Determination of 13 carotenoids in fruits and vegetables by high performance liquid chromatography-mass spectrometry

Abstract

The application discloses a kind of 13 kinds of carotenoid monomer high-efficiency liquid chromatography-mass spectrometry detection methods in fruits and vegetables, belongs to analytical chemistry and food detection technical field, fruits and vegetables are extracted after solvent, gradient elution separation is used chromatographic column, with methanol-acetonitrile and ultrapure water (containing 0.1% formic acid, 5 mmol / L ammonium acetate) as mobile phase, using mass spectrometry detection, acquisition 13 kinds of carotenoid monomer characteristic ion pair.The application can simultaneously detect 13 kinds of carotenoid monomers: lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene, lycopene, gamma-carotene, new xanthin, purple xanthin, (E / Z)-octahydro lycopene, anther xanthin, beta-apo-8'-carotene aldehyde and capsicum red, with mild conditions, instrument applicable range is wide, sensitivity is high, selectivity is good, analysis time is short, qualitative and quantitative accurate, suitable for the analysis and detection of carotenoid in various fruits and vegetables samples.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and food testing technology. More specifically, this invention relates to a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the detection of 13 carotenoid monomers in fruits and vegetables. Background Technology

[0002] Carotenoids are a class of natural pigments widely found in plants, animals, and algae, and usually refer to C. 40 Carotenoids are a collective term for hydrocarbons (carotene) and their oxidation derivatives (lutein), structurally composed of eight isoprene units condensed together. Carotenoids not only give fruits and vegetables their vibrant colors but also possess excellent antioxidant and anticancer activities, playing a vital role in preventing chronic human diseases such as cancer, cardiovascular disease, osteoporosis, and diabetes. They are also high-quality natural pigments, widely used as colorants and feed additives in the food industry. Accurately determining the composition and content of carotenoids in fruits and vegetables is of great significance for fruit and vegetable quality evaluation, nutritional component analysis, and the development of functional foods.

[0003] However, the diverse types and similar structures of carotenoids, along with significant differences in polarity, thermal stability, and matrix distribution among different carotenoid monomers, pose considerable challenges to their simultaneous detection. Several existing methods for detecting carotenoids have been reported, but all have varying degrees of limitations.

[0004] A rapid detection method for carotenoids in food (CN116297920A) employs a chloroform-methanol-water system for extraction, followed by purification using a hydrophilic-lipophilic balance (HLB) solid-phase extraction column. However, chloroform, as one of the extraction solvents, easily degrades carotenoids containing conjugated carbonyl groups, such as capsanthin, affecting the accuracy of the detection results. Furthermore, this method requires multiple steps including liquid-liquid partitioning, solid-phase extraction loading, rinsing, and elution, resulting in a cumbersome and time-consuming pretreatment process. In addition, while high-resolution orbital trap mass spectrometry (HLS) is used as the detector and data is acquired in full-scan mode, it can record ion information over a wide mass range. However, due to signal dispersion effects in full-scan mode, the detection sensitivity for low-content components is insufficient. Moreover, extensive data processing and peak extraction are required after data acquisition, making it difficult to meet the rapid detection needs of large batches of fruit and vegetable samples. The carotenoids covered by this method include astaxanthin and its esterified derivatives. These components are present in very low or almost non-existent amounts in fresh fruits and vegetables. Capsaicin, β-apo-8′-carotene aldehyde, and (E / Z)-hydrolycopene, which are characteristic of fresh fruits and vegetables, were not included in the detection system in this method.

[0005] A method for determining the content of 13 carotenoids in sweet potatoes (CN115792018A) uses a C30 chromatographic column combined with a UV detector for the separation and determination of carotenoids. This method relies solely on retention time and UV absorption spectroscopy for component identification. Due to the complex composition of fruit and vegetable matrices, interfering components with similar polarities in the co-elutions also produce absorption signals on the UV detector, easily leading to false positive results and insufficient qualitative accuracy. Furthermore, this method only uses room temperature or low temperature extraction in sample pretreatment, without incorporating low-temperature freeze-drying. Thermosensitive carotenoids such as neoxanthin and apoxanthin are prone to degradation and structural transformation during extraction and concentration, resulting in detected values ​​lower than the actual content. The carotenoid components detected by this method also lack capsanthin, β-apo-8′-carotene aldehyde, and (E / Z)-hydrolycopene, failing to meet the needs of comprehensive analysis of carotenoids in fruits and vegetables.

[0006] A method for detecting seven types of carotenoids (CN112557536A) uses ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to detect carotenoids. However, this method only targets lutein compounds in algal samples, covering only seven types. It completely lacks core carotenoid components abundant in fruits and vegetables, such as lycopene, β-carotene, and γ-carotene, and also excludes characteristic carotenoids from fruits and vegetables, such as capsanthin and β-apo-8′-carotene aldehyde. The pretreatment method and chromatographic conditions of this method are optimized based on the characteristics of the algal matrix. However, algal matrices differ significantly from fresh fruits and vegetables in terms of water content, cell wall structure, pigment composition, and types of interfering substances. Therefore, the technical solution cannot be directly transferred to the detection of fruit and vegetable samples.

[0007] Furthermore, traditional high-performance liquid chromatography (HPLC) methods for carotenoid detection typically require analysis times exceeding 40 minutes, resulting in low throughput and difficulty in meeting the analytical needs of large-volume samples. In some existing methods, a saponification step is required to convert carotenoid esters into their free state for detection. This saponification process is not only time-consuming, but the strong alkalinity and heating conditions can easily lead to the degradation and structural transformation of thermosensitive carotenoids such as neoxanthin and azoxanthin, causing the detection results to deviate from the true values.

[0008] In summary, no existing technology has established a method for detecting carotenoids that simultaneously meets the following requirements: (1) applicability to fresh fruit and vegetable sample matrices; (2) no need for saponification treatment, avoiding degradation of heat-sensitive components; (3) coverage of 13 characteristic monomers of major carotenoids and lutein in fruits and vegetables, including capsanthin, β-apo-8′-carotene aldehyde, and (E / Z)-hydrolycopene; (4) short analysis time and high throughput; and (5) high sensitivity and high selectivity, capable of accurately distinguishing isomers and co-eluting components. Therefore, developing a method for detecting multiple carotenoid monomers in fruits and vegetables that simultaneously meets the above requirements is a technical problem urgently needing to be solved in this field. Summary of the Invention

[0009] This invention provides a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the detection of 13 carotenoid monomers in fruits and vegetables. It solves the problems of existing carotenoid detection methods, such as long analysis time, limited number of simultaneously detectable monomers, difficulty in baseline separation of isomers, easy degradation of some heat-sensitive components during saponification, and low detection sensitivity. This invention achieves rapid separation and highly selective detection of 13 carotenoid monomers by optimizing HPLC conditions, using a dedicated C18 column and gradient elution program, combined with multiple reaction monitoring (MRM) mass spectrometry. This method offers the following advantages: the total analysis time is only 15–22 min, significantly shorter than the 40+ min required by traditional HPLC methods; it can simultaneously detect 13 carotenoid monomers, covering the major carotenoids and lutein components in fruits and vegetables; the separation between monomers is excellent, with baseline separation achieved even for difficult-to-separate isomers such as lutein and zeaxanthin; the mass spectrometry detection limit is 0.09–1.5 μg / L, demonstrating high sensitivity; detection is performed using characteristic precursor ion-daughter ion pairs, resulting in good selectivity and accurate quantification even for co-elutants that were not completely separated chromatographically; no saponification treatment is required, avoiding degradation of heat-sensitive components; method validation shows that its precision, repeatability, stability, and spiked recovery all meet analytical requirements, making it suitable for the qualitative and quantitative detection of carotenoids in various fruit and vegetable samples, including sweet potatoes, carrots, pumpkins, tomatoes, mangoes, and citrus fruits.

