CN111707761B - Application of high-content 2-isopropyl malic acid in evaluation of authenticity of pine pollen - Google Patents

Application of high-content 2-isopropyl malic acid in evaluation of authenticity of pine pollen Download PDF

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CN111707761B
CN111707761B CN202010853509.6A CN202010853509A CN111707761B CN 111707761 B CN111707761 B CN 111707761B CN 202010853509 A CN202010853509 A CN 202010853509A CN 111707761 B CN111707761 B CN 111707761B
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pollen
mobile phase
sample
authenticity
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CN111707761A (en
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杨术鹏
李熠
蔡冬梅
周金慧
杨宇晖
张金震
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Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
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Abstract

The invention relates to the field of food detection, in particular to application of high-content 2-isopropyl malic acid in authenticity evaluation of pine pollen. Meanwhile, the invention provides a pine pollen authenticity evaluation method, which takes 2-isopropyl malic acid as a characteristic compound, and when the content of the 2-isopropyl malic acid is between 18 and 35 mg/kg, a sample can be judged as pine pollen; otherwise, the pollen is judged to be other pollen or adulterated pollen pini. The method provided by the invention is simple, efficient and strong in stability, and is suitable for accurately measuring the content of the 2-isopropylmalic acid in the pine pollen. The method is suitable for evaluating the authenticity and purity of the pine pollen, and has important practical significance for protecting the legal rights and interests of consumers and maintaining the healthy development of the pine pollen industry.

Description

Application of high-content 2-isopropyl malic acid in evaluation of authenticity of pine pollen
Technical Field
The invention relates to the field of food detection, in particular to application of high-content 2-isopropyl malic acid in authenticity evaluation of pine pollen.
Background
Pine pollen is pollen produced by Pinaceae plants such as Pinus massoniana lamb, Chinese pine, etc. and is a natural food and health-care product obtained by the steps of picking, drying, removing impurities, etc., and is widely applied to the fields of food health care, skin care, beauty treatment, trauma treatment, etc. Particularly, the pine pollen contains rich various proteins, carbohydrates, minerals, nucleotides, flavonoids, monosaccharides, polysaccharides and the like, and can comprehensively supplement and balance nutrition required by a human body, so that the pine pollen is popular with people (especially the elderly) and has good application in foods or health care products. Meanwhile, the pine pollen has certain effects of skin care, hemostasis, convergence and the like, and is widely applied to the aspects of female beautifying, infant skin care, trauma treatment and the like. However, the current pine pollen has limited yield, larger market gap and higher selling price, which is usually more than several times of other pollen, such as rape pollen, camellia pollen and the like. In order to chase after high profit, some bad merchants often add starch or other varieties of pollen into the pine pollen for adulteration and selling, which not only damages the rights and interests of consumers, but also hinders the healthy and orderly development of the pine pollen industry. Currently, China does not set up a method for evaluating the authenticity and purity of the pine pollen, and how to evaluate the authenticity and purity of the pine pollen becomes a problem which is acknowledged by vast practitioners and consumers and needs to be solved urgently.
Like other types of pollen, pollen pini is a gamete produced by the stamen of plants, and contains abundant endogenous compounds. Because endogenous substances in different varieties of pollen have larger variety and content difference, part of compounds exist in certain plant pollen only or the content of the compounds is obviously different from that of other varieties of pollen. For such compounds, they are generally referred to as characterizing or indicative substances for the authenticity or purity evaluation of the pollen. The invention aims to adopt the technologies such as liquid phase tandem mass spectrometry and the like to carry out recognition and identification research on endogenous substances in the pine pollen and aims to discover characteristic substances of the pine pollen so as to evaluate the authenticity and purity of the pine pollen.
