Synchronous detection method for multiple carotenoids in wolfberry

Abstract

The invention provides a method for synchronously detecting various carotenoids in medlar, which comprises the following steps: preparing a medlar sample into a to-be-detected sample solution, and detecting the to-be-detected sample solution by adopting an ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technology, the conditions of ultra-high performance liquid chromatography are as follows: a chromatographic column is a core-shell type C30 chromatographic column, the mobile phase comprises an A-phase solvent and a B-phase solvent for gradient elution, the A-phase solvent contains acetonitrile and a 0.4 g / L ammonium formate aqueous solution in a volume ratio of 80: 20, and the B-phase solvent contains isopropanol and acetonitrile in a volume ratio of 60: 40; and performing simultaneous qualitative and / or quantitative detection on the multiple carotenoids contained in the medlar sample through at least one piece of information of retention time, chromatographic peak area, parent ions and characteristic fragment ions. The method provided by the invention can be used for simultaneously, quickly and efficiently separating and accurately quantifying nine carotenoid compounds in wolfberry samples with different germplasm.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method for the simultaneous detection of multiple carotenoids in wolfberry. Background Technology

[0002] Carotenoids are a class of natural pigments widely found in plants, algae, and certain microorganisms. They have important nutritional and physiological functions, such as antioxidation, anti-inflammation, immune regulation, and prevention of chronic diseases. Goji berries ( Lycium barbarum Goji berries (L.) are a traditional medicinal and edible plant rich in various carotenoids, including β-carotene, lutein, zeaxanthin, and their esterified derivatives (such as lutein dipalmitate and zeaxanthin dispalmitate). These components are not only the main contributors to the color of goji berries but also important material bases for their health benefits. Therefore, establishing accurate and efficient methods for detecting carotenoids in goji berries is of great significance for goji berry quality evaluation, processing technology optimization, and functional food development.

[0003] The carotenoids in wolfberries mainly fall into two categories: free and esterified forms. Free carotenoids include β-carotene, lutein, zeaxanthin, β-cryptoxanthin, lycopene, and (E / Z)-hydrolycopene, while esterified forms include β-cryptoxanthin palmitate, lutein dipalmitate, and zeaxanthin dipalmitate. Esterified carotenoids account for over 80% of wolfberries, making them a characteristic component that distinguishes them from other fruits and vegetables. Studies have shown that different wolfberry varieties (such as Ningqi No. 1, Ningqi No. 7, Ningqi No. 7-8, and Ningqi No. 14-02) and processing methods (freeze-drying, hot-air drying, and freshness-locking) significantly affect the composition and content of carotenoids. For example, Ningxia wolfberries are predominantly composed of β-carotene, zeaxanthin, and their esters. Furthermore, factors such as heat, light, and oxygen during processing can lead to the degradation or isomerization of carotenoids, thereby affecting their bioactivity and product quality. Therefore, establishing a method that can simultaneously detect free and esterified carotenoids is of great significance for scientifically evaluating the quality of wolfberries, optimizing processing technology, and guiding consumer choices.

[0004] Currently, the main methods for detecting carotenoids include ultraviolet-visible spectrophotometry (UV-Vis), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). However, these methods still have the following problems when applied to the analysis of carotenoids in wolfberry. UV-Vis was the earliest method used for carotenoid detection, and its principle is based on the quantitative analysis of carotenoids at specific wavelengths (such as around 450 nm). However, UV-Vis can only determine the total content of carotenoids and cannot provide information on individual components; it also has low sensitivity, insufficient detection capability for low-content components (such as lycopene); and it suffers from significant matrix interference, as other pigments in wolfberry (such as anthocyanins) may interfere with the measurement results. Compared to UV-Vis methods, HPLC can achieve multi-component separation, but it still has the following shortcomings: traditional C18 columns have poor separation effects on carotenoids with similar polarities (such as lutein and zeaxanthin), especially esterified carotenoids (such as lutein dipalmitate and zeaxanthin dipalmitate), which are prone to co-elution; UV detectors have low sensitivity, making it difficult to detect trace components; and relying solely on retention time and UV spectroscopy is insufficient to accurately identify structurally similar carotenoids. LC-MS combines the high separation capability of chromatography with the high sensitivity and specificity of mass spectrometry, making it a cutting-edge technology for carotenoid detection. However, existing LC-MS methods still face the following challenges in the analysis of wolfberry: carotenoids, especially non-polar esterified carotenoids (such as zeaxanthin dipalmitate), have low ionization efficiency in electrospray ionization (ESI).

[0005] Sample pretreatment is a crucial step in carotenoid detection, directly affecting extraction efficiency and detection accuracy. Currently used extraction methods include organic solvent extraction, saponification, and emerging subcritical fluid extraction, but all have certain drawbacks: traditional solvents (such as acetone and n-hexane) have low extraction efficiency for esterified carotenoids and are prone to oxidative degradation; KOH-methanol saponification is commonly used in fresh fruit analysis to remove lipids, but strongly alkaline conditions may destroy alkali-labile components such as β-carotene; although C... Extraction is widely used in the extraction of active plant components, but its extraction efficiency for polar carotenoids (such as lutein) is not ideal, and the equipment cost is high. Furthermore, existing sample pretreatment methods are mostly designed for single forms (such as dried fruit or fresh fruit), lacking universal pretreatment schemes applicable to different processing methods (dried fruit, fresh fruit, pulp, extract). The coexisting substances such as sugars and phenols in wolfberries may inhibit or enhance the ionization efficiency of the target analyte, leading to quantitative bias.

