected by utilizing a chromatography-tandem mass spectrum, and the detection accuracy and sensitivity are high.

The method for detecting the crocin is suitable for detecting the quality of the crocin on the market, and has diversified instrument selection and simple operation.

In order to facilitate understanding of the method for detecting crocin according to an embodiment of the present invention, which is explained herein according to an embodiment of the present invention, the method includes:

s100 extraction processing

According to the embodiment of the invention, the sample to be detected is subjected to extraction treatment so as to obtain the extracting solution. Therefore, the crocin is extracted from the sample to be detected, and the subsequent detection is convenient.

S200 detection processing

According to an embodiment of the present invention, said extract is subjected to a detection treatment by means of chromatography-tandem mass spectrometry in order to obtain said crocin content. Therefore, the crocin can be quantitatively detected by utilizing the chromatography-tandem mass spectrometry, the detection accuracy and sensitivity are high, the instrument is diversified in selection, and the operation is simple.

According to an embodiment of the present invention, the extraction process includes: mixing the sample to be tested with an extraction solvent to obtain a mixture; subjecting the mixture to ultrasonic extraction to obtain an ultrasonic product; and centrifuging the ultrasonic product to extract a supernatant, wherein the supernatant is the extracting solution. Therefore, the ultrasonic extraction is favorable for fully extracting the crocin in the sample.

According to the embodiment of the invention, the extraction solvent is an ethanol aqueous solution, the crocin has high solubility in water, a proper amount of organic solvent is added into the extraction solvent to facilitate the re-dissolution of subsequent extracts, the subsequent treatment process is simplified, the crocin has good solubility in ethanol, the crocin can be conveniently extracted from the object to be detected by utilizing the ethanol aqueous solution, preferably, the 50 volume percent ethanol aqueous solution is used, and the extraction efficiency of the crocin is better.

According to the embodiment of the invention, the temperature condition of ultrasonic extraction is 35-50 ℃, and the time condition is 30-50 minutes. The inventor finds that crocin is easier to separate from saffron at high temperature, and based on the characteristics of ultrasonic instruments, the crocin can be controlled within a certain temperature range so as not to be denatured but can be extracted to a greater extent.

According to an embodiment of the invention, the chromatography in the chromatography-tandem mass spectrometry is high performance liquid chromatography. Because the selected mobile phase and the set flow rate are relative to the crocin, the pressure upper limit of the chromatographic column is easily exceeded in the classical liquid chromatography, therefore, the inventor selects the high performance liquid chromatography, and can meet the requirements of the saffron detection on the flow rate.

According to the embodiment of the invention, the chromatographic column of the high performance liquid chromatography is an ACQUITY UPLC HSS T3 chromatographic column. The crocin is polar substance, and the chromatographic column can improve the retention of crocin.

According to the embodiment of the invention, the mobile phase A of the high performance liquid chromatography is water containing 5mM ammonium acetate, and the mobile phase B is methanol. Further, according to an embodiment of the present invention, the elution of the high performance liquid chromatography is a gradient elution. Specifically, according to an embodiment of the present invention, the gradient elution conditions are: 0-0.2 min, and 20% of mobile phase B: 0.2-2 min, and the mobile phase B is 50%: 2-4 min, wherein the mobile phase B is 100%; 4-6 min, wherein the mobile phase B is 20%; 6-6.2 min, and 20% of mobile phase B. Therefore, the method is beneficial to separating the crocin from other impurities, and the detection accuracy is higher.

According to an embodiment of the present invention, the chromatographic conditions of the chromatography-tandem mass spectrometry are: column temperature: 34-35 ℃; sample introduction amount: 3 mu L of the solution; flow rate: 0.3 ml/min. According to the characteristics of polar substances, the single-needle sample injection is not less than 2 mu L, and the high performance liquid chromatography has the characteristic of accurately analyzing small-volume samples, so that the sample injection volume is selected to be 3 mu L, and the other conditions are basic parameters of the instrument.

According to an embodiment of the present invention, the mass spectrometry conditions of the chromatography-tandem mass spectrometry are: ESI⁺(ii) a FULL MASS dd MS2 mode; the scanning range is 200-1200 m/z; the collision energy is 35V; the electrospray voltage was 4500V. According to the properties of the instrument and the material, the optimal collision energy of the two and fragment ions is 35V, and the high pressure value is about 5000V.

According to another aspect of the present invention, the present invention provides a kit for detecting crocin. According to an embodiment of the present invention, the kit comprises reagents, standards, auxiliary materials or a combination of at least one of them used in the aforementioned method for detecting crocin. Therefore, the kit provided by the embodiment of the invention is used for detecting crocin, and the crocin can be quantitatively detected by using chromatography-tandem mass spectrometry, so that the detection accuracy and sensitivity are high.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.