Disclosure of Invention
In order to solve the technical problems, the invention firstly carries out scanning analysis on a large number of real pine pollen and other common pollen by a positive mode and a negative mode of liquid chromatogram tandem high-resolution mass spectrum, finds that the pine pollen has a chromatographic peak with higher abundance together in the negative mode, and the peak-off time is 5.60 min (see fig. 2, wherein, a picture A is a total ion current chromatogram (TIC) of the pine pollen detected by LC-HRMS, and a picture B is an extracted ion current chromatogram (EIC) of 2-isopropylmalic acid in the pine pollen detected by LC-HRMS), and is obviously different from other common pollen. The precise mass number of the substance ism/z 175.06120, the major fragments thereof containingm/z115.03852 (see fig. 3, wherein A is compound in pollen Pini)m/z175 Full Mass diagram, B diagram is the compound in pine pollenm/zMS/MS mass spectrum of 175). Based on the accurate mass number provided by the high-resolution mass spectrum, the element composition C of the substance is obtained7H12O5With major debrism/z115.03832 the elemental composition has a reduced C content as compared to the elemental composition2H4O2And guesses about the possible fragmentation process of the compound (see figure 4). Based on the above analysis, it was found that the elemental composition of the substance was C7H12O5Mainly constituted as C5H8O3And C5H9And O. And obtaining the MS/MS spectrum with accurate mass number based on the high-resolution mass spectrum by adopting Compound discover 2.0 softwarem/z Cloud、Chem Bank, m/z Multiple chemical database searches are performed in Vault et al databases. Based on the above data, the compound was preliminarily identified as 2-isopropylmalic acid (chemical structure of which is shown in FIG. 1). To further confirm the compound, the inventors purchased 20 mg of a standard 2-isopropylmalic acid. Through comparison verification, the compound is further determined to be 2-isopropylmalic acid.
Because the content of the 2-isopropylmalic acid in the pine pollen is higher and is obviously different from that of common pollen, the 2-isopropylmalic acid can be used as a potential index for identifying the authenticity and evaluating the purity of the pine pollen and is used for evaluating whether other pollen is mixed in the pine pollen or not or whether the pine pollen is mixed in the pine pollen. Through analysis of a large number of pollen pini samples, the 2-isopropylmalic acid content in the pollen pini in different years and production places is found to be stable and is between 18 and 35 mg/kg.
According to the findings, the invention firstly provides the application of high-content 2-isopropylmalic acid as a characteristic marker of pine pollen, wherein the high content is 18-35 mg/kg, and the chemical structure of the 2-isopropylmalic acid is as follows:
Figure 522656DEST_PATH_IMAGE001
the invention further provides a method for evaluating the authenticity of the pollen pini, which takes 2-isopropylmalic acid as a characteristic compound, and judges that the pollen sample is the pollen pini if the content of the 2-isopropylmalic acid in the pollen sample is between 18 and 35 mg/kg; and otherwise, judging the pollen sample to be other pollen or adulterated pollen pini.
Preferably, the linear relation between the 2-isopropylmalic acid content and the peak area is confirmed by blank pollen added with 2-isopropylmalic acid, and the content is calculated according to the peak area of the 2-isopropylmalic acid in the pollen pini sample.
Preferably, the pollen pini sample is detected by UHPLC-Q-Orbitrap (ultra high liquid chromatography-high resolution mass spectrometry) and/or LC-MS/MS.
Preferably, the 2-isopropylmalic acid contains an excimer ion peak in a precise extraction ion current chromatogram of UHPLC-Q-Orbitrapm/z 175.06120 ([M-H]-) The error of the exact mass number should be less than 5 ppm; preferably, the retention time of the chromatographic peak of the 2-isopropylmalic acid is 5.60 min, and the deviation of the retention time is less than 0.2 min.
In addition, in order to improve the discrimination capability of the pine pollen, the accurate mass number and retention time of the 2-isopropylmalic acid meet the conditions, besides the conditions, the MS/MS (sub-ion) spectrum of the substance should contain the main fragment ions, and the invention further determines the characteristic fragment ions (the MS/MS spectrum of the 2-isopropylmalic acid should contain one or more of the following fragment ion bees):m/z 115.03832、m/z 85.06421、m/z113.05926, the error in the exact mass number should be less than 10 ppm. Based on the main fragment ions provided in the MS/MS spectrum, even when no 2-isopropylmalic acid standard substance exists, whether the pollen sample contains the pollen pini can be judged through the fragment ion information, and the pollen pini content can be calculated by integrating the peak area of the parent ion.
