CN112147254B

CN112147254B - Method for rapidly and simultaneously determining 35 effective components in wolfberry wine by using UPC2-PDA-Q-Tof/MS

Info

Publication number: CN112147254B
Application number: CN202011020958.9A
Authority: CN
Inventors: 马金同; 曹润洁; 何宏魁; 李安军; 刘国英; 汤有宏; 丁峰; 王录; 孟涛; 秦黎明; 牛培育
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: 2022-04-12
Anticipated expiration: 2040-09-25
Also published as: CN112147254A

Abstract

The invention discloses a method for rapidly and simultaneously determining 35 effective components in medlar wine by using UPC2-PDA-Q-Tof/MS, which rapidly determines 35 effective components in medlar by the steps of preparation of a standard substance, pretreatment of a sample, establishment of chromatographic conditions, mass spectrum conditions, qualitative and quantitative methods and the like. 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 the effective components in the medlar wine and can provide important reference significance for the quality control of the medlar wine.

Description

Method for rapidly and simultaneously determining 35 effective components in wolfberry wine by using UPC2-PDA-Q-Tof/MS

Technical Field

The invention belongs to the field of functional wine, and particularly relates to a method for rapidly and simultaneously determining 35 effective components in wolfberry wine by using UPC 2-PDA-Q-Tof/MS.

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. Along with the concern of people on the health, various low-degree medicinal liquors are produced at the same time, and the wolfberry wine is one of the medicinal liquors. From Shen nong Ben Cao Jing (Shen nong's herbal), Gou Qi Zi is listed as the top grade, and has the effects of nourishing liver and kidney, replenishing vital essence to improve eyesight, cooling blood and removing heat from the body, clearing lung-heat and reducing internal heat. The lycium ruthenicum murr is called black pearl in the west of China, is rich in anthocyanin and has remarkable anti-oxidation effect. Modern scientific researches find that the chemical components in the medlar are various, and mainly relate to compounds such as alkaloids, flavonoids, terpenoids and the like, and in addition, polysaccharide and carotenoid derivatives are common components in the medlar. People combine the medlar and the wine together to prepare the medlar wine, the medlar wine is simple to prepare, has the nutrient components of the medlar, and has certain health-care effect after being drunk for a long time in a small amount.

However, in the current process of producing the medlar wine, various processes of eight-door five-flower are appeared, including direct soaking, crushing soaking, high-degree 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 the effective components of the medlar are various, the dissolution conditions of various substances are different, and the development of the medlar wine is also hindered.

The existing data show that the detection method of the effective components of the medlar is mainly High Performance Liquid Chromatography (HPLC), thin layer chromatography, mass spectrometry and the like, has low detection flux, can not integrally react the content of the effective components of the medlar, 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. The limit of traditional Chromatography is compensated by a novel chromatographic separation technology represented by ultra performance conversion Chromatography (UPC 2)And the GC and LC technologies are combined on the mobile phase, and the separation and analysis capability has the advantages of both 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 problems in the prior art, the invention provides a method for rapidly and simultaneously determining 35 effective components in medlar wine by using UPC 2-PDA-Q-Tof/MS. The method can accurately, quickly, stably and efficiently carry out qualitative and quantitative analysis on the effective components in the wolfberry fruit wine, and provides important reference significance for quality control of the wolfberry fruit wine.

The invention relates to a method for rapidly and simultaneously determining 35 effective components in medlar wine by using UPC2-PDA-Q-Tof/MS, which comprises the steps of firstly freezing and drying a wine sample to be determined 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, performing gradient elution separation through an UPC2BEH column, simultaneously collecting UPC2-PDA mode and MS mode data, respectively monitoring under an ESI positive and negative ion mode, comparing the obtained spectrogram of a sample to be determined 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 invention relates to a method for rapidly and simultaneously determining 35 effective components in medlar wine by using UPC2-PDA-Q-Tof/MS, which comprises the following steps:

step 1: pretreatment

Freeze drying fructus Lycii wine sample to be tested to solid, dissolving with 0.4% formic acid-5% water-94.6% methanol mixed solution, and filtering with 0.22 μm filter membrane;

step 2: preparation of standard substance

Respectively preparing standard solutions of 1G/L beta-carotene, alpha-carotene, stigmasterol, stearic acid, crataegolic acid, oleic acid, linoleic acid, daucosterol, palmitic acid, apigenin, luteolin, kaempferol, quercetin, atropine, L-henbane, cinnamic acid, scopoletin acetate, myricetin, ferulic acid, p-hydroxycinnamic acid, dihydroferulic acid, caffeic acid, nicotinamide, protocatechuic acid, nicotinic acid, malvidin chloride, petunidin, rutin, betaine, riboflavin, cornflower-3-O-glucoside, delphin glucoside, hyoscyami and cornflower-3-O-2G-glucose rutinoside, mixing and gradually diluting the standard solutions of 35 effective components of the barbary wolfberry fruit, obtaining mixed standard substance solutions 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 standard spectrograms of 35 effective component standard products of the medlar, taking the peak area of each effective component as a vertical coordinate and the concentration of the standard product as a horizontal coordinate to obtain corresponding standard curves, wherein a linear regression equation and a relative standard deviation are shown in the following table 1:

TABLE 1 Standard Curve of 35 effective ingredients of Lycium barbarum

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

And (2) carrying out gradient elution separation on the wine sample to be detected after pretreatment in the step (1) through a UPC2BEH column, simultaneously collecting data of a UPC2-PDA mode and MS mode, respectively monitoring in an ESI positive and negative ion mode, comparing the obtained spectrogram of the sample to be detected with a standard spectrogram, carrying out qualitative determination by using retention time and molecular weight, and carrying out quantitative determination through a standard curve linear regression equation.