[0010] To achieve these objectives and other advantages according to the present invention, a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method is provided for the detection of 13 carotenoid monomers in fruits and vegetables. The 13 carotenoid monomers are endogenous free characteristic monomers from fruits and vegetables, specifically comprising: neoxanthin, apoxanthin, antheroxin, capsanthin, lutein, zeaxanthin, β-cryptoxanthin, lycopene, γ-carotene, α-carotene, β-carotene, (E / Z)-epoxy-lycopene, and β-apo-8'-carotene aldehyde. The method requires no saponification treatment, does not use HLB solid-phase extraction for purification, and does not introduce chloroform or halogenated solvents. The detection method specifically includes the following steps: S1. Sample pretreatment: After crushing and homogenizing the fruit and vegetable samples, they were extracted in the dark using a mixed organic solvent containing 0.01 wt% 2,6-di-tert-butyl-p-cresol in hexane, acetone and anhydrous ethanol. The extract was centrifuged and filtered, and then redissolved in acetonitrile to obtain the test solution. S2: Liquid chromatography separation: A C18 column with a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm was used to separate the test solution at a flow rate of 0.3 mL / min, a column temperature of 40℃, and an injection volume of 2 μL. Mobile phase A is ultrapure water containing 0.1% formic acid and 5 mmol / L ammonium acetate by volume, and mobile phase B is a mixed solution of methanol and acetonitrile by volume ratio of 4:6. The following gradient elution program was used: 0–1 min: mobile phase A volume fraction 15%, mobile phase B volume fraction 85%; 1–8 min: mobile phase A was linearly gradiented from 15% to 0%, mobile phase B was linearly gradiented from 85% to 100%; 8–22 min: mobile phase A remained at 0%, mobile phase B remained at 100%. S3: Mass spectrometry detection: A triple quadrupole mass spectrometer was used with an electrospray ionization source in positive ion mode and multiple reaction monitoring mode to collect characteristic parent ion-daughter ion pairs of the above 13 carotenoid monomers: neoxanthin, azoxanthin, antheroxin, capsanthin, lutein, zeaxanthin, β-cryptoxanthin, lycopene, γ-carotene, α-carotene, β-carotene, (E / Z)-octahydrolycopene and β-apo-8'-carotene aldehyde; S4: Results Analysis: The content of the 13 carotenoid monomers in the test solution was calculated based on the standard curves of each standard using the matrix-matched external standard method.

[0011] Preferably, in step S3, the mass spectrometry detection uses the following ion source parameters: nebulizing gas pressure of 45.0 psi, drying gas flow rate of 8.0 L / min, drying gas temperature of 250℃, sheath gas flow rate of 11.0 L / min, sheath gas temperature of 350℃, capillary voltage of 3000 V, and nozzle voltage of 500 V.

[0012] Preferably, in step S2, the total running time of the gradient elution procedure is 15 to 22 minutes.

[0013] Preferably, in step S1, the volume ratio of n-hexane, acetone, and anhydrous ethanol is 2:1:1.

[0014] Preferably, in step S3, the parent ion and daughter ion pairs of the 13 types of carotenoid monomers are specifically as follows: Neoxanthin: parent ion m / z 601.4, daughter ions m / z 221.1 and 565.5; Violet-yellow ion: parent ion m / z 601.4, daughter ions m / z 221.0, 565.5 and 565.8; Anther xanthophyll: parent ion m / z 585.4, daughter ions m / z 175.1, 493.5, 505.4 and 568.6; Capsaicin: parent ion m / z 585.4, daughter ions m / z 109.1, 175.2 and 209.1; Lutein: parent ion m / z 569.4, daughter ions m / z 175.0 and 338.3; Zeaxanthin: parent ion m / z 569.4, daughter ions m / z 174.8 and 477.5; β-apo-8′-carotene aldehyde: parent ion m / z 417.3, daughter ions m / z 161.2 and 325.2; β-cryptoxanthin: parent ion m / z 553.5, daughter ions m / z 105.1, 119.1 and 461.2; Lycopene: parent ion m / z 537.4, daughter ions m / z 69.1, 144.9 and 445.4; γ-Carotene: parent ion m / z 537.4, daughter ions m / z 104.9, 118.9 and 176.9; α-Carotene: parent ion m / z 537.4, daughter ions m / z 145.3 and 444.5; β-Carotene: parent ion m / z 537.4, daughter ions m / z 119.0, 177.1 and 445.5; (E / Z)-Hydroxylycopene: parent ion m / z 545.4, daughter ions m / z 69.0, 81.0 and 339.1.

[0015] Preferably, in step S3, the fragmentation voltage and impact energy of each monomer are as follows: Neoxanthine: fragmentation voltage 172 V, collision energy corresponding to daughter ion m / z 221.1 is 10 eV, collision energy corresponding to daughter ion m / z 565.5 is 18 eV; Violet-yellow matter: fragmentation voltage 142 V, collision energy corresponding to daughter ion m / z 221.0 is 15 eV, collision energy corresponding to daughter ion m / z 565.5 is 20 eV, and collision energy corresponding to daughter ion m / z 565.8 is 22 eV; Anther xanthophyll: Fragmentation voltage 130 V, collision energy corresponding to daughter ion m / z 175.1 is 18 eV, collision energy corresponding to daughter ion m / z 493.5 is 18 eV, collision energy corresponding to daughter ion m / z 505.4 is 20 eV, collision energy corresponding to daughter ion m / z 568.6 is 20 eV; Capsaicin: Fragmentation voltage 178 V, collision energy corresponding to daughter ion m / z 109.1 is 25 eV, collision energy corresponding to daughter ion m / z 175.2 is 20 eV, and collision energy corresponding to daughter ion m / z 209.1 is 20 eV; Lutein: Fragmentation voltage 168 V, collision energy corresponding to daughter ion m / z 175.0 is 22 eV, collision energy corresponding to daughter ion m / z 338.3 is 18 eV; Zeaxanthin: fragmentation voltage 138 V, collision energy corresponding to daughter ion m / z 174.8 is 22 eV, and collision energy corresponding to daughter ion m / z 477.5 is 15 eV; β-apo-8′-carotene aldehyde: fragmentation voltage 118 V, collision energy corresponding to daughter ion m / z 161.2 is 20 eV, collision energy corresponding to daughter ion m / z 325.2 is 5 eV; β-cryptoxanthin: fragmentation voltage 175 V, collision energy corresponding to daughter ion m / z 105.1 is 75 eV, collision energy corresponding to daughter ion m / z 119.1 is 40 eV, and collision energy corresponding to daughter ion m / z 461.2 is 12 eV; Lycopene: Fragmentation voltage 152 V, collision energy corresponding to daughter ion m / z 69.1 is 48 eV, collision energy corresponding to daughter ion m / z 144.9 is 38 eV, and collision energy corresponding to daughter ion m / z 445.4 is 10 eV; γ-Carotene: Fragmentation voltage 165 V, collision energy corresponding to daughter ion m / z 104.9 is 65 eV, daughter ion m / z 118.9 is 40 eV, daughter ion m / z 176.9 is 18 eV; α-Carotene: Fragmentation voltage 175 V, collision energy corresponding to daughter ion m / z 145.3 is 42 eV, collision energy corresponding to daughter ion m / z 444.5 is 15 eV; β-Carotene: Fragmentation voltage 135 V, collision energy corresponding to daughter ion m / z 119.0 is 45 eV, collision energy corresponding to daughter ion m / z 177.1 is 22 eV, and collision energy corresponding to daughter ion m / z 445.5 is 15 eV; (E / Z)-Hydroxylycopene: Fragmentation voltage 142 V, collision energy corresponding to daughter ion m / z 69.0 is 62 eV, collision energy corresponding to daughter ion m / z 81.0 is 28 eV, and collision energy corresponding to daughter ion m / z 339.1 is 32 eV.