Preferably, when the UHPLC-Q-Orbitrap is adopted to detect the pollen pini sample, the liquid phase conditions are as follows:
separating by using a C18 chromatographic column and using 0.1% formic acid water as a mobile phase A and 0.1% formic acid acetonitrile as a mobile phase B by adopting a gradient elution procedure: 0-2.0 min, 5% of mobile phase B; 2.0-7.0 min, 5-30% of mobile phase B; 7.0-14.0min, 30-95% of mobile phase B; 14.0-18.0 min, 95% mobile phase B; 18.0-18.1 min, 95-5% of mobile phase B; 18.1-20.0 min, 5% of mobile phase B.
Preferably, the flow rate of the liquid phase is 0.30 mL/min.
The preferable sample amount is 5.0 μ L.
Preferably, when the UHPLC-Q-Orbitrap is adopted to detect the pollen pini sample, the mass spectrum conditions are as follows:
ion source parameters: the flow rate of sheath gas 45 arb; flow rate of the auxiliary gas 10 arb; the flow rate of cone blocking gas is 0 arb; the electrospray voltage was 3.5 kV; the temperature of the ion guide tube is 320 ℃; the S-lens RF level is set to 60V; the temperature of the ion source was 350 ℃.
The preferred collection mode is Full MS-ddMS in negative ion mode2Ensure that the scan range contains 175.06120 Da and the collision energy (NCE) is 35 eV. The specific parameters of Full MS are set as follows: resolution ratio: 70000; AGC Target: 3e 6; maximum IT: 100 ms; scan range: 80-1200 Da; spectrum data: a Centroid; wherein the specific parameter settings of dd-MS2 are as follows: resolution ratio: 17500 (mm); AGC Target: 1.6e 5; maximum IT: 50 ms; loop count: 1; isolation window: 2.0 Da; NCE: 35 eV; spectrum data: a Centroid; in dd settings, Minimum AGC: 8.0e 3; apex trigger: 2-6 s; exclude isotope: on; dynamic exclusion: 8.0 s.
When the LC-MS/MS is adopted to detect the pollen pini sample, the difference between the liquid phase and mass spectrum setting conditions of the LC-MS/MS and the UHPLC-Q-Orbitrap is larger due to the difference of detection instruments.
Preferred liquid phase conditions are as follows: adopting a C18 chromatographic column, wherein the column temperature is 25 ℃, 0.1% formic acid water is used as a mobile phase A, and 0.1% formic acid acetonitrile is used as a mobile phase B; separation was performed using a gradient elution procedure: 0-0.8 min, 5% of mobile phase B; 0.8-1.2 min, 5-40% of mobile phase B; 1.2-2.5 min, 40-90% of mobile phase B; 2.5-3.5 min, 90% of mobile phase B; 3.5-3.6 min, 90-5% of mobile phase B; 3.6-6.0 min, 5% of mobile phase B.
Preferably, the flow rate of the liquid phase is 0.30 mL/min.
Preferably, the sample size is 5.0 muL.
Preferably, in order to accurately quantify the 2-isopropylmalic acid in the pollen pini, when the pollen pini sample is detected by adopting LC-MS/MS, the collected mode is multi-reaction monitoring (MRM) in an anion mode, and the key parameters of the MRM are set as follows: 175.1>115.0 (quantitation ion), 9 eV; 175.1>85.1 (quantitation ion), 16 eV.
The mass spectrometry conditions were as follows: electrospray ion source (ESI); the scanning mode is as follows: scanning negative ions; ion spray voltage: 3500V; atomizing gas pressure: 45 psi; temperature of the drying gas: 300 ℃; flow rate of drying gas: 5L/min; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 11L/min.