[0006] Therefore, it is urgent to establish a highly sensitive simultaneous detection method covering both free and esterified carotenoids; develop differentiated pretreatment processes for wolfberry (dried fruit, fresh fruit, pulp, extract) adapted to different processing methods; and optimize chromatographic-mass spectrometry conditions to solve the problem of separating ester isomers. Summary of the Invention

[0007] To solve all or part of the above-mentioned technical problems, the present invention provides the following technical solutions: The main objective of this invention is to provide a method for the simultaneous detection of multiple carotenoids in wolfberry, comprising: Pretreatment of wolfberry samples includes: extracting carotenoids from wolfberry samples to obtain wolfberry extract; adding butylated hydroxytoluene and acetic acid to wolfberry extract to obtain a mixture; concentrating and drying the mixture; dissolving the precipitate obtained from drying in a mixed solvent of isopropanol and acetonitrile to obtain the sample solution to be tested. The sample solution to be tested was detected by ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry to obtain an ultra-high performance liquid chromatography-mass spectrum; The conditions for ultra-high performance liquid chromatography include: the chromatographic column is a core-shell C30 column, the mobile phase includes A-phase solvent and B-phase solvent for gradient elution, wherein the A-phase solvent contains acetonitrile and 0.4 g / L ammonium formate aqueous solution in a volume ratio of 80:20, and the B-phase solvent contains isopropanol and acetonitrile in a volume ratio of 60:40. The simultaneous qualitative and / or quantitative detection of multiple carotenoids in wolfberry samples was performed using at least one of the following information: retention time, chromatographic peak area, parent ion, and characteristic fragment ions.

[0008] The ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technology provided by this invention can achieve simultaneous, rapid, and efficient separation and accurate quantitative detection of nine carotenoid compounds in wolfberry samples. The method provided by this invention has good sensitivity and accuracy, and is applicable to wolfberry samples with various processing methods such as dried fruit, fresh fruit, pulp, and extract, and has good versatility.

[0009] Carotenoids are often found in complex plant or food matrices. Compared to ESI ion sources, APCI ion sources (atmospheric pressure chemical ionization) are less affected by matrix effects. As a "soft ionization" source, APCI provides abundant molecular ion information; combined with MS... n The function is to induce collisional dissociation of target ions, generating characteristic fragments. For carotenoid isomers, although they have the same molecular weight, their fragmentation patterns differ. This can be achieved by analyzing MS... 1 or MS 2Characteristic fragment ions (such as those that lose toluene and m-xylene) and their abundance ratios in the spectrum can effectively distinguish and identify cis / trans isomers.

[0010] In some embodiments, the amount of butylated hydroxytoluene added is 0.05wt% to 0.2wt% of the wolfberry extract.

[0011] In some embodiments, the amount of acetic acid added is such that the pH of the mixture is 4.5.

[0012] In some embodiments, the volume ratio of isopropanol to acetonitrile in the mixed solvent is 80:20.

[0013] In some embodiments, the precipitate is dissolved in a mixed solvent of isopropanol and acetonitrile to obtain a test solution, which is then filtered using a filter membrane before detection.

[0014] In some embodiments, the core-shell C30 column is a Waters Acquity BEH C30 column.

[0015] In some embodiments, the column temperature of the chromatographic column is 40°C to 50°C.

[0016] In some embodiments, the sample solution to be tested is 3 μL to 10 μL.

[0017] In some embodiments, the flow rate of the mobile phase is 0.3 mL / min. -1 .

[0018] In some embodiments, the detection wavelength is 450 nm.

[0019] In some embodiments, the gradient elution includes: 0~11min: The volume percentage of phase A solvent is 15%~80%, and the volume percentage of phase B solvent is 20%~85%; 11~20min: The volume percentage of phase A solvent is 8%~15%, and the volume percentage of phase B solvent is 85%~92%; 20~27min: The volume percentage of phase A solvent is 6%~8%, and the volume percentage of phase B solvent is 92%~94%; 27~37min: The volume percentage of phase A solvent is 5%~6%, and the volume percentage of phase B solvent is 94%~95%; 37~42min: The volume percentage of phase A solvent is 0~5%, and the volume percentage of phase B solvent is 95%~100%; 42~45min: The volume percentage of phase A solvent is 0~20%, and the volume percentage of phase B solvent is 80%~100%.

[0020] In a typical embodiment, the gradient elution specifically includes: at 0 min, the volume ratio of phase A solvent to phase B solvent is 80:20; at 2 min, the volume ratio of phase A solvent to phase B solvent is 55:45; at 11 min, the volume ratio of phase A solvent to phase B solvent is 15:85; at 20 min, the volume ratio of phase A solvent to phase B solvent is 8:92; at 22 min, the volume ratio of phase A solvent to phase B solvent is 6:95; at 27 min, the volume ratio of phase A solvent to phase B solvent is 6:94; at 37 min, the volume ratio of phase A solvent to phase B solvent is 5:95; at 40 min, the volume ratio of phase A solvent to phase B solvent is 0:100; at 42 min, the volume ratio of phase A solvent to phase B solvent is 0:100; and at 43-45 min, the volume ratio of phase A solvent to phase B solvent is 80:20.