[0016] Preferably, in step S1, the sample pretreatment specifically includes: cutting the fruit and vegetable samples into small pieces, freezing them at -80℃ for 8 h, then placing them in a freeze dryer and freeze-drying them in the dark at -50℃ to -40℃ for 24 h, and then pulverizing them through a 60-mesh sieve to obtain sample powder; extracting the sample in the dark using a mixed organic solvent containing 0.01wt% 2,6-di-tert-butyl-p-cresol in hexane, acetone, and anhydrous ethanol; adding 1 mL of ultrapure water after extraction and stirring magnetically for 10 min; repeating the extraction 3 times; centrifuging the extract at 4℃ and 12000 rpm for 10 min; drying the extract with nitrogen and then redissolving it with acetonitrile, vortexing for 3 min, and filtering it through a 0.22 μm organic filter membrane.

[0017] Preferably, in step S1, the liquid-to-solid ratio of the mixed organic solvent containing 0.01 wt% 2,6-di-tert-butyl-p-cresol in hexane, acetone and anhydrous ethanol for extraction in the dark is 10:1, mL / g, and the extraction time is 20 min.

[0018] Preferably, the limits of detection (LODs) for the 13 carotenoid monomers are 0.09–1.5 μg / L, and the limits of quantification (LOQs) are 0.2–4.5 μg / L; the precision RSD is 1.2%–2.5%, and the spiked recovery rate is 85.2%–106.2%. Specifically, the LOD for β-apo-8′-carotene aldehyde is 0.09 μg / L, and the LOD for quantification is 0.2 μg / L; the LODs for antheroxin, capsanthin, lutein, and zeaxanthin are 0.2 μg / L; the LOD for β-cryptoxanthin is 0.3 μg / L; the LODs for neoxanthin and amethystoxanthin are 0.5 μg / L; the LOD for β-carotene is 0.8 μg / L; the LODs for γ-carotene, α-carotene, and (E / Z)-hydrolycopene are 1.0 μg / L; and the LOD for lycopene is 1.5 μg / L. μg / L.

[0019] Preferably, the fruits and vegetables include yellow peaches, sweet potatoes, carrots, pumpkins, tomatoes, mangoes, and citrus fruits.

[0020] The present invention has at least the following beneficial effects: 1. Multiple detection types: It can simultaneously detect 13 types of carotenoid monomers, covering the main carotenoids (such as α-carotene, β-carotene, γ-carotene, lycopene, (E / Z)-hydrolycopene) and lutein (such as lutein, zeaxanthin, β-cryptoxanthin, neoxanthin, amethystin, antheroxanthin, capsanthin, β-apo-8'-carotene aldehyde) in fruits and vegetables, meeting the comprehensive analytical needs of carotenoids in fruits and vegetables.

[0021] 2. Short analysis time: With the optimized gradient elution program, the total analysis time (total gradient elution run time) is only 15 to 22 minutes, which is much shorter than the 40 minutes or more of the traditional HPLC method, significantly improving the detection throughput.

[0022] 3. Excellent separation effect: Using an optimized mobile phase composition (phase A is ultrapure water containing 0.1% formic acid and 5 mmol / L ammonium acetate, phase B is methanol:acetonitrile = 4:6) and a dedicated C18 column, the separation between monomers is good, and the minimum resolution (Rs) of adjacent chromatographic peaks is greater than 1.5. Baseline separation can be achieved for difficult-to-separate isomers such as lutein and zeaxanthin. The peaks are symmetrical and sharp, with no overlapping interference.

[0023] 4. High sensitivity and selectivity: Employing triple quadrupole mass spectrometry in multiple reaction monitoring mode, it performs targeted detection through characteristic precursor ion-daughter ion pairs, exhibiting strong anti-interference capabilities. The limits of detection (LOD) for each monomer ranges from 0.09 to 1.5 μg / L, and the limits of quantitation (LOQ) range from 0.2 to 4.5 μg / L, with the LOD for β-apo-8'-carotene aldehyde as low as 0.09 μg / L. Even co-elutings that are not completely separated by chromatography can be accurately quantified.

[0024] 5. The method is accurate and reliable: The method validation results show that the precision RSD is 1.2% to 2.5%, the repeatability RSD is 2.5% to 4.1%, the stability RSD is 3.2% to 5.2%, the spiked recovery rate is 85.2% to 106.2%, and the linear correlation coefficient R² is greater than 0.99, which meets the requirements for analysis and detection.

[0025] 6. No saponification required, protecting heat-sensitive components: This invention eliminates the need for a time-consuming saponification step, allowing direct detection of free and naturally occurring carotenoids. This avoids the degradation and structural transformation of heat-sensitive carotenoids (such as neoxanthin and auroxin) during the saponification process, resulting in more accurate and reliable test results.

[0026] 7. Wide applicability: Utilizing mild mobile phase reagents and a standard C18 column, this detection condition is achievable with all mass spectrometry instruments. It has been validated for use in the detection of carotenoids in various fruits and vegetables, including yellow peaches (such as Jinxiu Yellow Peach, Golden Honey No. 1, and Frederica), sweet potatoes, carrots, pumpkins, tomatoes, mangoes, and citrus fruits, as well as their processed products.

[0027] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0028] Figure 1 The total ion current chromatogram of a mixed standard of 13 types of carotene detected using the method of this invention; Figure 2 The chromatogram of carotenoid monomers in Prunus cerasifera using the detection method of this invention is shown. Figure 3 The chromatogram of carotenoid monomers in Golden Honey No. 1 yellow peaches detected using the method of this invention is shown. Figure 4 The chromatogram of carotenoid monomers in Frederica yellow peaches detected using the method of this invention is shown. Figure 5 The total ion chromatograms of 13 carotenoid monomers were obtained using a comparative detection method. Figure 6 The chromatograms of liquid phase separation of 13 types of carotenoids are shown when mobile phase A is only ultrapure water. Figure 7 The liquid phase chromatogram of 13 carotenoids when mobile phase A is ultrapure water containing only 0.1% (v / v) formic acid; Figure 8 The chromatogram shows the liquid phase separation of 13 carotenoids when mobile phase A is ultrapure water containing only 5 mmol / L ammonium acetate. Figure 9 The image shows the liquid phase chromatograms of 13 carotenoids separated in mobile phase B with a methanol:acetonitrile ratio of 3.5:6.5. Figure 10 The image shows the liquid phase chromatograms of 13 carotenoids separated in mobile phase B with a methanol:acetonitrile ratio of 4.5:5.5. Figure 11 The liquid phase chromatograms show the separation of 13 types of carotenoids at a flow rate of 0.2 mL / min. Figure 12 The chromatograms show the liquid phase separation of 13 types of carotenoids at a flow rate of 0.4 mL / min. Detailed Implementation

[0029] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0030] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0031] Instruments and equipment: Triple quadrupole mass spectrometer (6495, Agilent Technologies); analytical balance; high-speed grinder; ultrasonic cleaner; centrifuge; nitrogen blower.

[0032] Reagents and standards: Methanol, acetonitrile, acetone, n-hexane, and anhydrous ethanol are all chromatographic grade; 2,6-di-tert-butyl-p-cresol (BHT) and formic acid are mass spectrometry grade; ultrapure water, and 13 types of carotene standards with a purity ≥96%.

