CN112147249A

CN112147249A - UPC2-PDA-Q-Tof/MS detection method for 31 effective components in waxberry wine

Info

Publication number: CN112147249A
Application number: CN202011019579.8A
Authority: CN
Inventors: 马金同; 何宏魁; 曹润洁; 李安军; 汤有宏; 刘国英; 张严; 丁峰; 王录; 孟涛; 秦杰杰
Original assignee: Anhui Ruisiweier Technology Co Ltd
Current assignee: Anhui Ruisiweier Technology Co Ltd
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2020-12-29
Anticipated expiration: 2040-09-25
Also published as: CN112147249B

Abstract

The invention discloses a UPC2-PDA-Q-Tof/MS detection method of 31 effective components in waxberry wine, which comprises the steps of firstly freezing and drying a wine sample to be detected of the waxberry wine into a solid, dissolving the solid with a formic acid-water-methanol mixed solution, then passing the solid through a 0.22 mu m filter membrane, carrying out gradient elution separation through an UPC2 BEH column, simultaneously collecting UPC2-PDA mode and MS mode data, respectively monitoring an ESI positive and negative ion mode, comparing the obtained spectrogram of the sample to be detected with a standard spectrogram, obtaining corresponding substances with the same retention time, and quantifying each effective component through an MS mode standard curve linear regression equation. The method has simple pretreatment, linear correlation coefficient of more than 0.99, recovery rate of 85-115% and relative standard deviation within 10%.

Description

UPC2-PDA-Q-Tof/MS detection method for 31 effective components in waxberry wine

Technical Field

The invention belongs to the field of functional wine, and particularly relates to a UPC2-PDA-Q-Tof/MS detection method for 31 effective components in waxberry wine.

Background

China has a long brewing history and a plurality of varieties, and most of the varieties are white spirit, fruit wine and rice wine, but the alcohol has higher degree generally, and the excessive drinking of the alcohol can cause harm to human bodies. With the concern of people on the health of people, various low-degree medicinal liquors are produced at the same time, and the waxberry wine is one of the medicinal liquors. Recorded in Ben Cao gang mu, the red bayberry has the efficacies of promoting the production of body fluid, quenching thirst, regulating five internal organs, cleaning intestines and stomach, and relieving restlessness and muddlehead and foul breath. The waxberry is a fruit variety which is extremely intolerant to storage and transportation, and the fruit ripening period is concentrated in the month of Juqi with high temperature and raininess, so the saying that the fruit becomes changed in taste, color and quality in two days and three days is provided. The method for processing the waxberries into the waxberry wine is the most feasible method for fully utilizing the waxberries and improving the added value of the waxberries, people combine the waxberries and the wine together to prepare the waxberry wine, the waxberry wine is simple to prepare, has the nutritional ingredients of the waxberries, and has certain health-care effect after being drunk for a long time in a small amount.

However, in the current process of making the waxberry wine, various processes of eight flowers and eight doors appear, including direct soaking, boiling, crushing soaking, high and low degree soaking, reduced pressure extraction and additional fermentation, but the extraction rate of effective components is greatly different in various extraction modes, the types of effective components of the waxberries are various, the dissolution conditions of various substances are different, and the development of the waxberry wine is also hindered.

The existing data show that the detection method of the effective components of the waxberries mainly comprises High Performance Liquid Chromatography (HPLC), mass spectrometry, thin-layer chromatography and the like, has low detection flux, cannot integrally react the content of the effective components of the waxberries, and has low efficiency and time consumption.

Supercritical Fluid Chromatography (SFC) technique is based on Supercritical CO₂CO in supercritical state as mobile phase₂The method has the advantages of low viscosity coefficient, good mass transfer performance, high separation efficiency and environmental protection, and the limitations of the traditional liquid chromatography are broken through. A novel chromatographic separation technology represented by ultra performance conversion Chromatography (UPC 2) preferably makes up the limitation of the traditional Chromatography, GC and LC technologies are combined on a mobile phase, and the separation and analysis capability has two advantages of GC and LC. The Time of Flight Mass Spectrometer (TOF) has the advantages of high resolution, high speed, high upper limit of quality, high analysis throughput and the like, and can realize the acquisition and analysis of hundreds of substances.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a UPC2-PDA-Q-Tof/MS detection method for 31 effective components in waxberry wine. The method can accurately, quickly, stably and efficiently carry out qualitative and quantitative analysis on the effective components in the waxberry wine, and provides important reference significance for quality control of the waxberry wine.