In order to verify the scientific reasonability of the established LC-MS/MS method, the accuracy and precision of the method are examined, the variation coefficient is less than 10 percent, and the method meets the standard requirement.
Based on the instruments and published parameters adopted by the method, different analysis laboratories and detection mechanisms can carry out certain adjustment on the parameters according to the relevant knowledge of the liquid chromatogram tandem high-resolution mass spectrum or the liquid chromatogram tandem triple quadrupole mass spectrum technology.
Preferably, before the detection, the method further comprises the step of extracting the pollen pini sample;
in some embodiments, the pollen pini is blended at a weight of 1 g: dissolving 10 mL of the mixture in methanol according to the mass-volume ratio, and extracting; then diluted 100 times with pure water.
As a preferred scheme, the extraction specifically comprises the following steps: mixing the pollen pini sample with 5 mL of methanol in a ratio of 0.5 g pollen pini sample, and after the pollen pini is fully dissolved, 20194xgAnd centrifuging at 4 ℃ for 20 min, taking 10 muL of extracting solution, and adding the extracting solution into 990 muL of pure water for dilution.
The reagents and standards involved in the present invention are commercially available, and the operations involved are all conventional in the art unless otherwise specified.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The invention has the beneficial effects that:
the invention discloses a high-content substance 2-isopropylmalic acid in pollen pini for the first time, provides an index that the high-content 2-isopropylmalic acid can be used for evaluating the authenticity and purity of the pollen pini, and establishes and optimizes a method for detecting the 2-isopropylmalic acid in the pollen pini by liquid phase tandem mass spectrometry (UHPLC-Q-Orbitrap or LC-MS/MS). The method has high specificity and sensitivity, and the detection limit is 2 mug/kg.
In addition, in order to accurately quantify the content of the 2-isopropylmalic acid in the pine pollen, the LC-MS/MS detection method and key detection parameters of the substance are optimized.
Meanwhile, the method disclosed by the invention has the advantages of simplicity, high efficiency and the like, is convenient to operate and popularize, and has important practical significance for protecting the legal rights and interests of pollen pini consumers and maintaining the healthy development of the pollen pini industry.
Drawings
FIG. 1 shows the chemical structure of 2-isopropylmalic acid as a biomarker in pollen Pini.
FIG. 2 is a Total Ion Chromatogram (TIC) of pollen Pini by LC-HRMS detection, and is an Extracted Ion Chromatogram (EIC) of 2-isopropylmalic acid in pollen Pini by LC-HRMS detection.
FIG. 3, A is the compound in pollen Pinim/z175 Full Mass diagram, B diagram is the compound in pine pollenm/zMS/MS mass spectrum of 175.
FIG. 4. 2-cleavage pathway possible when isopropylmalic acid was subjected to LC-MS collision induced cleavage.
FIG. 5 shows UHPLC-Q-Orbitrap detection of accurate extraction ion current chromatograms of 2-isopropylmalic acid in pollen Pini and other pollen samples, respectively, wherein deviation of extracted mass window is less than 5 ppm. A diagram is pollen Pini EIC diagram, B diagram is rape pollen EIC diagram, C diagram is radix Codonopsis pollen EIC diagram, D diagram is corn pollen EIC diagram, E diagram is lotus pollen EIC diagram, F diagram is flos Lonicerae powder EIC diagram, G diagram is flos Rosae Rugosae pollen EIC diagram.
FIG. 6 MRM mode of LC-MS/MS quantifies the content of 2-isopropylmalic acid in different pollen samples.
FIG. 7 shows ion current chromatogram of quantitative and qualitative ion extraction of 2-isopropylmalic acid in MRM mode of LC-MS/MS.
FIG. 8 shows the content of 2-isopropylmalic acid in 40 pollen pini samples on the market.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.