[0033] Example 1 Sample pretreatment: Take fresh fruit and vegetable (yellow peach) samples, remove inedible parts, cut into small pieces, freeze at -80˚C for 8 h, then freeze-dry in a freeze dryer at -50℃ in the dark for 24 h, then pulverize and sieve (60 mesh). Accurately weigh 0.05 g of sample powder and place it in a brown centrifuge tube. In a dark, room temperature environment, add a mixture of n-hexane, acetone and anhydrous ethanol (containing 0.01 wt% 2,6-di-tert-butyl-p-cresol, BHT) at a liquid-to-solid ratio of 10:1 (mL / g). Stir magnetically for 20 min (200 rpm), add 1 mL of ultrapure water and continue stirring magnetically for 10 min. After standing, aspirate the supernatant solution. Repeat the extraction 3 times in the dark until the extract is colorless. Combine the extracts. During this process, the extracts are stored in an ice pack environment. After centrifugation (4˚C, 12000 rpm, 10 min), the supernatant was collected and dried under nitrogen using a nitrogen blower in a dark, room temperature environment. The extract was then reconstituted with acetonitrile (mass spectrometry grade), vortexed for 3 min to ensure complete dissolution, and then filtered through a 0.22 μm organic filter membrane to obtain the test solution. The test solution was stored at -20˚C for later testing or immediately measured, ensuring that the storage time at -20˚C did not exceed 12 h.

[0034] Example 2 Establishment of liquid chromatography conditions: This embodiment is used to illustrate the process of establishing liquid chromatography conditions in the detection method of the present invention. In particular, by optimizing parameters such as mobile phase composition, gradient elution program, column type and column temperature, efficient separation of 13 carotenoid monomers was achieved within 22 min, overcoming the technical problems of long analysis time, poor resolution and difficulty in distinguishing isomers in the prior art.

[0035] 1) Preliminary screening of chromatographic conditions: First, an Agilent PAHS column (2.1 mm × 150 mm, 1.8 µm) was selected, which exhibits good retention and separation performance for nonpolar and weakly polar compounds. In the preliminary experiment, mobile phase A was ultrapure water (containing 0.1% (v / v) formic acid), mobile phase B was methanol:acetonitrile (1:1, v / v), the flow rate was 0.3 mL / min, the column temperature was 35℃, and the injection volume was 2 μL. The results showed that the chromatographic peaks of some carotenoid monomers (such as lutein and zeaxanthin) overlapped significantly, with a resolution lower than 1.0. Furthermore, nonpolar components such as lycopene, γ-carotene, α-carotene, and β-carotene eluted concentratedly after 15 min, failing to achieve baseline separation.

[0036] 2) Optimization of mobile phase composition: To improve the separation effect, the present invention systematically optimizes mobile phases A and B: Optimization of mobile phase A: Different concentrations of ammonium acetate (2, 5, 10 mmol / L) were added to phase A. The results showed that adding 5 mmol / L ammonium acetate significantly improved the peak shape of polar carotenoids such as xanthophyll and neoxanthophyll, reducing the tailing factor from 1.48 to 1.12. Simultaneously, the addition of ammonium acetate improved the stability of the mass spectrometry response, especially enhancing the [M+H]+ ion signal. Furthermore, 0.1% (v / v) formic acid was maintained in mobile phase A to provide the acidic environment required for protonation. The final mobile phase A was determined to be: ultrapure water + 0.1% formic acid + 5 mmol / L ammonium acetate.

[0037] Optimization of mobile phase B: Different volume ratios of methanol to acetonitrile (7:3, 6:4, 5:5, 4:6, 3:7) were investigated. When the methanol ratio was too high (>50%), the retention time of strongly hydrophobic components such as lycopene was prolonged to over 20 min, affecting the detection throughput; when the acetonitrile ratio was too high (>70%), the separation degree between neoxanthin and zeaxanthin decreased. Experiments showed that when methanol:acetonitrile = 4:6 (v / v), the components achieved optimal equilibrium: polar components separated well within 5-7 min, non-polar components eluted sequentially within 15-18 min, and the separation degree between the isomers lutein and zeaxanthin reached over 1.5. The final determined mobile phase B ratio was methanol:acetonitrile = 4:6 (v / v).

[0038] In this invention, mobile phase A is simultaneously supplemented with 0.1% formic acid and 5 mmol / L ammonium acetate, which produce a synergistic effect: formic acid provides the acidic environment required for protonation, enhancing the [M+H]+ ion signal; ammonium acetate, as a buffer salt, improves the peak shape of polar carotenoids (such as neoxanthin and azoxanthin), reducing the tailing factor from 1.48 to 1.12. Neither formic acid nor ammonium acetate alone can simultaneously achieve high mass spectrometry response and good peak shape (see comparative example). The optimal ratio of methanol to acetonitrile in mobile phase B, 4:6, was obtained through systematic optimization: an excessively high methanol ratio (>50%) leads to a prolonged retention time of strongly hydrophobic components such as lycopene exceeding 20 min; an excessively high acetonitrile ratio (>70%) causes the separation of neoxanthin and azoxanthin to drop below 1.0. This specific ratio achieved complete separation of 13 compounds within 22 min.

[0039] 3) Based on the optimized mobile phase composition, this invention further designed a gradient elution program, aiming to shorten the analysis time as much as possible while ensuring separation. After multiple trials and adjustments of the gradient curves, the program shown in Table 1 was finally established: 0-1 min maintains 15% A / 85% B, 1-8 min B phase increases from 85% to 100%, and 8-22 min maintains 100% B. The characteristics of this program are: The initial high proportion of phase B (85%) allows highly polar carotenoids such as neoxanthin and azoxanthin to be rapidly eluted within 5-7 minutes, preventing them from remaining on the column for too long and causing degradation.

[0040] Mid-term rapid gradient (1-8 min): Phase B is increased to 100% within 7 min, effectively compressing the elution window of moderately polar components such as capsanthin, lutein, and zeaxanthin, and reducing the total analysis time.

[0041] Later isocratic elution (8-22 min): Maintain 100% B phase for 14 min to ensure complete elution of strongly hydrophobic components such as lycopene, γ-carotene, α-carotene, β-carotene and phytohexene, and avoid residues affecting subsequent sample injection.

[0042] Table 1. Gradient elution program of the present invention Experimental results showed that all 13 target compounds were eluted within 22 min, and the minimum resolution (Rs) of adjacent chromatographic peaks was greater than 1.5. The resolution of the isomers lutein and zeaxanthin reached 1.8, which is superior to the HPLC method reported in the literature (usually Rs < 1.2).

[0043] 4) The effect of column temperature: This invention investigated four column temperature conditions: 30℃, 35℃, 40℃, and 45℃. The results showed that increasing the column temperature helped reduce the viscosity of the mobile phase and increase the mass transfer rate, thereby shortening the retention time and improving peak shape. However, when the column temperature reached 45℃, neoxanthin and violet-xanthin showed partial degradation peaks (peak area decreased by >10%), indicating that thermosensitive carotenoids are unstable at high temperatures. At 40℃, the peak shapes of each component were symmetrical, and the retention times were moderate (the separation between γ-carotene and α-carotene was optimal). Therefore, the column temperature was determined to be 40℃.

[0044] In summary, the liquid chromatography separation conditions of this invention are as follows: a C18 column with a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm is used for separation at a flow rate of 0.3 mL / min, a column temperature of 40 °C, and an injection volume of 2 μL. Mobile phase A is ultrapure water containing 0.1% formic acid and 5 mmol / L ammonium acetate by volume, and mobile phase B is a mixed solution of methanol and acetonitrile by volume ratio of 4:6. The following gradient elution program was used: 0–1 min: mobile phase A volume fraction 15%, mobile phase B volume fraction 85%; 1–8 min: mobile phase A was linearly gradiented from 15% to 0%, mobile phase B was linearly gradiented from 85% to 100%; 8–22 min: mobile phase A remained at 0%, mobile phase B remained at 100%. Orthogonal optimization proof: (1) Optimization and validation of mobile phase A: Under the condition that other liquid chromatographic separations remain unchanged, the separation effects of different mobile phases A were investigated: ultrapure water only, ultrapure water containing only 0.1% (v / v) formic acid, and ultrapure water containing only 5 mmol / L ammonium acetate. The separation chromatograms are shown below. Figure 6 (Ultrapure water only) Figure 7 (Contains only 0.1% formic acid) Figure 8 (Contains only 5 mmol / L ammonium acetate). From Figures 6-8 It is known that the separation of various carotenoid monomers is incomplete regardless of whether ultrapure water is used alone, formic acid is added alone, or ammonium acetate is added alone. Some isomers (such as lutein and zeaxanthin) completely overlap. Therefore, it is necessary to add 0.1% formic acid and 5 mmol / L ammonium acetate to mobile phase A at the same time.