The chromatographic conditions of 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: 360 nm; 540 nm;

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.

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 quercetin, kaempferol and rutin at 360nm is 1.55, 1.63 and 4.16 respectively, and is consistent with that of ESI ion source detection.

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 the effective components in the medlar wine and can provide important reference significance for the quality control of the medlar wine.

Drawings

FIG. 1 is a diagram of the ES + mode total ion flow of wolfberry wine.

FIG. 2 is a diagram of the ES-mode total ion flow of wolfberry wine.

FIG. 3 is a chromatogram of dihydroferulic acid extract ions.

FIG. 4 is a chromatogram of quercetin, kaempferol, and rutin at 360 nm.

FIG. 5 is a chromatogram at 450nm of β -carotene or α -carotene.

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

35 effective constituent standard substances (purity > 95%, Shanghai' an spectrum/Shanghai source leaf) of fructus Lycii, 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. mu.lAnd m is selected. 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 wavelength of 360nm and 450nm by the 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 500g of wolfberry fruit purchased from the market in 5000mL of white spirit, stirring once a day, after 30 days, taking 50mL of wolfberry fruit 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 35 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, after the Ningxia wolfberry fruit is soaked in the white spirit for 30 days, the Chinese wolfberry fruit is detected to contain 429.55 mu g/L of beta-carotene, 102.56 mu g/L of stearic acid, 254.16 mu g/L of stigmasterol, 226.59 mu g/L of daucosterol, 489.29 mu g/L, L-henbane 489.57 mu g/L of luteolin, 568.12 mu g/L of cinnamic acid, 245.15 mu g/L of ferulic acid, 145.25 mu g/L of p-hydroxycinnamic acid, 867.45 mu g/L of dihydroferulic acid, 669.58 mu g/L of rutin, 1784.45 mu g/L of betaine, 449.15 mu g/L of cornflower-3-O-glucoside, 187.45 mu g/L of delphinidin glucoside, and other substances are not detected, so that the soaking effect of the whole plant is judged to be relatively common.

Example 2: measurement of extract

Preparing an angelica sinensis extracting solution: soaking 4000g of fructus Lycii purchased from market in 45kg of 65% (V/V) alcohol water solution overnight, heating at 85 deg.C, vacuum reflux extracting for 4h, heating at 80 deg.C, concentrating for 3h to obtain extractive solution 3.5kg,

pretreatment of a medlar extracting solution: filtering 20mL of fructus Lycii extractive solution 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 35 effective components in a medlar extracting solution, and injecting samples to be measured according to a liquid phase condition and a mass spectrum condition of 1.4, wherein the results are as follows:

through detection, the angelica sinensis extract contains 5446.32 mu g/L of beta-carotene, 6934.56 mu g/L of stearic acid, 1145.5 mu g/L of stigmasterol, 1452.64 mu g/L of daucosterol, 2254.33 mu g/L, L-hyoscyamine 44781.5 mu g/L of luteolin, 554.36 mu g/L of cinnamic acid, 1147.52 mu g/L of ferulic acid, 5647.36 mu g/L of p-hydroxycinnamic acid, 7746.35 mu g/L of dihydroferulic acid, 55471.35 mu g/L of rutin, 88461, 36 mu g/L of betaine, 224.11 mu g/L of cyanidin-3-O-glucoside, 50.26 mu g/L of delphinidin glucoside and other substances which are not detected, the content of each effective component of the extract is obviously more than 10 times higher than that of the soaked wine sample on the whole, only cyanidin-3-O-glucoside, and the like, The delphinidin glucoside content is lower than that of the soaked wine sample, and the delphinidin glucoside is decomposed probably due to heating, so that the ideal extraction can be judged.

Claims

1. The method for rapidly and simultaneously determining 35 effective components in the medlar wine by using UPC2-PDA-Q-Tof/MS is characterized by comprising the following steps:

firstly, freeze-drying a wine sample to be tested into a solid, dissolving the solid with a formic acid-water-methanol mixed solution, filtering the solid with a 0.22 mu m filter membrane, performing gradient elution separation by using a UPC2BEH column, simultaneously acquiring UPC2-PDA mode and MS mode data, respectively monitoring the sample in an ESI positive and negative ion mode, comparing an obtained spectrogram of the sample to be tested with a standard spectrogram, obtaining corresponding substances when the retention time is the same, and quantifying each effective component by using an MS mode standard curve linear regression equation;

the method comprises the following steps:

step 1: pretreatment

Freeze drying fructus Lycii wine sample to be tested to solid, dissolving with formic acid-water-methanol mixed solution, and filtering with 0.22 μm filter membrane;

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 and MS mode to obtain a standard spectrogram of 35 effective component standard products of the medlar, 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

Carrying out gradient elution separation on the wine sample to be detected after pretreatment in the step 1 through a UPC2BEH column, simultaneously acquiring data of a UPC2-PDA mode and MS mode, respectively monitoring under an ESI positive and negative ion mode, comparing the obtained spectrogram of the sample to be detected with a standard spectrogram, carrying out qualitative determination by using retention time and molecular weight, and carrying out quantitative determination through a standard curve linear regression equation;

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;

the chromatographic conditions were 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.

2. The method according to claim 1, wherein the detection condition is set as follows:

PDA detector wavelength: 360 nm; 540 nm;

MS mode;

ESI-ionization mode barA piece: 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.

3. The method of claim 1, wherein:

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.