Instruments and reagents:
1. mass spectrometer (Q-exact Plus), Thermo Fisher Scientific, USA;
2.1200 series liquid chromatography-6460 triple quadrupole mass spectrometry, Agilent Technologies, USA;
3. desk top cryogenic centrifuge (1-15Pk), Sigma, Germany;
4. electronic analytical balance (PL203), mettleteledo, germany;
5. ultra pure water machines (Milli-Q Gradient), Millipore Inc. of USA;
6. vortexer (G560E), Scientific Industries, USA;
2-isopropylmalic acid (chemical structure shown in FIG. 1) was purchased from Sigma Aldrich trade company, Inc.; formic Acid (FA) was purchased from shanghai' an spectral laboratory science and technology limited (CNW); acetonitrile (ACN) and methanol (MeOH) were purchased from Fisher corporation.
Example 1
1. Sample source
50 common real pollen pini samples are purchased from markets or pine growers and used for detecting the content of the 2-isopropyl malic acid in various pollen pini, and the samples are derived from the following pollen pini in different production places: jilin, Liaoning, Jiangsu, Shandong, Yunnan, Zhejiang and Fujian, and comprises a real pollen pini sample (10 parts) and other common pollen, and specifically comprises the following components: rape pollen (10 parts), codonopsis pilosula pollen (5 parts), corn pollen (7 parts), lotus pollen (8 parts), mountain pollen (5 parts) and rose pollen (5 parts).
2. Solution preparation
2-isopropylmalic acid standard solution: weighing 10 mg of 2-isopropylmalic acid standard substance, and diluting the methanol to 10 mL. Storing at 4 deg.C for 2 months.
3. The Q active plus detection conditions are as follows:
(1) sample treatment:
weighing 0.5 g of pollen pini sample, 5 mL of methanol, mixing, fully dissolving pollen pini, 20194xgAnd centrifuging at 4 ℃ for 20 min, taking 10 muL of extracting solution, and adding the extracting solution into 990 muL of pure water for dilution. Transferring to a sample bottle, mixing evenly by vortex, and waiting for analysis on a computer.
(2) Quality control samples:
0.5 g of blank pollen (without 2-isopropylmalic acid) was weighed out, added to 5 mL of methanol, vortexed at room temperature until completely dissolved, and vortexed at 20194 gCentrifugation was carried out at 4 ℃ for 20 min. Taking 10 muL of extracting solution, adding the extracting solution into 990 muL of pure water to dilute the extracting solution into blank matrix, carrying out vortex mixing, moving 20 muL of 1 mg/L of 2-isopropyl malic acid standard solution into 980 muL of blank matrix, carrying out vortex mixing, and waiting for on-machine analysis.
(3) Q active plus instrument settings
The chromatographic conditions are as follows: the analytical column is a C18 chromatographic column. Using 0.1% formic acid water as mobile phase A and 0.1% formic acid acetonitrile as B, and adopting gradient elution procedure to separate: 0-2.0 min, 5% of mobile phase B; 2.0-7.0 min, 5-30% of mobile phase B; 7.0-14.0min, 30-95% of mobile phase B; 14.0-18.0 min, 95% mobile phase B; 18.0-18.1 min, 95-5% of mobile phase B; 18.1-20.0 min, 5% of mobile phase B; flow rate: 0.30 mL/min; sample introduction amount: 5.0 muL.
Ion source parameters: sheath gas flow rate 45 arb; auxiliary gas flow rate 10 arb; cone baffle airflow rate 0 arb; the electrospray voltage was 3.5 kV; the temperature of the ion guide tube is 320 ℃; s-lens RF level is set to 60; the temperature of the ion source was 350 ℃.
The collection mode is Full MS-ddMS in a negative ion mode2。
The specific parameters of Full MS are set as follows: resolution ratio: 70000; AGC Target: 3e 6;maximum IT: 100 ms; scan range: 80-1200 Da; spectrum data: centroid. Wherein dd-MS2The specific parameter settings are as follows: resolution ratio: 17500 (mm); AGC Target: 1.6e 5; maximum IT: 50 ms; loop count: 1; isolation window: 2.0 Da; NCE: 35 eV; spectrum data: centroid. And in dd settings, Minimum AGC: 8.0e 3; apex trigger: 2-6 s; exclude isotope: on; dynamic exclusion: 8.0 s.