[0021] In a typical embodiment, the conditions for the ultra-high performance liquid chromatography specifically include: Column: Waters Acquity BEH C30 column; Column temperature: 35℃ Flow rate: 0.30 mL / min -1 Sample solution injection volume: 3~10 uL; Detection wavelength: 450 nm; Gradient elution: At 0 min, the volume ratio of phase A solvent to phase B solvent was 80:20; at 2 min, it was 55:45; at 11 min, it was 15:85; at 20 min, it was 8:92; at 22 min, it was 6:95; at 27 min, it was 6:94; at 37 min, it was 5:95; at 40 min, it was 0:100; at 42 min, it was 0:100; and from 43 to 45 min, it was 80:20.

[0022] In some embodiments, the quantitative detection includes: taking standard samples of carotenoids, preparing a series of standard sample solutions of different concentrations for each type of carotenoid, measuring the standard sample solutions under the same conditions as the test sample solution, obtaining a standard curve of peak area versus concentration by plotting the peak area of ​​the measured chromatographic peak as the ordinate and the concentration of the corresponding carotenoid in the standard sample solution as the abscissa, and calculating the content of the corresponding carotenoid in the wolfberry sample based on the standard curve.

[0023] In some embodiments, the conditions for atmospheric pressure chemical ionization mass spectrometry include: Modes: APCI+ mode and APCI- mode; Ionization source scanning quality: 300-1200 m / z; Atomizer pressure: 60 psi; Drying temperature 250℃; Flow rate: 3.8 mL / min; The secondary mass spectrometer was analyzed using automatic acquisition mode, and the strongest ion peak was selected as the parent ion of the secondary mass spectrometer fragmentation. dd-MS was then used. 2 Data-related acquisition parameters are used to obtain the product ion spectrum; Resolution: 35000 FWHM, m / z 200, cycle time 128 ms; using a quality inclusion list including the m / z of the precursor ions for each target carotenoid and their expected chromatographic retention time windows; The sheath gas flow rate is 35 arbitrary units, the auxiliary gas flow rate is 10 arbitrary units, and the purge gas flow rate is 10 arbitrary units; the spray voltage is 3.80 kV, the capillary temperature is 300℃, and the S-lens RF level is 50 arbitrary units. In full mass spectrometry scanning mode, the precursor ion m / z value (mass error ≤ 5 × 10⁻⁶) 6 () is used for quantitative detection, and the target MS / MS mode is used for structural confirmation of carotenoids.

[0024] In some embodiments, the mass spectrometry data is further processed using the software Xcalibur: the efficient screening software Xcalibur is used to compare the m / z values ​​of precursor ions (mass error ≤ 5 × 10⁻⁶). 6 Qualitative analysis was performed using retention time, isotopic distribution, and MS / MS spectra. The ion information displayed on the TIC chromatogram and mass spectrum were identified one by one after comparison with retention time, maximum absorbance wavelength, protonated molecules [M+H]-, MS fragments, and relevant literature. The fragmentation patterns obtained from UPLC-APCI-MSn (APCI-) show the presence of protonated molecular weight [M+H]-, characteristic MS / MS fragments, and high-abundance fragments corresponding to the neutral-loss fatty acid portion, which is the main method for identifying fatty acids in carotenoids.

[0025] In some embodiments, the wolfberry sample contains free carotenoids and esterified carotenoids. The free carotenoids include one or more of β-carotene, lutein, zeaxanthin, β-cryptoxanthin, lycopene, and (E / Z)-hydrolycopene. The esterified carotenoids include one or more of β-cryptoxanthin palmitate, lutein dipalmitate, and zeaxanthin dipalmitate.

[0026] In some embodiments, the method can simultaneously perform quantitative and / or qualitative detection of β-carotene, lutein, zeaxanthin, β-cryptoxanthin, lycopene, (E / Z)-hydrolycopene, β-cryptoxanthin palmitate, lutein dipalmitate, and zeaxanthin dipalmitate in wolfberry samples, that is, achieve simultaneous detection of 9 types of carotenoids.

[0027] In some embodiments, the wolfberry sample includes dried wolfberry fruit, fresh wolfberry fruit, wolfberry pulp, or wolfberry subcritical extract. Differences in the content and quantity of carotenoid compounds are determined by liquid chromatography-mass spectrometry (LC-MS). Different methods can be used to extract carotenoids from different wolfberry samples to obtain the wolfberry extract. For example, carotenoids can be extracted from dried wolfberry fruit using THF-BHT liquid nitrogen grinding, from fresh wolfberry fruit using petroleum ether-KOH saponification purification, from wolfberry pulp using dichloromethane (DCM) centrifugal extraction, or from wolfberry subcritical extract using subcritical butane extraction.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects: The ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technology provided by the present invention can achieve simultaneous, rapid and efficient separation of nine carotenoid compounds in wolfberry samples and accurate quantitative detection. The method provided by the present invention has good sensitivity and accuracy, and it is suitable for the detection of wolfberry samples of different varieties, such as dried fruit, fresh fruit, pulp, extract and other wolfberry samples with different processing methods, and has good universality. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The chromatograms of carotenoids in wolfberries processed using four different methods (L1, X1, Y1, and T1) are shown in the examples. Figure 2a The colorimetric diagrams are of the carotenoid extracts of wolfberries processed using four different methods (L1, X1, Y1, and T1) in the examples. Figure 2b The graph shows the content of carotenoids in wolfberries processed using four different methods (L1, X1, Y1, and T1) in the examples. Figure 3Here is a structural diagram of β-carotene, and the primary ( ) measured in the examples. Figure 3 (A) Level 2 ( Figure 3 (B) Mass spectrum; Figure 4 Here is a structural diagram of lycopene, and the primary ( ) measured in the examples. Figure 4 (A) Secondary mass spectrum ( Figure 4 (Middle B) Figure 5 The structural diagram of (E / Z)-hydrolycopene and the primary ( ) measured in the examples. Figure 5 (A) Level 2 ( Figure 5 (B) Mass spectrum; Figure 6 The diagram shows the structure of zeaxanthin and the primary ( ) measured in the examples. Figure 6 (A) Level 2 ( Figure 6 (B) Mass spectrum; Figure 7 Here is a structural diagram of lutein, and the primary ( ) measured in the examples. Figure 7 (A) Level 2 ( Figure 7 (B) Mass spectrum; Figure 8 The structural diagram of β-cryptoxanthin and the primary ( ) measured in the examples. Figure 8 (A) Level 2 ( Figure 8 (B) Mass spectrum; Figure 9 The diagram shows the structure of β-cryptoxanthin palmitate and the primary fraction measured in the examples. Figure 9 (A) Level 2 ( Figure 9 (B) Mass spectrum; Figure 10 The diagram shows the structure of zeaxanthin dipalmitate and the primary fraction measured in the examples. Figure 10 (A) Level 2 ( Figure 10 (B) Mass spectrum; Figure 11 The diagram shows the structure of lutein dipalmitate and the primary ( ) measured in the examples. Figure 11 (A) Level 2 ( Figure 11 (B) Mass spectrum.