The invention relates to a UPC2-PDA-Q-Tof/MS detection method of 31 effective components in waxberry wine, which is characterized in that a wine sample to be detected of waxberry wine is frozen and dried into solid, dissolved by a formic acid-water-methanol mixed solution and then passes through a 0.22 mu m filter membrane, gradient elution separation is carried out by a UPC2 BEH column, simultaneously UPC2-PDA mode and MS mode data are collected, ESI positive and negative ion modes are respectively monitored, the obtained spectrogram of the sample to be detected is compared with a standard spectrogram, corresponding substances with the same retention time are obtained, and each effective component is quantified by an MS mode standard curve linear regression equation.

The UPC2-PDA-Q-Tof/MS detection method of 31 effective components in the waxberry wine comprises the following steps:

step 1: pretreatment

Freezing and drying the waxberry wine sample to be tested into solid, dissolving the solid with a mixed solution of 0.4% formic acid, 5% water and 94.6% methanol, and filtering the solid with a 0.22 mu m filter membrane;

step 2: preparation of standard substance

Respectively preparing 1g/L of standard solutions of squalene, myricetic acid C, beta-sitosterol, myricetic acid B, beta-elemene, euscaphic acid, tormentic acid, quercetin, myricetin, kaempferol, myricetin, linalool, alpha-terpineol, 6, 7-dimethoxy coumarin, myricetin, scopoletin, piceidin F1, (+) -S-myricetin glucoside, caffeic acid, dihydromyricetin, gallic acid, myricitrin and myricetin 3-O-galactoside, wherein the solvent is formic acid-water-methanol solution, and the standard solutions of 31 effective components of myrica rubra are mixed and diluted step by step to obtain mixed standard solution with different concentrations;

and step 3: drawing standard spectrogram and standard curve

Performing chromatographic elution separation on the mixed standard solution prepared in the step 2, collecting by using a PDA and MS mode to obtain a standard spectrogram of 31 waxberry effective component standard products, taking the peak area of each effective component as a vertical coordinate, taking the concentration of the standard product as a horizontal coordinate, and obtaining corresponding standard curves, wherein a linear regression equation and relative standard deviation are shown in a table 1:

TABLE 1 Standard Curve of 31 effective components of fructus Myricae Rubrae

And 4, step 4: detection of a sample to be tested

And (3) carrying out chromatographic elution separation on the wine sample to be detected after pretreatment in the step (1), then carrying out data acquisition by utilizing a PDA and MS mode, comparing the obtained spectrogram of the sample to be detected with the standard spectrogram obtained in the step (3), carrying out qualitative determination by utilizing retention time and molecular weight, and carrying out quantitative determination by utilizing a standard curve linear regression equation.

The conditions for chromatographic elution separation adopted by the invention are set as follows:

a chromatographic column: ACQUITY UPC2, BEH, 2.1X 50mm, 1.7 μm.

Sample introduction amount: 2 mu L of the solution;

column temperature: mobile phase at 55 ℃: mobile phase a CO₂And the mobile phase B formic acid-water-methanol mixed solution has the flow rate: 1.5 mL/min; the gradient elution procedure was: initial conditions were 2% mobile phase B. Mobile phase B increased to 50% in 2min and remained for 1.5min after reaching 50%. Then mobile phase B was returned to 2% in 0.1min, and the system was rebalanced for 3.0 min. The total cycle time was 10.0 min.