And collecting and storing data generated by mass spectrum by using Xcalibur software, analyzing raw data acquired by mass spectrum by using a Qualitive scanner of the Xcalibur, comparing Full MS and MS/MS spectrums of 2-isopropylmalic acid in a sample and a blank standard-added sample, and determining that the sample contains the 2-isopropylmalic acid. Detecting the accurate extraction ion current chromatograms of 2-isopropylmalic acid in blank pollen and pollen Pini samples respectively, wherein the deviation of the extracted mass window is less than 5 ppm, to obtain figure 5, wherein A is pollen Pini EIC, B is rape pollen EIC, C is radix Codonopsis pollen EIC, D is corn pollen EIC, E is lotus pollen EIC, F is flos Lonicerae powder EIC, and G is flos Rosae Rugosae pollen EIC.
4. The conditions for Agilent1200 liquid chromatography-6460 triple quadrupole mass spectrometry were as follows:
in the liquid phase condition, a short C18 chromatographic column is adopted for separation, a C18 chromatographic column is adopted, the column temperature is 25 ℃, 0.1% formic acid water is used as a mobile phase A, and 0.1% formic acid acetonitrile is used as a mobile phase B; separation was performed using a gradient elution procedure: 0-0.8 min, 5% of mobile phase B; 0.8-1.2 min, 5-40% of mobile phase B; 1.2-2.5 min, 40-90% of mobile phase B; 2.5-3.5 min, 90% of mobile phase B; 3.5-3.6 min, 90-5% of mobile phase B; 3.6-6.0 min, 5% of mobile phase B.
The flow rate of the liquid phase was 0.30 mL/min. The sample size is 5.0 muL.
The mass spectrum conditions for LC-MS/MS are as follows: electrospray ion source (ESI); the scanning mode is as follows: scanning negative ions; the monitoring mode is as follows: multiple Reaction Monitoring (MRM); ion spray voltage: 3500V; atomizing gas pressure: 45 psi; temperature of the drying gas: 300 ℃; flow rate of drying gas: 5L/min; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 11L/min.
The collection mode is the MRM mode in the negative ion mode. MRM parameters: 175.1>115.0 (quantitation ion), 9 eV; 175.1>85.1 (counterion), 16 eV.
FIG. 7 shows the quantitative and qualitative ion extraction of 2-isopropylmalic acid in MRM mode of LC-MS/MS.
5. The detection process is as follows:
(1) sample treatment:
weighing 0.5 g of pollen Pini sample, adding into 5 mL of methanol, mixing, and dissolving pollen Pini completely, 20194xgAnd centrifuging at 4 ℃ for 20 min, taking 10 muL of extracting solution, and adding the extracting solution into 990 muL of pure water for dilution. Transferring to a sample bottle, mixing evenly by vortex, and waiting for analysis on a computer.
(2) Drawing a standard curve: a series of 2-isopropyl malic acid solutions (5, 10, 20, 50, 100, 200, 500 ng/mL) were prepared in blank pollen sample extract, diluted blank matrix solution. And blank additions of 20 ng/ml were assigned as quality control samples. Samples and blank spiked samples were analyzed by Agilent's Mass Hunter quantitative software. Quantifying by an external standard method to obtain a standard curve formula of Y =15230.0X-87340.2, R2=0.9961(X is concentration Y quantitative ion response integral).
The content of 2-isopropylmalic acid in pollen Pini is obtained by concentration dilution calculation, and is shown in FIG. 6. When the content of the 2-isopropylmalic acid in the sample is between 18 and 35 mg/kg, the sample is considered to be the pine pollen.
Example 2
1. Sample source
40 portions of pollen pini samples with various grades or brands and labeled pollen pini are purchased from the market.