[0045] (2) Optimization and verification of mobile phase B ratio: Under the condition that other liquid chromatographic separations remain unchanged, the separation effects of different mobile phase B ratios of methanol:acetonitrile = 3.5:6.5 (v / v) and methanol:acetonitrile = 4.5:5.5 (v / v) were investigated. The separation chromatograms are shown below. Figure 9 (Methanol:acetonitrile = 3.5:6.5) and Figure 10 (Methanol:acetonitrile = 4.5:5.5) as shown. Figure 9 The high proportion of acetonitrile in the solution caused the resolution between neoxanthin and violet-xanthin to drop below 1.0, making baseline separation impossible. Figure 10 An excessively high methanol ratio leads to prolonged retention times of non-polar components such as lycopene and gamma-carotene, exceeding 20 minutes, thus affecting detection throughput. Therefore, a methanol:acetonitrile ratio of 4:6 is the optimal mobile phase B ratio.

[0046] (3) Flow rate optimization verification: Under the condition that other liquid chromatography separations remain unchanged, the separation effect of different flow rates: 0.2 mL / min and 0.4 mL / min was investigated. The separation chromatograms are shown below. Figure 11 and Figure 12 As shown, Figure 11 As shown, when the flow rate is 0.2 mL / min, the retention time of each component is prolonged, the total analysis time exceeds 30 min, and the peak broadening is obvious, affecting the separation efficiency; for example... Figure 12 As shown, when the flow rate is 0.4 mL / min, although the analysis time is shortened to less than 18 min, the resolution of some components with similar polarity (such as lutein and zeaxanthin) drops below 1.2, which cannot meet the quantitative requirements. Therefore, 0.3 mL / min was determined to be the optimal flow rate, balancing resolution and analytical efficiency.

[0047] 5) Comparison of different chromatographic conditions: Comparative liquid chromatography separation conditions: A C18 column with a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm was used for separation at a flow rate of 0.3 mL / min, a column temperature of 40 °C, and an injection volume of 2 μL. Mobile phase A is a mixed solution of methanol and acetonitrile in a volume ratio of 3:1, and mobile phase B is methanol tert-butyl ether. The gradient elution procedure is shown in Table 2 below.

[0048] Table 2. Comparative Liquid Chromatography Gradient Elution Program Table Separation results as follows Figure 1 (This invention) and Figure 5As shown in the (Comparative Example), comparing the liquid chromatography separation results of this invention with those of the comparative example reveals the following: First, in terms of analysis time, the total run time of the HPLC method in the comparative example is as long as 40 min, while the LC-MS / MS method of this invention only requires 22 min, reducing the analysis time by nearly half and significantly improving the detection throughput. Second, in terms of separation effect, baseline separation of 13 carotenoid monomers was not achieved in the comparative example. Among them, lutein and zeaxanthin showed complete overlap of chromatographic peaks at 15 to 16 min, with a resolution of less than 0.8; lycopene, γ-carotene, α-carotene, and β-carotene formed continuous "coupling peaks" in the range of 25 to 35 min, making it impossible to distinguish individual components; the highly polar neoxanthin and azadirachtin showed asymmetrical peak shapes at 5 to 8 min, with tailing factors greater than 1.5. This invention employs a PAHS-specific chromatographic column, an optimized mobile phase composition (phase A is ultrapure water containing 0.1% formic acid and 5 mmol / L ammonium acetate, phase B is methanol:acetonitrile = 4:6, and phase B contains no additives), and a gradient elution program, ensuring complete elution of all 13 monomers within 22 minutes. Adjacent peak resolutions are greater than 1.5, peaks are symmetrical and sharp, and isomers such as lutein / zeaxanthin and γ- / α-carotene are baseline separated. Furthermore, regarding sensitivity, the comparative example uses a UV detector (450 nm), where some low-content components, such as neoxanthin and antheroxin, have a signal-to-noise ratio below 10, making accurate quantification difficult. In contrast, this invention uses triple quadrupole mass spectrometry in multiple reaction monitoring mode, detecting characteristic precursor ion-daughter ion pairs. This provides strong anti-interference capabilities, with a detection limit of 2 μg / L, allowing accurate quantification even of co-elutants that are not completely separated on the chromatogram. Finally, regarding method applicability, the comparative method requires a time-consuming and degradation-prone saponification step to detect esterified carotenoids; while this invention eliminates the need for saponification, directly detecting free and naturally occurring carotenoids, avoiding degradation and structural transformation of heat-sensitive components. In summary, conventional HPLC methods cannot meet the requirements for simultaneous, rapid, and high-resolution detection of 13 carotenoid monomers. This invention, through systematic optimization of chromatographic conditions, achieves significant improvements in analysis time, resolution, sensitivity, and method applicability. Specifically, as... Figure 5 As shown, lutein and zeaxanthin completely overlapped at 15–16 min, lycopene, γ-, α-, and β-carotene formed continuous peaks in the range of 25–35 min, and neoxanthin and zeaxanthin peaks were severely tailed at 5–8 min.

[0049] Example 3 Establishment and detection of mass spectrometry analysis conditions Instrument and ion source conditions: An Agilent 6495 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was used. The analyte solution separated by chromatography in Example 2 was directly introduced into the mass spectrometer for detection.

[0050] The ion source parameters were systematically optimized to achieve stable ionization efficiency while ensuring sensitivity. The optimization process examined key parameters such as atomizing gas pressure, drying gas flow rate and temperature, sheath gas flow rate and temperature, capillary voltage, and nozzle voltage. The final determined ion source conditions are as follows: Ion source mode: Electrospray ionization source, positive ion mode (ESI+). Due to the presence of multiple conjugated double bonds in the structure of carotenoid molecules, [M+H]+ or [M]+• ions are easily formed in positive ion mode, with a response intensity much higher than that in negative ion mode.

[0051] Atomizing gas pressure: 45.0 psi. At this pressure, the atomization effect of the gas flow is optimal, the droplet size is moderate, and it is beneficial for desolvation.

[0052] Drying gas flow rate: 8.0 L / min; drying gas temperature: 250℃. The drying gas is used to assist solvent evaporation. Too low a temperature will reduce ionization efficiency, while too high a temperature may cause degradation of heat-sensitive carotenoids. 250℃ is the optimal balance point.

[0053] Sheath gas flow rate: 11.0 L / min; sheath gas temperature: 350℃. The sheath gas surrounds the ion flow, further promoting the volatilization of neutral solvent molecules and reducing background noise.

[0054] Capillary voltage: 3000 V. At this voltage, [M+H] + Once the ion signal intensity reaches a plateau, further increasing the voltage will increase background noise.

[0055] Nozzle voltage: 500 V. Appropriate nozzle voltage aids in ion focusing and transport.

[0056] The above ion source parameters were obtained through sequential optimization using single-factor experiments. Taking the nebulizer gas pressure as an example, the response intensity was investigated under conditions of 30, 35, 40, 45, and 50 psi. The results showed that at 45 psi, the [M+H] of each monomer... + The ion signal was strongest at 250°C, but further increases in pressure resulted in no significant signal gain. The drying gas temperature was optimized within the range of 200–300°C. At 250°C, the degradation rate of thermosensitive carotenoids (such as neoxanthin and auroxin) was the lowest (<5%), and the ionization efficiency was the highest. Sheath gas temperature and flow rate, capillary voltage, and nozzle voltage were all optimized item by item using the same method, and the above parameter combination was ultimately determined to be the optimal condition.