[0031] Figure 12 The Sankey diagram for identifying compounds in four different types of extracts (L1, X1, Y1, and T1) represents the differences in the types of carotenoid compounds found in the extracts. Detailed Implementation

[0032] The technical solutions of the present invention will be described in detail below with reference to specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention. The specific functional details disclosed herein should not be construed as limiting, but are merely intended to form the basis of the claims and to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0033] In addition, unless otherwise specified, all raw materials used in the following embodiments can be purchased from the market, and all production and testing equipment used are known in the art. The chromatograph used in the embodiments of the present invention is an ultra-high performance liquid chromatograph, and the mass spectrometer is a liquid chromatography-quadrupole electrostatic field track trap mass spectrometer.

[0034] The wolfberry samples used in the embodiments of this invention are shown in Table 1: Table 1. Information on wolfberry samples

[0035] Example

[0036] 1. Preparation of the sample solution and chromatographic and mass spectrometric conditions

[0037] (1) Preparation method of the sample solution to be tested: The extraction method of carotenoids from dried wolfberry is as follows: Weigh 5 g of dried wolfberry sample (L1 in Table 1), add 0.01 wt% butylated hydroxytoluene and grind with liquid nitrogen; use 25 mL tetrahydrofuran (THF) for ultrasonic extraction (frequency 6 Hz, power 8W) for 20 min, collect the supernatant, and continue to extract the residue until colorless; combine the extracts, concentrate and evaporate to dryness by rotary evaporation at 36℃, and dissolve and make up to 25 mL with dichloromethane (DCM) to obtain the wolfberry dried fruit extract, which is stored at -80 ℃ for analysis.

[0038] The extraction method of carotenoids from fresh wolfberry is as follows: 5g of fresh wolfberry (X1 in Table 1) was ground and then 75 mL of THF and 0.01wt% butylated hydroxytoluene were added. The mixture was ultrasonically extracted 3 times, and the extracts were combined. The mixture was concentrated at 36 °C. 75 mL of petroleum ether and 25 mL of 10% NaCl aqueous solution were added to a separatory funnel. The organic layer was washed with water until colorless. The organic layers were combined and dehydrated with anhydrous sodium sulfate and concentrated to 30 mL. 30 mL of 10% KOH-methanol solution was added, and N2 was introduced. The mixture was saponified in the dark for 8 h. 25 mL of petroleum ether and 25 mL of 10% NaCl were added. The organic layer was washed with pure water until neutral (pH 7.0) and concentrated to dryness in the dark. The residue was dissolved in dichloromethane (DCM) and the volume was adjusted to 25 mL to obtain the wolfberry extract.

[0039] The extraction method of carotenoids from wolfberry pulp is as follows: Take a sample of wolfberry pulp (Y1 in Table 1), shake it well, accurately weigh 5 g (accurate to 0.0001 g) into a 50 mL centrifuge tube, add 25 mL of dichloromethane (DCM), vortex at 3000 r / min for 5 min, then centrifuge at 4000 r / min for 10 min, take the lower layer solution, and obtain the wolfberry pulp extract.

[0040] The subcritical extraction method for carotenoids in wolfberry is as follows: Take the subcritical extract of mixed wolfberry varieties (T1 in Table 1), use subcritical butane extraction, the temperature is 50 ℃, the material-liquid ratio is 1:1.1, and the extraction time is 40 minutes 4 times to obtain wolfberry subcritical extract.

[0041] The method for preparing the above extract into a test sample solution is as follows: 0.01 wt% butylated hydroxytoluene is added to the extract, and a 2 wt% aqueous acetic acid solution is added to adjust the pH to 4.5 to inhibit oxidation, resulting in a mixed solution; the mixed solution is concentrated and dried to obtain a precipitate, which is then precipitated with isopropanol and acetonitrile solvent in a volume ratio of 80:20 and brought to a final volume of 10.0 ml. The solution is then filtered through a filter membrane to obtain the test sample solution.