2. Experimental procedure
(1) Solution preparation
2-isopropylmalic acid standard solution: weighing 10 mg of 2-isopropylmalic acid standard substance, and diluting the methanol to 10 mL. Storing at 4 deg.C for 2 months.
(2) Sample treatment:
weighing 0.5 g of pollen Pini sample, adding into 5 mL of methanol, mixing, and dissolving pollen Pini completely, 20194g，4 ℃Centrifuging for 20 min, then taking 100 muL of extracting solution, adding the extracting solution into 900 muL of pure water for dilution, then taking 100 muL of diluent, and adding the diluent into 900 muL of pure water for secondary dilution. Transferring to a sample bottle, mixing evenly by vortex, and waiting for analysis on a computer.
(2) Quality control samples:
0.5 g of blank pollen (without 2-isopropylmalic acid) was weighed out, added to 5 mL of methanol, vortexed at room temperature until completely dissolved, and vortexed at 20194gCentrifugation was carried out at 4 ℃ for 20 min. Taking 10 muL of extracting solution, adding the extracting solution into 990 muL of pure water to dilute the extracting solution into blank matrix, carrying out vortex mixing, then moving 20 muL of 1 mg/L of 2-isopropyl malic acid standard solution into 980 muL of blank matrix, carrying out vortex mixing, and waiting for on-machine analysis.
3. The conditions for Agilent1200 liquid chromatography-6460 triple quadrupole mass spectrometry were as follows:
in the liquid phase condition, a short C18 chromatographic column is adopted for separation, a C18 chromatographic column is adopted, the column temperature is 25 ℃, 0.1% formic acid water is used as a mobile phase A, and 0.1% formic acid acetonitrile is used as a mobile phase B; separation was performed using a gradient elution procedure: 0-0.8 min, 5% of mobile phase B; 0.8-1.2 min, 5-40% of mobile phase B; 1.2-2.5 min, 40-90% of mobile phase B; 2.5-3.5 min, 90% of mobile phase B; 3.5-3.6 min, 90-5% of mobile phase B; 3.6-6.0 min, 5% of mobile phase B.
The flow rate of the liquid phase was 0.30 mL/min. The sample size is 5.0 muL.
The mass spectrum conditions for LC-MS/MS are as follows: electrospray ion source (ESI); the scanning mode is as follows: scanning negative ions; the monitoring mode is as follows: multiple Reaction Monitoring (MRM); ion spray voltage: 3500V; atomizing gas pressure: 45 psi; temperature of the drying gas: 300 ℃; flow rate of drying gas: 5L/min; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 11L/min.
The collection mode is the MRM mode in the negative ion mode. MRM parameters: 175.1>115.0 (quantitation ion), 9 eV; 175.1>85.1 (counterion), 16 eV.
FIG. 7 shows the quantitative and qualitative ion extraction of 2-isopropylmalic acid in MRM mode of LC-MS/MS.
4. Drawing a standard curve: prepared in blank matrix solution extracted and diluted from blank pollen sampleA series of 2-isopropyl malic acid solutions (5, 10, 20, 50, 100, 200, 500 ng/mL) were prepared. And blank addition of 100 mug/L is designated as a quality control sample. Samples and blank spiked samples were analyzed by Agilent's Mass Hunter quantitative software. Quantifying by an external standard method to obtain a standard curve formula of Y =18230.0X-87340.2, R2=0.9961(X is concentration Y quantitative ion response integral).
The 2-isopropylmalic acid content in the pollen pini sample can be analyzed through quantitative software. The result is shown in fig. 8, wherein the content of 2-isopropylmalic acid in 45% of samples is less than 18 mg/kg, which indicates that commercial pine pollen sold in the market has serious quality problems, and the purity and authenticity problems are worth paying attention.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. The application of the high-content 2-isopropylmalic acid in the evaluation of the authenticity of the pine pollen is characterized in that the high content is 18-35 mg/kg.