The detection conditions of the invention are as follows:

PDA detector wavelength: 250nm and 290 nm;

MS mode;

ESI + ionization mode conditions: capillary voltage: 3.0 kV; sampling the taper hole voltage: 21V; ion source temperature: 120 ℃; desolventizing temperature: at 450 ℃; flow rate of desolventizing gas: 1000L/H; reference mass: leucine enkephalin [ M + H ]]⁺＝556.2766；

ESI-ionization mode conditions: capillary voltage: 2.5 kV; sampling the taper hole voltage: 21V; ion source temperature: 120 ℃; desolventizing temperature: at 450 ℃; flow rate of desolventizing gas: 1000L/H; reference mass: leucine enkephalin [ M-H]^-554.2615. The collection range is as follows: m/z is 50 to 1200.

In order to ensure qualitative accuracy, PDA detection data can be extracted through a TIC window, and a standard substance and a sample are compared by using three reference values of the retention time of PDA detection, the retention time of ESI detection and molecular weight, so that the qualitative accuracy is ensured. The retention time of myricetin, dihydromyricetin and ESI ion source detection is consistent.

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

the method has simple pretreatment, the linear correlation coefficient is more than 0.99, the recovery rate is between 85 and 115 percent, and the relative standard deviation is within 10 percent, which shows that the method can be applied to qualitative and quantitative analysis of effective components in the waxberry wine and can provide important reference significance for quality control of the waxberry wine.

Drawings

FIG. 1 is a total ion flow diagram of an ES + mode of waxberry wine.

FIG. 2 is an ES-mode total ion flow diagram of waxberry wine.

FIG. 3 is a chromatogram of myricetin and myricetin.

FIG. 4 is a chromatogram of dihydromyricetin.

Detailed Description

In order to facilitate understanding of the present invention, the technical solutions of the present invention will be further described with reference to specific examples.

1.1 reagents, drugs

31 effective component standard substances (purity > 95%, Shanghai' an spectrum/Shanghai source leaf) of fructus Myricae Rubrae, and single-label stock solution (400ug/L) prepared from 50% methanol water, and storing at-20 deg.C in dark place.

1.2 instrumentation

Waters ACQUITY

I-Class system

G2-XS QTof mass spectrometer equipped with photodiode matrix (PDA) detector, electrospray ion source (ESI); waters ACQUITY

I-Class System UPC2 compatible with phase chromatograph, was equipped with 515 pump.

1.3 sample pretreatment

Freeze-drying the sample to be tested, adding 2mL of formic acid-water-methanol mixed solution, mixing uniformly, and filtering with a 0.22 mu m filter membrane to be tested.

1.4 UPC2-PDA-Q-TOF/MS detection

Separating each compound from the liquid to be detected by an ultra-high performance combined phase chromatography system, and sequentially collecting by adopting two modes, namely ES + and ES-respectively, by adopting UPC2-Q-Tof matched with an electrospray ion source; chromatographic conditions are as follows: a chromatographic column: ACQUITY UPC2, BEH, 2.1X 50mm, 1.7 μm. Mobile phase: mobile phase a CO₂Mobile phase B methanol with 0.4% formic acid, 2% water, flow rate: 1.5mL/min, setting two channels with the wavelengths of 250nm and 290nm by PDA, and carrying out gradient elution; initial conditions were 2% mobile phase B. Mobile phase B increased to 50% in 2min and remained for 1.5min after reaching 50%. Then mobile phase B was returned to 2% in 0.1min, and the system was rebalanced for 3.0 min. The total cycle time was 10.0 min. Sample introduction amount: 2 mu L of the solution; column temperature: at 55 ℃.

Mass spectrum conditions:

G2-XS QTof mass spectrometer; an acquisition mode: MS mode; ESI + ionization mode conditions: capillary voltage: 3.0 kV; sampling the taper hole voltage: 40V; ion source temperature: 120 ℃; desolventizing temperature: at 450 ℃; flow rate of desolventizing gas: 1000L/H; reference mass: leucine enkephalin [ M + H ]]⁺556.2766; ESI-ionization mode conditions: capillary voltage: 2.5 kV; sampling the taper hole voltage: 40V; ion source temperature: 120 ℃; desolventizing temperature: at 450 ℃; flow rate of desolventizing gas: 1000L/H; reference mass: leucine enkephalin [ M-H]^-554.2615. The collection range is as follows: m/z is 50 to 1200.