[0042] (2) Preparation of standard sample solutions

[0043] Take lutein, β-carotene, lycopene, (E / Z)-hydrolycopene, β-cryptoxanthin, zeaxanthin, β-cryptoxanthin palmitate, zeaxanthin dipalmitate, and lutein dipalmitate standards. Dissolve the above nine carotenoid standards separately in a 1:1 volume ratio of methanol and dichloromethane (DCM) to prepare 500 ppm carotenoid standard stock solutions. All stock solutions are stored at -20°C.

[0044] The above-mentioned carotenoid standard stock solutions were diluted with a 1:1 volume ratio of methanol and dichloromethane (DCM) mixed solvent. For each type of carotenoid, a series of standard sample solutions with concentrations of 0.05-25 ppm were prepared.

[0045] (3) Ultra-high performance liquid chromatography (UPLC) conditions

[0046] Column: Waters Acquity BEH C30 column (4.6 mm × 150 mm, 2.6 μm); Temperature: 35℃; Flow rate: 0.30 mL / min -1 ; Injection volume: 3 uL; Detection wavelength: 450 nm; Mobile phase: Phase A solvent is an aqueous solution of acetonitrile and ammonium formate with a volume ratio of 80:20, wherein the concentration of ammonium formate in the aqueous solution is 0.4 g / L. Phase B solvent is isopropanol and acetonitrile with a volume ratio of 60:40. Gradient elution is performed, and the changes in mobile phase during gradient elution are shown in Table 2.

[0047] Table 2. Mobile phase gradient elution program

[0048] (4) Atmospheric pressure chemical ionization mass spectrometry conditions (MS / MS): Modes: APCI+ mode and APCI- mode; Ionization source scanning quality: 300-1200 m / z; Atomizer pressure: 60 psi; Drying temperature 250 ℃; Flow rate: 3.8 L / min.

[0049] Secondary mass spectrometry analysis was performed using automatic acquisition mode, and the strongest ion peak was selected as the parent ion for secondary mass spectrometry fragmentation. Data correlation acquisition (dd-MS) was also performed. 2 The parameters are used to obtain the ion spectrum of the product.

[0050] Resolution: 35,000 FWHM, m / z 200 (cycle time 128 ms), using a quality-included list including the precursor ion m / z and acquisition window (based on the expected retention time of each chromatographic peak) for each target compound.

[0051] The APCI source parameters are: sheath gas flow rate of 35 arbitrary units, auxiliary gas flow rate of 10 arbitrary units, purge gas flow rate of 10 arbitrary units, spray voltage of 3.80 kV, capillary temperature of 300°C; RF level of the S-lens of 50 arbitrary units; and precursor ion m / z value (mass error ≤ 5 × 10⁻⁶) in full mass spectrometry scanning mode. 6 ) is used for quantification, and the target MS / MS mode is used for structural confirmation of each compound.

[0052] 2. Ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (UPLC-APCI-MS) was used. n Nine types of carotenoids were tested.

[0053] (1) Linear investigation range

[0054] Preparation of mixed standard solutions: The following standard samples are divided into the following three categories: Hydrocarbon carotenoids (nonpolar functional groups): β-carotene, lycopene, (E / Z)-octahydrolycopene; Free lutein (containing hydroxyl groups): zeaxanthin, lutein, β-cryptoxanthin; Esterified lutein (containing fatty acid esters): β-cryptoxanthin palmitate, lutein dipalmitate, zeaxanthin dipalmitate.

[0055] Dissolving esterified lutein: Weigh 1 g of β-cryptoxanthin palmitate, lutein dipalmitate, and zeaxanthin dipalmitate standards, add about 1 mL of dichloromethane (DCM), vortex, and sonicate until completely dissolved.

[0056] Dissolving carotenoids: Weigh 1 g of β-carotene, lycopene, and (E / Z)-octahydrolycopene standards, add about 1 mL of dichloromethane (DCM), vortex and sonicate until completely dissolved. Lycopene dissolves more slowly, so sonicate for 30 min.

[0057] Dissolving free lutein: Weigh 1 g of zeaxanthin, lutein, and β-cryptoxanthin standards, add about 1 mL of dichloromethane (DCM), vortex, and sonicate until completely dissolved.

[0058] Purge a 10 mL brown volumetric flask with nitrogen. Using a pipette, quantitatively transfer all the completely dissolved solutions from the above mixture into this flask. Rinse each original vial several times with dichloromethane (DCM), transferring all the rinsing solution into the volumetric flask to ensure complete transfer. Continue to dilute to the 10 mL mark with the above mixed solvent. Vortex the volumetric flask vigorously for at least 1 minute to ensure thorough mixing, preparing a mixed standard solution.

[0059] Take an appropriate amount of the mixed standard solution and serially dilute it with dichloromethane (DCM) by 2, 4, 8, 16, 32, and 64 times. Shake well to prepare mixed standard solutions with six concentration gradients. Inject 10 μL of each solution into the UPLC-APCI-MS. Perform UPLC-APCI-MS under the chromatographic and mass spectrometric conditions described above. n Detect and record the peak area of ​​the chromatographic peak; Linear regression was performed with the peak area (Y) of the corresponding carotenoid as the ordinate and the concentration (X, μg / mL) of the carotenoid in the mixed standard solution as the abscissa to obtain the detection limit and quantitation limit of the nine carotenoids at signal-to-noise ratios of 3 and 10. The results are shown in Table 3.

[0060] Table 3. Linear relationships, detection limits, and quantitation limits of nine types of carotenoids.