2. The authenticity evaluation method of the pollen pini is characterized in that 2-isopropylmalic acid is taken as a characteristic compound, and if the content of the 2-isopropylmalic acid in a pollen sample is between 18 and 35 mg/kg, the pollen sample is judged to be the pollen pini; and if the content of the 2-isopropylmalic acid in the pollen sample is lower than 18 mg/kg, judging that the pollen sample is other pollen or adulterated pollen pini.
3. The authenticity assessment method according to claim 2, characterized in that it comprises, before the detection, a step of extraction of said pollen sample with methanol.
4. The authenticity assessment method according to claim 2, characterized in that the pollen sample is tested with UHPLC-Q-Orbitrap and/or LC-MS/MS.
5. The authenticity assessment method according to claim 4, wherein 2-isopropylmalic acid contains an excimer ion peak in the UHPLC-Q-Orbitrap's exact extraction ion chromatogramm/z 175.06120 ([M-H]-) The error of the exact mass number should be less than 5 ppm; the retention time of the chromatographic peak of the 2-isopropylmalic acid is 5.60 min, and the deviation of the retention time is less than 0.2 min.
6. The authenticity evaluation method according to claim 4, wherein the MS/MS spectrum of 2-isopropylmalic acid containsm/z 115.03832、m/z 85.06421、m/z113.05926, the error in the exact mass number should be less than 10 ppm.
7. The authenticity evaluation method according to claim 4 or 5, wherein when the pollen sample is detected by UHPLC-Q-Orbitrap, the liquid chromatography conditions are as follows:
separating by using a C18 chromatographic column and using 0.1% formic acid water as a mobile phase A and 0.1% formic acid acetonitrile as a mobile phase B by adopting a gradient elution procedure: 0-2.0 min, 5% of mobile phase B; 2.0-7.0 min, 5-30% of mobile phase B; 7.0-14.0min, 30-95% of mobile phase B; 14.0-18.0 min, 95% mobile phase B; 18.0-18.1 min, 95-5% of mobile phase B; 18.1-20.0 min, 5% of mobile phase B.
8. The authenticity evaluation method according to claim 4 or 5, wherein when the pollen sample is detected by UHPLC-Q-Orbitrap, the mass spectrum conditions are as follows:
ion source parameters: sheath gas flow rate 45 arb; flow rate of the auxiliary gas 10 arb; the flow rate of cone blocking gas is 0 arb; the electrospray voltage was 3.5 kV; the temperature of the ion guide tube is 320 ℃; the S-lens RF level is set to 60V; the temperature of the ion source is 350 ℃; the collection mode is Full MS-ddMS in a negative ion mode2The scanning range is 80-1200 Da, and the collision energy is 35 eV.
9. The authenticity evaluation method according to claim 4, wherein when LC-MS/MS is adopted to detect the pollen pini sample, the liquid phase conditions are as follows: adopting a C18 chromatographic column, wherein the column temperature is 25 ℃, 0.1% formic acid water is used as a mobile phase A, and 0.1% formic acid acetonitrile is used as a mobile phase B; separation was performed using a gradient elution procedure: 0-0.8 min, 5% of mobile phase B; 0.8-1.2 min, 5-40% of mobile phase B; 1.2-2.5 min, 40-90% of mobile phase B; 2.5-3.5 min, 90% of mobile phase B; 3.5-3.6 min, 90-5% of mobile phase B; 3.6-6.0 min, 5% of mobile phase B.
10. The authenticity evaluation method according to claim 4, wherein when the pollen sample is detected by LC-MS/MS, the mass spectrum conditions are as follows: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the monitoring mode is as follows: monitoring multiple reactions; ion spray voltage: 3500V; atomizing gas pressure: 45 psi; temperature of the drying gas: 300 ℃; flow rate of drying gas: 5L/min; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 11L/min; the acquisition mode is MRM in a negative ion mode, and the parameters are as follows: 175.1>115.0, 9 eV, 175.1>85.1, 16 eV.
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