[0057] For the 13 carotenoid monomers, mass spectrometry parameters were optimized using standard solutions. First, the precursor ion of each monomer was determined using full scan mode (Q1 Scan). Since carotenoids mainly form [M+H]+ or [M]+• ions in ESI+ mode, the m / z values ​​of the precursor ions for each monomer were as follows: neoxanthin 601.4, amethysthin 601.4, antheroxin 585.4, capsanthin 585.4, lutein 569.4, zeaxanthin 569.4, β-apo-8′-carotene aldehyde 417.3, β-cryptoxanthin 553.5, lycopene 537.4, γ-carotene 537.4, α-carotene 537.4, β-carotene 537.4, and (E / Z)-octahydrolycopene 545.4.

[0058] Then, product ion scans were performed on each parent ion, and 2–4 product ions with the strongest response and least background interference were selected as quantitative and qualitative ion pairs. Simultaneously, the response of the target product ions was maximized by optimizing the fragmentation voltage and collision energy. After system optimization, the mass spectrometry detection parameters for the 13 carotenoid monomers are shown in Table 3 below.

[0059] Table 3. Complete set of parameters for MRM mass spectrometry of free carotenoids in 13 kinds of fruits and vegetables. Note: In Table 3, the first daughter ion of each monomer is the quantitative ion, and the remaining daughter ions are the qualitative ions. In actual detection, the external standard method is used for quantification based on the peak area of ​​the quantitative ion, and the qualitative ions are used to assist in qualitative confirmation. For example, the quantitative ion for neoxanthin is m / z 221.1, for violet-xanthin it is m / z 221.0, for anther-xanthin it is m / z 175.1, for capsanthin it is m / z 109.1, for lutein it is m / z 175.0, for zeaxanthin it is m / z 174.8, for β-apo-8′-carotene aldehyde it is m / z 161.2, for β-cryptoxanthin it is m / z 105.1, for lycopene it is m / z 69.1, for γ-carotene it is m / z 104.9, for α-carotene it is m / z 145.3, for β-carotene it is m / z 119.0, and for (E / Z)-hydrolycopene it is m / z 69.0.

[0060] Based on the retention times and characteristic ion pairs of each monomer determined in Table 3, this invention employs multiple reaction monitoring (MRM) mode for data acquisition. In this mode, the mass spectrometer only detects preset mother ion-daughter ion pairs and does not acquire signals from other non-target ions. Specifically, for neoxanthin, the instrument monitored the parent ion m / z 601.4 and its characteristic daughter ions m / z 221.1 and 565.5 around the retention time of 4.96 min; for purpuric xanthin, it monitored the parent ion m / z 601.4 and its characteristic daughter ions m / z 221.0, 565.5, and 565.8 around 6.59 min; for anther xanthin, it monitored the parent ion m / z 585.4 and its characteristic daughter ions m / z 175.1, 493.5, 505.4, and 568.6 around 7.47 min; for capsanthin, it monitored the parent ion m / z 585.4 and its characteristic daughter ions m / z 109.1, 175.2, and 209.1 around 7.59 min; and for lutein, it monitored the parent ion m / z 569.4 and its characteristic daughter ions m / z 221.0, 565.5, and 565.8 around 8.89 min. For zeaxanthin, the parent ion m / z 569.4 and its characteristic daughter ions m / z 174.8 and 477.5 were monitored around 9.39 min; for β-apo-8′-carotene aldehyde, the parent ion m / z 417.3 and its characteristic daughter ions m / z 161.2 and 325.2 were monitored around 10.47 min; for β-cryptoxanthin, the parent ion m / z 553.5 and its characteristic daughter ions m / z 105.1, 119.1 and 461.2 were monitored around 11.98 min; for lycopene, the parent ion m / z 537.4 and its characteristic daughter ions m / z 69.1, 144.9 and 445.4 were monitored around 13.81 min; for γ-carotene, the parent ion m / z 537.4 and its characteristic daughter ions m / z 69.1, 144.9 and 445.4 were monitored around 14.82 min. The m / z values ​​were 104.9, 118.9, and 176.9. For α-carotene, the m / z of the parent ion was monitored around 15.05 min, along with its characteristic daughter ions m / z 145.3 and 444.5. For β-carotene, the m / z of the parent ion was monitored around 15.29 min, along with its characteristic daughter ions m / z 119.0, 177.1, and 445.5. For (E / Z)-hydrolycopene, the m / z of the parent ion was monitored around 16.36 min, along with its characteristic daughter ions m / z 69.0, 81.0, and 339.1. This targeted acquisition method effectively eliminates interference from the sample matrix, improves the signal-to-noise ratio and selectivity, and ensures high sensitivity and selectivity for the detection of 13 carotene monomers.

[0061] Example 4 Standard curve plotting This embodiment illustrates the process of establishing the standard curve in the detection method of the present invention.

[0062] Thirteen carotenoid standards were accurately weighed, including neoxanthin, azoxanthin, antheroxin, capsanthin, lutein, zeaxanthin, β-apo-8′-carotene aldehyde, β-cryptoxanthin, lycopene, γ-carotene, α-carotene, β-carotene, and (E / Z)-hydrolycopene. The purity of each standard was not less than 95.8% (see Table 4 for details). Each standard was dissolved and diluted to volume with acetonitrile (mass spectrometry grade) to prepare appropriate concentrations of single-standard stock solutions, which were stored at -80°C protected from light.

[0063] When using, take an appropriate amount of each individual standard stock solution, dilute it stepwise with acetonitrile, and mix to prepare a series of mixed standard working solutions of different concentrations. The concentration ranges of each monomer in the mixed standard working solution are as follows: neoxanthin 2–300 μg / L, amethystin 2–1000 μg / L, antheroxin 2–1000 μg / L, capsanthin 2–1000 μg / L, lutein 2–500 μg / L, zeaxanthin 2–300 μg / L, β-apo-8′-carotene aldehyde 2–500 μg / L, β-cryptoxanthin 2–500 μg / L, lycopene 2–500 μg / L, γ-carotene 2–300 μg / L, α-carotene 2–500 μg / L, β-carotene 2–500 μg / L, and (E / Z)-hydrolycopene 2–300 μg / L.

[0064] The mixed standard working solutions of the above series of concentrations were injected and analyzed under the chromatographic conditions of Example 2 and the mass spectrometry conditions of Example 3, and the peak areas of each monomer quantitative ion pair were recorded. A standard curve was plotted with the peak area of ​​each monomer as the ordinate (Y) and the corresponding standard concentration as the abscissa (X, in μg / L), and the regression equation and correlation coefficient were calculated.