[0061] Based on the standard curves of the nine carotenoids mentioned above, the content of the corresponding carotenoids in the wolfberry sample can be calculated using the following formula: X = C × V / m × 1000; Wherein, X represents the content of the target carotenoid in the wolfberry sample, in mg / kg; C represents the concentration of the target carotenoid in the sample solution, calculated from the standard curve, in μg / L; V represents the final volume of the sample solution, in mL; and m represents the mass of the wolfberry sample to be tested, in g.

[0062] (2) Stability test

[0063] Weigh 5g of freeze-dried wolfberry (L1 in Table 1) and prepare it into a sample solution according to the above-described "Preparation Method for Sample Solution". Place it at room temperature and perform chromatographic and mass spectrometric analysis at 0, 2, 4, 6, 8, 10, 12, and 24 hours. Calculate the RSDs of the peak areas of β-carotene, lycopene, (E / Z)-hydrolycopene, zeaxanthin, lutein, β-cryptoxanthin, β-cryptoxanthin palmitate, zeaxanthin dipalmitate, and lutein dipalmitate, which are 1.40%, 1.20%, 2.10%, 1.80%, 1.50%, 2.20%, 2.60%, 2.10%, and 1.30%, respectively. The results show that the sample is stable within 24 hours.

[0064] (2) Repeatability test

[0065] Six samples of 5.0 g of freeze-dried wolfberry (L1 in Table 1) from the same batch were prepared according to the above-mentioned "Preparation Method of Sample Solution". The samples were injected and analyzed under the above-mentioned chromatographic and mass spectrometric conditions. The average contents of the nine carotenoids were calculated as follows: lutein 0.053 mg / g, β-carotene 127.55 mg / g, lycopene 82.02 mg / g, (E / Z)-hydrolycopene 0.058 mg / g, β-cryptoxanthin 0.089 mg / g, zeaxanthin 0.051 mg / g, β-cryptoxanthin palmitate 0.30 mg / g, zeaxanthin dipalmitate 17.28 mg / g, and lutein dipalmitate 10.65 mg / g. The RSD range of the nine carotenoids was between 0.89% and 1.95%, indicating that the method has good repeatability.

[0066] 3. Determination of the content of nine carotenoid compounds in wolfberry samples processed by different methods

[0067] After preparing the wolfberry samples from the four different processing methods in Table 1 into test solutions, the UPLC-APCI-MS established above was used. nThe method involved determining the content of nine types of carotenoids in wolfberry samples processed using four different methods. The results are shown in Table 4.

[0068] The content of nine carotenoids in wolfberry samples processed using different methods was calculated using the standard curve method, as shown in Table 4. The content of the same component varied significantly across different processing methods, and the content of each component also differed to varying degrees. Therefore, the quality of dried fruit, fresh fruit, pulp, and extracts cannot be evaluated based on a single or two components; rather, the content of multiple components in the sample should be considered simultaneously for a comprehensive evaluation of the sample quality. Dried wolfberry had the highest β-carotene content, while fresh wolfberry had relatively high levels of zeaxanthin and zeaxanthin dipalmitate. Wolfberry pulp and subcritical extract had the highest zeaxanthin dipalmitate content. Most notably, β-cryptoxanthin was the least abundant among wolfberries processed using different methods.

[0069] Table 4. Content of 9 compounds in wolfberry samples processed by different methods

[0070] 4. Identification and differences of carotenoid compounds in wolfberry samples processed by different methods

[0071] After preparing the wolfberry samples from the four different processing methods in Table 1 into test solutions, the UPLC-APCI-MS established above was used. n The method involved identifying compounds using Xcalibur software, and the results are shown in [Figure number missing]. Figure 12 .

[0072] L1 and T1 samples showed abundant zeaxanthin isomers, such as (3S, 3'S)-zeaxanthin, (3R, 3'S)-zeaxanthin, and cis-zeaxanthin. Significant esterified lutein (lutein-myristate, lutein-palmitate) and zeaxanthin dipalmitate were detected in X1, while other samples showed less. Significant differences in the zeaxanthin isomer composition were observed among different wolfberry samples, which can be used to identify the processing technology and freshness of wolfberry. The distribution patterns of free and esterified carotenoids in wolfberry were clearly distinguished, providing a key indicator for evaluating the maturity and nutritional value of wolfberry fruit. This method can sensitively detect metabolic intermediates or stress indicators such as lycopene and epoxidized zeaxanthin, indicating its great application potential in monitoring the growth environment, physiological state, and variety selection of wolfberry. Therefore, this invention is not only suitable for quality control and authenticity identification of wolfberry products, but also provides a powerful analytical tool for in-depth metabolomics research on wolfberry.

[0073] Figure 1 The chromatograms of carotenoids in wolfberries processed using four different methods (L1, X1, Y1, and T1) are shown in the examples.

[0074] Figure 2a The image shows the colorimetric diagrams of carotenoid extracts from wolfberries processed using four different methods (L1, X1, Y1, and T1) in the examples. Figure 2b The graph shows the content of carotenoids in wolfberries processed using four different methods (L1, X1, Y1, and T1) in the examples.