1.5 calculation of the content

And preparing each standard curve according to each effective component standard product, and calculating the content of the effective components in each sample by using the standard curves.

The information of each effective component and the standard curve are shown in the following table.

Example 1: measurement of soaked wine sample

Pretreatment of soaking wine samples: soaking 1000g of waxberry purchased in the market in 5000mL of white spirit, stirring once a day, after 30 days, taking 50mL of waxberry wine, freeze-drying in a 50mL centrifuge tube, adding 2mL of formic acid-water-methanol mixed solution, uniformly mixing, and filtering with a 0.22 mu m filter membrane to be detected;

and (3) measuring 31 effective components in the soaking wine sample, and injecting the sample to be measured according to the liquid phase condition and the mass spectrum condition of 1.4, wherein the results are as follows:

through detection, 1000g of waxberry is directly soaked in 5000mL of white spirit for 30 days, and the red bayberry wine is detected to contain 58.6 mu g/L squalene, 58.6 mu g/L myricetic acid C481.39 mu g/L myricetic acid, 189.36 mu g/L beta-sitosterol, 587.49 mu g/L myricetic acid B, 481.11 mu g/L beta-elemene, 491.22 mu g/L euscaphic acid, 73.19 mu g/L tormentic acid, 257.49 mu g/L quercetin, 4483.36 mu g/L myricetin, 781.49 mu g/L kaempferol, 4479.34 mu g/L myricetin, linalool 5581.26 mu g/L linalool, alpha-terpineol 447.36 mu g/L6, 7-dimethoxycoumarin 449.35 mu g/L myricetin 10225.36 mu g/L myricetin, scopoletin 443.36 mu g/L rubus glycoside F1445.36 mu g/L and (+) -S-glucosyl 446.39 mu g/L myricetin, The general soaking effect can be judged by the fact that the caffeic acid is 884.95 mu g/L, the dihydromyricetin is 6694.36 mu g/L, the gallic acid is 851.39 mu g/L, the myricitrin is 11269.34 mu g/L, the myricetin 3-O-galactoside is 4486.39 mu g/L, and other substances are not detected.

Example 2: measurement of extract

Preparing a waxberry extracting solution: soaking commercially available Notoginseng radix 5000g in 80% (V/V) ethanol water 45kg overnight, heating at 85 deg.C, vacuum reflux extracting for 4 hr, heating at 80 deg.C, concentrating for 3 hr to obtain extractive solution 3.5kg,

pre-treating a waxberry extracting solution: filtering 20mL of Notoginseng radix extract with 8 layers of gauze, centrifuging at 8000r/min for 5min, freeze drying 10mL of supernatant in a centrifuge tube, adding 50mL of formic acid-water-methanol mixed solution, mixing, and filtering with 0.22 μm filter membrane.

The method comprises the following steps of (1) measuring 31 effective components in waxberry extract, and carrying out sample injection on a sample to be measured according to a liquid phase condition and a mass spectrum condition of 1.4, wherein the results are as follows:

the detection shows that the extract contains 994.21 mu g/L squalene, C4576.45 mu g/L myricetic acid, beta-sitosterol 457.53 mu g/L myricetic acid, B1563.47 mu g/L myricetic acid, ursolic acid 551.23 mu g/L, beta-elemene 481.11 mu g/L carotene 1241.25 mu g/L, euscaphic acid 1753.65 mu g/L myricetic acid, tormentic acid 476.34 mu g/L, quercetin 447.22 mu g/L, caryophyllene 245.25 mu g/L, calamene 343.4 mu g/L, myricetin 56734.2 mu g/L, kaempferol 4574.56 mu g/L, myricetin 75645.23 mu g/L, linalool 74133.11 mu g/L, alpha-terpineol 4753.52 mu g/L, 6, 7-dimethoxy coumarin 4471.31 mu g/L, myricetin 45634.22 mu g/L, scopoletin 4631.44 mu g/L lactone 4631.44 mu g/L, Rubusoside F11244.31 μ g/L, (+) -S-myricetin glucoside 753.45 μ g/L, caffeic acid 1536.53 μ g/L, dihydromyricetin 10543.53 μ g/L, gallic acid 1454.25 μ g/L, myricitrin 25436.3 μ g/L, myricetin 3-O-galactoside 9846.32 μ g/L, scopoletin 441.25 μ g/L, and other substances were not detected, and compared with direct soaking, ursolic acid, daucosterol, caryophyllene, calamenene, and scopoletin detected in the extracting solution were not dissolved by soaking, so that it was determined that the dissolving effect of the extracting solution was more desirable than that of direct soaking.

Claims

1. A UPC2-PDA-Q-Tof/MS detection method for 31 effective components in waxberry wine is characterized in that:

firstly, freeze-drying a wine sample to be detected of the waxberry wine into a solid, dissolving the solid by using a formic acid-water-methanol mixed solution, then passing the solid through a 0.22 mu m filter membrane, performing gradient elution separation by using a UPC2 BEH column, simultaneously acquiring data of a UPC2-PDA mode and MS mode, respectively monitoring the data in positive and negative ion modes, comparing an obtained spectrogram of a sample to be detected with a standard spectrogram, obtaining corresponding substances when retention time is the same, and quantifying each effective component by using a linear regression equation of a standard curve of the MS mode.

2. The detection method according to claim 1, characterized by comprising the steps of:

step 1: pretreatment

step 2: preparation of standard substance

and step 3: drawing standard spectrogram and standard curve

Carrying out chromatographic elution separation on the mixed standard solution prepared in the step 2, collecting by utilizing a PDA (personal digital Assistant) and MS (Mass Spectrometry) mode, obtaining a standard spectrogram of 31 waxberry effective component standard products, and obtaining a corresponding standard curve and a linear regression equation by taking the peak area of each effective component as a vertical coordinate and the concentration of the standard product as a horizontal coordinate;

and 4, step 4: detection of a sample to be tested

3. The detection method according to claim 1 or 2, characterized in that:

in the formic acid-water-methanol mixed solution, the concentration of formic acid is 0.4%, the concentration of methanol is 94.6%, V/V and the balance is water.

4. The detection method according to claim 2, wherein the conditions for the chromatographic elution separation are set as follows:

a chromatographic column: ACQUITY UPC2, BEH, 2.1X 50mm, 1.7 μm;

sample introduction amount: 2 mu L of the solution;

column temperature: mobile phase at 55 ℃: mobile phase a CO₂And the mobile phase B formic acid-water-methanol mixed solution has the flow rate: 1.5 mL/min; the gradient elution procedure was: the initial condition is 2% of mobile phase B, the mobile phase B is increased to 50% in 2min, the mobile phase B is maintained for 1.5min after reaching 50%, then the mobile phase B returns to 2% in 0.1min, and the system is rebalanced for 3.0 min; the total cycle time was 10.0 min.

5. The detection method according to claim 2, wherein the detection condition is set as follows:

PDA detector wavelength: 250nm and 290 nm;

MS mode;

ESI-ionization mode conditions: capillary voltage: 2.5 kV; sampling the taper hole voltage: 21V; ion source temperature: 120 ℃; desolventizing temperature: at 450 ℃; flow rate of desolventizing gas: 1000L/H; reference mass: leucine enkephalin [ M-H]^-554.2615; the collection range is as follows: m/z is 50 to 1200.

6. The detection method according to claim 2, characterized in that:

in step 4, in order to ensure qualitative accuracy, PDA detection data can be extracted through a TIC window, and a standard substance and a sample are compared by using three reference values of the retention time of PDA detection, the retention time of ESI detection and the molecular weight, so that the qualitative accuracy is ensured.