[0065] The results showed that all 13 carotenoid monomers exhibited good linear relationships within their respective linear ranges, with correlation coefficients (R0 and R0) of low value. 2 All values ​​are greater than 0.99. The specific regression equations and correlation coefficients are shown in Table 4 below: The regression equation for the neoxanthin is Y = 116.1688X, R0 2 = 0.9963, linear range 2–300 μg / L, standard purity 98.9%; The regression equation for xanthocyanin is Y = 1835.9377X, R0 2 = 0.9995, linear range 2–1000 μg / L, standard purity 97.9%; The regression equation for the xanthophyll in anthers is Y = 1293.2079X, R 2 = 0.9996, linear range 2–1000 μg / L, standard purity 97.7%; The regression equation for capsanthin is Y = 3570.6937X, R0 2 = 0.9999, linear range 2–1000 μg / L, standard purity 95.8%; The regression equation for lutein is Y = 90.9824X, R0 2 = 0.9999, linear range 2–500 μg / L, standard purity 97.9%; The regression equation for maize xanthine is Y = 1209.8492X, R0 2 = 0.9958, linear range 2–300 μg / L, standard purity 97.3%; The regression equation for β-apo-8′-carotene aldehyde is Y = 824.8908X, R0 2 = 0.9901, linear range 2–500 μg / L, standard purity 96.3%; The regression equation for β-cryptoxanthin is Y = 1847.7330X, R0 2 = 0.9985, linear range 2–500 μg / L, standard purity 98.3%; The regression equation for lycopene is Y = 1394.3552X, R0 2 = 0.9932, linear range 2~500 μg / L, standard purity 98.9%; The regression equation for γ-carotene is Y = 858.4749X, R0 2 = 0.9988, linear range 2–300 μg / L, standard purity 98.54%; The regression equation for α-carotene is Y = 36.9480X, R0 2 = 0.9928, linear range 2–500 μg / L, standard purity 98.1%; The regression equation for β-carotene is Y = 2784.2201X, R0 2 = 0.9986, linear range 2–500 μg / L, standard purity 97.2%; The regression equation for (E / Z)-phytopenic acid is Y = 6.2453X, R 2 = 0.9937, linear range 2~300 μg / L, standard purity 98.0%.

[0066] The above standard curves are used for the quantitative calculation of the content of various carotenoid monomers in subsequent actual samples.

[0067] Table 4. Regression equations and correlation coefficients for 13 types of carotenoid monomers. Note: y represents the peak area, and x represents the concentration of the standard (μg / L).

[0068] Example 5 Method Validation This embodiment is used to illustrate the reliability verification of the detection method described in this invention, including precision, repeatability, stability, spike recovery rate, and matrix effect tests.

[0069] 1) Precision test The same mixed standard working solution was injected six times consecutively within one day (n=6) under the chromatographic conditions of Example 2 and the mass spectrometric conditions of Example 3. The peak areas of each monomer quantitative ion pair were recorded, and the relative standard deviation (RSD%) was calculated to evaluate the precision of the method. The results showed that the RSD of the precision of the 13 carotenoid monomers was between 1.2% and 2.5%, specifically: neoxanthin 1.8%, azoxanthin 1.9%, antheroxin 1.2%, capsanthin 1.3%, lutein 1.5%, zeaxanthin 1.6%, β-cryptoxanthin 2.1%, lycopene 2.5%, γ-carotene 2.2%, α-carotene 2.3%, β-carotene 1.9%, β-apo-8′-carotene aldehyde 2.0%, and (E / Z)-hydrolycopene 2.2%. These results indicate that the method of the present invention has good precision.

[0070] 2) Repeatability test Six (n=6) test solutions were prepared in parallel using the same batch of yellow peach sample powder, following the pretreatment method of Example 1. These solutions were then analyzed under the conditions of Examples 2 and 3. The peak area of ​​each monomer was recorded, and the content was calculated. The repeatability of the method was evaluated using the relative standard deviation (RSD%). The results showed that the repeatability RSD for the 13 carotenoid monomers ranged from 2.5% to 4.1%, specifically: neoxanthin 3.2%, amethysthin 3.4%, antheroxin 2.5%, capsanthin 2.6%, lutein 2.9%, zeaxanthin 3.0%, β-cryptoxanthin 3.6%, lycopene 4.1%, γ-carotene 3.7%, α-carotene 3.8%, β-carotene 3.3%, β-apo-8′-carotene aldehyde 3.5%, and (E / Z)-hydrolycopene 3.7%. These results indicate that the method of the present invention has good repeatability.

[0071] 3) Stability test The same yellow peach sample solution was analyzed at 0, 2, 4, 8, 12, and 24 h after preparation, under the conditions of Examples 2 and 3. The peak area of ​​each monomer was recorded, and the relative standard deviation (RSD%) was calculated to evaluate the stability of the sample solution within 24 h. The results showed that the RSD of the stability of the 13 carotenoid monomers ranged from 3.2% to 5.2%, specifically: neoxanthin 4.1%, amethysthin 4.3%, antheroxin 3.2%, capsanthin 3.4%, lutein 3.6%, zeaxanthin 4.5%, β-cryptoxanthin 4.8%, lycopene 3.7%, γ-carotene 4.5%, α-carotene 4.2%, β-carotene 4.4%, β-apo-8′-carotene aldehyde 4.6%, and (E / Z)-hydrolycopene 5.2%. These results indicate that the monomers in the sample solution have good stability within 24 h of storage at -20℃, meeting the detection time requirements.

[0072] 4) Spike recovery test Powdered yellow peach samples with known content were added to mixed standard solutions of low, medium, and high concentrations, with three parallel preparations for each concentration level. The samples were treated according to the pretreatment method of Example 1, and analyzed under the conditions of Examples 2 and 3. The spiked recoveries of each monomer were calculated. The results showed that the recoveries of the 13 carotenoid monomers ranged from 85.2% to 106.2%, specifically: neoxanthin 92.5%–104.3%, amethysthin 91.8%–103.7%, antheroxin 85.2%–99.6%, capsanthin 94.9%–105.8%, lutein 93.7%–105.1%, zeaxanthin 93.2%–104.6%, β- Cryptoxanthin 89.1%–102.3%, lycopene 95.6%–106.2%, γ-carotene 88.4%–101.8%, α-carotene 86.7%–100.5%, β-carotene 90.3%–103.4%, β-apo-8′-carotene aldehyde 88.5%–101.2%, (E / Z)-hydrolycopene 88.9%–102.1%. These results indicate that the method of the present invention has good accuracy.

[0073] 5) Matrix effect test Solvent-matched standard curves (dissolved in pure acetonitrile) and matrix-matched standard curves (dissolved in the matrix extract of blank samples) were prepared separately. The slopes of the two standard curves were compared, and the matrix effect was calculated using the following formula: Matrix effect (%) = (Slope of matrix standard curve / Slope of solvent standard curve) × 100%. The closer the matrix effect is to 100%, the smaller the matrix interference. The results showed that the matrix effects of the 13 carotenoid monomers ranged from 72.5% to 92.4%, specifically: neoxanthin 86.2%, amethystin 85.6%, antheroxin 92.4%, capsanthin 91.5%, lutein 89.7%, zeaxanthin 88.9%, β-cryptoxanthin 79.1%, lycopene 72.5%, γ-carotene 78.2%, α-carotene 75.8%, β-carotene 80.3%, β-apo-8′-carotene aldehyde 78.4%, and (E / Z)-hydrolycopene 78.9%. The above results indicate that some monomers exhibit a certain matrix inhibition effect, but this is within an acceptable range and can be effectively corrected using a matrix-matched standard curve to ensure quantitative accuracy. Generally, a matrix effect between 80% and 120% is considered acceptable; exceeding this range requires quantitative correction using a matrix-matched standard curve. In this method, some monomers (such as lycopene, γ-carotene, and α-carotene) exhibit a matrix effect below 80%, demonstrating a significant matrix inhibition effect. Therefore, in actual sample quantitative analysis, a matrix-matched standard curve is uniformly used to eliminate matrix interference and ensure quantitative accuracy. Method validation data are shown in Table 5 below.

[0074] Table 5. Validation data of 13 types of carotene detection methods In summary, the results of Examples 4 and 5 fully demonstrate that the detection method established by this invention has good linearity, high precision, good repeatability, reliable stability, and a recovery rate that meets the requirements. Moreover, the matrix effect is controllable, which fully meets the requirements for simultaneous qualitative and quantitative detection of 13 kinds of carotenoid monomers in fruits and vegetables.

[0075] 6) The limits of detection (LOD) and quantitation (LOQ) for mass spectrometry are shown in Table 6 below.