[0075] Figure 3 Here is a structural diagram of β-carotene, and the primary ( ) measured in the examples. Figure 3 (A) Level 2 ( Figure 3 (B) Mass spectrum; Figure 4 Here is a structural diagram of lycopene, and the primary ( ) measured in the examples. Figure 4 (A) Secondary mass spectrum ( Figure 4 (Middle B) Figure 5 The structural diagram of (E / Z)-hydrolycopene and the primary ( ) measured in the examples. Figure 5 (A) Level 2 ( Figure 5 (B) Mass spectrum; Figure 6 The diagram shows the structure of zeaxanthin and the primary ( ) measured in the examples. Figure 6 (A) Level 2 ( Figure 6 (B) Mass spectrum; Figure 7 Here is a structural diagram of lutein, and the primary ( ) measured in the examples. Figure 7 (A) Level 2 ( Figure 7 (B) Mass spectrum; Figure 8 The structural diagram of β-cryptoxanthin and the primary ( ) measured in the examples. Figure 8 (A) Level 2 ( Figure 8 (B) Mass spectrum; Figure 9 The diagram shows the structure of β-cryptoxanthin palmitate and the primary fraction measured in the examples. Figure 9 (A) Level 2 ( Figure 9 (B) Mass spectrum; Figure 10 The diagram shows the structure of zeaxanthin dipalmitate and the primary fraction measured in the examples. Figure 10 (A) Level 2 ( Figure 10 (B) Mass spectrum; Figure 11 The diagram shows the structure of lutein dipalmitate and the primary ( ) measured in the examples. Figure 11 (A) Level 2 ( Figure 11 (B) Mass spectrum.

[0076] Figure 12 The Sankey diagram for identifying compounds in four different types of extracts (L1, X1, Y1, and T1) represents the differences in the types of carotenoid compounds found in the extracts.

[0077] Comparative Example 1

[0078] 1. Preparation of the sample solution to be tested

[0079] Take 0.01 g of the same freeze-dried wolfberry sample (L1 in Table 1) as in the above example, place it in a 50 mL Erlenmeyer flask, add 10 mL of methanol, and weigh it; then sonicate it (power 300 W, frequency 40 kHz) for 30 minutes; after cooling to room temperature, weigh it again, replenish the lost weight with methanol, and shake well; filter the resulting solution through a 0.22 μm microporous membrane, and take the filtrate as the test sample solution for the comparative example.

[0080] 2. Chromatographic conditions

[0081] Chromatograph: Agilent 1260 Infinity II High Performance Liquid Chromatograph; Chromatographic column: Phenomenex C30 column (4.6 mm × 250 mm, 5 μm); Column temperature: 35℃; Flow rate: 0.3 mL / min; Injection volume: 3 μL; Detection wavelength: 450 nm; Mobile phase: Phase A solvent is methanol-acetonitrile-water with a volume ratio of 81:14:5, and Phase B solvent is dichloromethane (DCM). Gradient elution is performed, and the gradient elution program is shown in Table 5.

[0082] The mass spectrometry and other conditions were controlled in the same way as in the example, and will not be repeated here.

[0083] Table 5 Comparative gradient elution program

[0084] 3. Results and Analysis of Comparative Example 1

[0085] Under the chromatographic conditions of Comparative Example 1, after running for 55 minutes, lutein, zeaxanthin, β-carotene, β-cryptoxanthin palmitate, and zeaxanthin dipalmitate were detected. However, the chromatographic conditions of Comparative Example 1 could not effectively detect or accurately quantify lycopene, (E / Z)-hydrolycopene, β-cryptoxanthin, and lutein dipalmitate. Specifically, the retention times of lycopene and β-carotene peaks were similar, the resolution (R) was less than 1.5, and the peaks severely overlapped, making integration calculation impossible. (E / Z)-hydrolycopene eluted very late under these gradient elution conditions, with a severely broadened peak shape, significantly reduced sensitivity, and the signal-to-noise ratio (S / N) did not meet the quantitative requirements. β-cryptoxanthin and zeaxanthin were not completely separated, showing a clear co-elution trend, making accurate quantitative analysis impossible. The polarity difference between lutein dipalmitate and zeaxanthin dipalmitate was smoothed out in this system, failing to achieve baseline separation.

[0086] Furthermore, the total time required to complete a single sample analysis (including gradient elution and system rebalancing to initial state) in Comparative Example 1 exceeds 60 minutes. Compared to the analysis method provided in this embodiment of the invention (less than 45 minutes), the single-needle sample analysis time is extended by at least 33%, resulting in low analysis efficiency and significantly limiting the application of this method in high-throughput screening of large batches of samples.

[0087] As can be seen, the method in Comparative Example 1 is only applicable to the detection of a limited number (5 types) of carotenoid compounds. It has significant technical limitations for the more structurally complex and polarity-range-widening Lycium barbarum carotenoid components (such as the 9 compounds in this invention), namely, insufficient separation capability and incomplete detection. Furthermore, the analysis cycle is too long (>60 minutes / injection), and the analysis efficiency is lower than the method provided by this invention, failing to meet the urgent needs of modern quality inspection processes for speed and efficiency.

[0088] In summary, this invention achieves simultaneous, rapid, efficient, and accurate separation and quantification of nine carotenoid compounds in wolfberry through innovative chromatographic conditions (including but not limited to column selection, gradient optimization, and mobile phase composition), significantly improving detection throughput and accuracy. It is also applicable to different wolfberry samples and has universality.

[0089] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0090] All aspects, embodiments, features, and examples of this invention should be considered illustrative and used to explain and illustrate the invention, but not to limit the invention. The scope of the invention is defined only by the claims.