Apparatus and materials

Thermo QE Orbitrap hplc-mass spectrometry system (Thermo Fisher, germany);

KQ5200V ultrasonic cleaner (kunshan ultrasonic instruments ltd);

Milli-Q Reference ultra pure water machine (Merck Millipore, Germany);

thermo Fisher Pico 17 high speed centrifuge (Thermo Fisher, Germany).

A crocin standard;

the methanol and the ethanol are pure in chromatography;

ammonium acetate is analytically pure;

the water is ultrapure water.

Saffron samples, purchased in tibet, were mainly classified into three different grades (first, top, second).

The mobile phase A is 5mmol/L ammonium acetate water solution, and the preparation method comprises the steps of taking 385.4mg ammonium acetate and diluting the ammonium acetate to 1L by ultrapure water; the mobile phase B is methanol.

Example 1

In this embodiment, the method for detecting crocin according to this embodiment is used to detect the crocin content in commercially available saffron and determine the quality of saffron, and specifically includes the following steps:

1. preparation of reference solution and drawing of standard curve

Accurately weighing 2.3mg crocin standard substance, dissolving in 23mL methanol to obtain 100ppm reference substance mother liquor, and diluting to 0.3ppm, 0.5ppm, 1.0ppm, 2.0ppm, 3.0ppm, 5.0ppm reference substance solution to be tested.

Detecting the reference substance solution in a high performance liquid chromatography-mass spectrum, wherein the conditions of the chromatogram and the mass spectrum are as follows:

a chromatographic column: an ACQUITY UPLC HSS T3 chromatography column;

column temperature: 35 ℃;

sample introduction amount: 3 μ L

The mobile phase ratios and flow rates are shown in table 1: the phase B is methanol, and the phase A is 5mmol/L ammonium acetate aqueous solution.

Table 1 mobile phase proportions and flow rate parameter settings

Mass spectrum conditions: the scanning range is 200-1200m/z, the collision energy is 35V, and the electrospray voltage is 4500V.

The mass spectrum second fragment ions of the control are shown in figure 3. The peak time of the crocin chromatogram is 3.96, as shown in fig. 5. The elution scheme of the C18 column chromatography for comparison is shown in FIG. 1. And (3) sequentially injecting different concentrations of the reference substance into the system, quantifying by an external standard method by using the QUAN BROWSER in the system, and drawing a standard curve, wherein the standard curve is shown in figure 7. The chromatographic diagram of the water extract of the sample is shown in FIG. 2.

2. Sample solution preparation

(1) Sample extraction:

(a) selecting 3 representative saffron crocus samples of different grades, respectively grinding into powder, respectively accurately weighing 100mg, placing into a test tube, adding a 50% ethanol aqueous solution (v: v ═ 1:1) with the volume of 10mL into the test tube with the sample by using a pipette, sealing by using a sealing film, and vortexing for 1min after confirming sealing;

(b) placing the vortexed sample in an ultrasonic instrument at 35-50 ℃ for ultrasonic extraction for 40min, placing the sample at room temperature for cooling and precipitation after the ultrasonic extraction is finished, and centrifuging 6mL of upper-layer solution (the centrifugation parameters are that the temperature is 4 ℃, the rotating speed is 9000rpm, and the time is 8 min);

(c) 3mL of supernatant of 3 samples after centrifugation are filtered through a 0.22 mu m filter membrane and collected, and the supernatant is filled in a brown sample bottle for standby;

(d) and (3) filling 1mL of sample into an injection bottle for nitrogen blowing, drying, re-dissolving 1mL of methanol, and vortexing for 1min to obtain a solution to be detected for later use.

(2) Sample detection

(a) Diluting the different samples to be tested in the step (1) by a proper amount by 2000 times to obtain different samples to be tested;

(b) sequentially feeding different sample solutions to be detected into the high performance liquid chromatography-mass spectrometry system, and quantifying the concentration x in the crocin sample to be detected in different samples according to a standard curve drawn in the system, wherein a reference substance is used for qualitative determination of crocin in the sample, the sample mass spectrum secondary fragment diagram is shown in figure 4, and the chromatographic peak diagram is shown in figure 6;

according to the formulaCalculating the content of crocin in the sample, wherein the concentration of crocin in the head stage sample is 1.196ppm, and the content of corresponding crocin is 23.92%; the content of crocin in the top sample is 1.036ppm, corresponding to the content of crocin is 20.72%; the concentration of crocin in the secondary sample was 609.740ppb, corresponding to a crocin content of 12.19%, which corresponds to the grade of the sample at the time of purchase.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.