[0091] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements in the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed, but rather to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. A method for simultaneous detection of multiple carotenoids in wolfberry, characterized in that, include: Pretreatment of wolfberry samples includes: extracting carotenoids from wolfberry samples to obtain wolfberry extract; adding butylated hydroxytoluene and acetic acid to wolfberry extract to obtain a mixture; concentrating and drying the mixture; dissolving the precipitate obtained from drying in a mixed solvent of isopropanol and acetonitrile to obtain the sample solution to be tested. The sample solution to be tested was detected by ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry to obtain an ultra-high performance liquid chromatography-mass spectrum; The conditions for ultra-high performance liquid chromatography include: the chromatographic column is a core-shell C30 column, the mobile phase includes A-phase solvent and B-phase solvent for gradient elution, wherein the A-phase solvent contains acetonitrile and 0.4 g / L ammonium formate aqueous solution in a volume ratio of 80:20, and the B-phase solvent contains isopropanol and acetonitrile in a volume ratio of 60:

40. The multiple carotenoids contained in wolfberry samples can be simultaneously qualitatively and / or quantitatively detected by using at least one of the following information: retention time, chromatographic peak area, parent ion, and characteristic fragment ions.

2. The method according to claim 1, characterized in that, The amount of butylated hydroxytoluene added is 0.05 wt% to 0.2 wt% of the wolfberry extract; And / or, the amount of acetic acid added makes the pH of the mixture 4.5; And / or, the volume ratio of isopropanol to acetonitrile in the mixed solvent is 80:20; And / or, after dissolving the precipitate in a mixed solvent of isopropanol and acetonitrile to obtain the test solution, the test solution is filtered using a filter membrane before detection.

3. The method according to claim 1, characterized in that: The core-shell C30 column is a Waters AcquityBEH C30 column.

4. The method according to claim 1, characterized in that: The specific conditions for the ultra-high performance liquid chromatography (UHPLC) include: a column temperature of 40℃~50℃; and / or, an injection volume of 3uL~10uL for the sample solution to be tested; and / or, a flow rate of 0.3mL / min for the mobile phase. -1 ; and / or, the detection wavelength is 450nm.

5. The method according to claim 1, characterized in that, The gradient elution specifically includes: 0~11min: The volume percentage of phase A solvent is 15%~80%, and the volume percentage of phase B solvent is 20%~85%; 11~20min: The volume percentage of phase A solvent is 8%~15%, and the volume percentage of phase B solvent is 85%~92%; 20~27min: The volume percentage of phase A solvent is 6%~8%, and the volume percentage of phase B solvent is 92%~94%; 27~37min: The volume percentage of phase A solvent is 5%~6%, and the volume percentage of phase B solvent is 94%~95%; 37~42min: The volume percentage of phase A solvent is 0~5%, and the volume percentage of phase B solvent is 95%~100%; 42~45min: The volume percentage of phase A solvent is 0~20%, and the volume percentage of phase B solvent is 80%~100%.

6. The method according to claim 1, characterized in that, The quantitative detection includes: taking carotenoid standard samples, preparing a series of standard sample solutions of different concentrations for each type of carotenoid, measuring the standard sample solutions under the same conditions as the test sample solution, and obtaining a standard curve of the relationship between peak area and concentration by plotting the peak area of ​​the measured chromatographic peak as the ordinate and the concentration of the corresponding carotenoid in the standard sample solution as the abscissa. The content of the corresponding carotenoid in the wolfberry sample is calculated based on the standard curve.

7. The method according to claim 1, characterized in that: The conditions for atmospheric pressure chemical ionization mass spectrometry include: Modes: APCI+ mode and APCI- mode; Ionization source scanning quality: 300-1200 m / z; Atomizer pressure: 60 psi; Drying temperature 250℃; Flow rate: 3.8 mL / min; The secondary mass spectrometer was analyzed using automatic acquisition mode, and the strongest ion peak was selected as the parent ion of the secondary mass spectrometer fragmentation. dd-MS was then used. 2 Data-related acquisition parameters are used to obtain the product ion spectrum; Resolution: 35000 FWHM, m / z 200, cycle time 128 ms; using a quality inclusion list including the m / z of the precursor ions for each target carotenoid and their expected chromatographic retention time windows; The sheath gas flow rate is 35 arbitrary units, the auxiliary gas flow rate is 10 arbitrary units, and the purge gas flow rate is 10 arbitrary units; the spray voltage is 3.80 kV, the capillary temperature is 300℃, and the S-lens RF level is 50 arbitrary units. In full mass spectrometry scanning mode, the precursor ion m / z value (mass error ≤ 5 × 10⁻⁶) 6 () is used for quantitative detection, and the target MS / MS mode is used for structural confirmation of carotenoids.

8. The method according to claim 1, characterized in that: The wolfberry sample contains free carotenoids and esterified carotenoids. The free carotenoids include one or more of β-carotene, lutein, zeaxanthin, β-cryptoxanthin, lycopene, and (E / Z)-hydrolycopene. The esterified carotenoids include one or more of β-cryptoxanthin palmitate, lutein dipalmitate, and zeaxanthin dipalmitate.

9. The method according to claim 8, characterized in that: The method can simultaneously perform quantitative and / or qualitative detection of β-carotene, lutein, zeaxanthin, β-cryptoxanthin, lycopene, (E / Z)-hydrolycopene, β-cryptoxanthin palmitate, lutein dipalmitate, and zeaxanthin dipalmitate in wolfberry samples.

10. The method according to claim 1, characterized in that, The goji berry samples included dried goji berries, fresh goji berries, goji berry juice, or subcritical extracts of goji berries. The differences in the content and quantity of carotenoid compounds were determined by liquid chromatography-mass spectrometry.

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