[0076] Table 6. LOD and LOQ of 13 types of carotenoids As shown in Table 6, the limits of detection (LOD) for the 13 carotenoid monomers ranged from 0.09 to 1.5 μg / L, and the limits of quantification (LOQ) ranged from 0.2 to 4.5 μg / L. This indicates that the method has high sensitivity and can meet the requirements for accurate quantification of low-content carotenoid monomers, and is especially suitable for the detection of trace components.

[0077] Example 6 Actual sample test results Samples of Jinxiu yellow peach, Golden Honey No. 1 yellow peach, and Frederica yellow peach were taken respectively and pretreated according to the method in Example 1. Detection was performed under the liquid chromatography conditions of Example 2 and the mass spectrometry conditions of Example 3 to obtain chromatograms of various carotenoid monomers, as shown below. Figure 2 , Figure 3 , Figure 4 As shown. By Figures 2-4 It is evident that there are significant differences in the composition and content of carotenoids among different varieties of yellow peaches, but each monomer can achieve baseline separation, with symmetrical peaks and no interference from impurities, further verifying the applicability of the method of the present invention to different fruit and vegetable samples.

[0078] This invention is particularly applicable to the detection of fruit and vegetable samples. Fruit and vegetable matrices differ significantly from processed food matrices (such as ham sausages, jams, and cookies) in terms of compositional complexity, the form of carotenoids (mainly free vs. mainly esterified), and the types of interfering substances. Carotenoids in fruits and vegetables mainly exist in the free form and contain a large number of coexisting pigments and phenolic substances with vastly different polarities, posing different technical requirements for pretreatment and chromatographic separation. This invention optimizes the extraction solvent (n-hexane / acetone / anhydrous ethanol = 2:1:1) and chromatographic conditions based on the characteristics of fruit and vegetable matrices, achieving accurate detection of carotenoids in various fruits and vegetables (yellow peaches, sweet potatoes, carrots, pumpkins, tomatoes, mangoes, and citrus fruits).

[0079] Comparative Example 1 Fresh yellow peach samples (same variety as in Example 1) were tested using the existing detection method (CN116297920A). The method involved chloroform-methanol-water extraction (1:2:0.8), followed by liquid-liquid partitioning and purification using an HLB solid-phase extraction column, and detection by high-resolution orbital trap mass spectrometry in full-scan mode.

[0080] Comparative Example 2 Fresh yellow peach samples (same variety as in Example 1) were tested using the existing detection method (CN115792018A). This method involved ethanol-acetone-n-hexane extraction, followed by detection using a YMC Carotenoid C30 column with a UV detector (450 nm).

[0081] Comparative Example 3 Fresh yellow peach samples (same variety as in Example 1) were tested using the existing detection method (CN112557536A). This method involves extraction with organic solvent grinding, followed by detection using an HSS T3 or BEH C18 column combined with triple quadrupole mass spectrometry (MRM mode).

[0082] The test results are shown in Tables 7 and 8 below.

[0083] Table 7. Comparison of results of different comparative methods for detecting 13 carotenoid monomers in fresh yellow peach samples Table 8. Comparison of core indicators of the detection methods of the present invention and Comparative Examples 1-3 As shown in Tables 7 and 8, Comparative Example 1, which used chloroform-methanol-water extraction combined with HLB solid-phase extraction purification and high-resolution orbital trap mass spectrometry full-scan detection, did not contain capsanthin, antheroxin, β-apo-8′-carotene aldehyde, or (E / Z)-hydrolycopene in its detection list. Furthermore, chloroform easily degrades carotenoids containing conjugated carbonyl groups, such as capsanthin. Simultaneously, the multi-step HLB purification resulted in recoveries of only 63.5%–72.4% and 58.9%–69.8% for thermosensitive components such as neoxanthin and azoxanthin, respectively. The signal dispersion effect of the full-scan mode led to insufficient response from low-content components such as lycopene (recovery rate 78.3%–86.4%). The total analysis time for a single sample was no less than 100 minutes. This method is mainly suitable for processing high-oil food matrices. Comparative Example 2 used a C30 column combined with a UV detector, but it also lacked the ability to detect capsanthin, antheroxin, β-apo-8′-carotene aldehyde, and (E / Z)-hydrolycopene. The lack of freeze-drying in the pretreatment resulted in severe degradation of neoxanthin and azoxanthin (recovery rate only 48.6%–58.7%). The UV detector had low sensitivity and relied solely on retention time for qualitative analysis, which easily led to false positives. The total analysis time for a single sample was no less than 45 min. The method is only applicable to sweet potato substrates. Comparative Example 3 uses UPLC-MS / MS (MRM mode) for detection, but this method only targets 7 lutein compounds in algal samples. Among the 13 target substances in this invention, only neoxanthin, zeaxanthin, and azathoxanthin can be detected. Carotenoids (lycopene, α-carotene, β-carotene, γ-carotene, and (E / Z)-hydrolycopene) cannot be detected at all. Its pretreatment scheme is based on algal powder substrate optimization, which has significant differences from the water content, cell wall structure, and pigment composition of fresh fruits and vegetables, and cannot effectively extract carotenoids from fruits and vegetables. In contrast, this invention employs triple quadrupole mass spectrometry (MRM) targeted detection, incorporating all 13 target compounds into the detection system, achieving full coverage of 5 carotenoids and 8 luteinoids; low-temperature freeze-drying pretreatment effectively protects thermosensitive components such as neoxanthin and auroxin; and through dedicated chromatographic separation and a complete set of MRM mass spectrometry parameters, the detection limit is as low as 0.09 μg / L, with a total analysis time of no more than 30 min for a single sample. It is suitable for a variety of fresh fruit and vegetable samples and outperforms the comparative example in terms of detection coverage, sensitivity, analytical efficiency, and matrix compatibility.

[0084] Unlike existing technologies that require a saponification step to convert carotenoid esters into a free state before detection, this invention directly detects free carotenoids without saponification. The beneficial effects of this feature include: (1) avoiding the degradation and structural transformation of heat-sensitive carotenoids such as neoxanthin and azoxanthin during saponification; (2) simplifying the pretreatment process and reducing the total analysis time to less than 22 minutes; and (3) providing more accurate results reflecting the actual form of carotenoids in the sample. While existing technologies (such as CN116297920A) also do not require saponification, they target esterified carotenoids that may exist in processed food products, whereas this invention targets naturally occurring free carotenoids in fruit and vegetable raw materials.

[0085] This invention, through systematic analysis of the carotenoid composition in various fruits and vegetables such as yellow peaches, sweet potatoes, carrots, pumpkins, tomatoes, mangoes, and citrus fruits, discovered that the aforementioned 13 compounds are the most common, abundant, and representative carotenoid monomers in fruits and vegetables. These 13 compounds cover two major categories: carotenoids (6 types) and luteinoids (7 types), comprehensively reflecting the carotenoid composition characteristics of fruits and vegetables. Not all carotenoids are suitable for inclusion in the same detection system. Some compounds (such as astaxanthin and canthaxanthin) are present in extremely low or almost non-existent amounts in fruits and vegetables. Including them in the detection system not only has no practical significance but may also increase the difficulty of chromatographic separation and interfere with mass spectrometry detection.

[0086] Although the technical solutions of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A high-performance liquid chromatography-mass spectrometry method for the detection of 13 carotenoid monomers in fruits and vegetables, characterized in that, The 13 carotenoid monomers are endogenous free characteristic monomers from fruits and vegetables, specifically: neoxanthin, apoxanthin, antheroxin, capsanthin, lutein, zeaxanthin, β-cryptoxanthin, lycopene, γ-carotene, α-carotene, β-carotene, (E / Z)-hydrolycopene, and β-apo-8'-carotene aldehyde. The method described herein requires no saponification, does not use HLB solid-phase extraction for purification, and does not introduce chloroform or halogenated solvents. The detection method specifically includes the following steps: S1. Sample pretreatment: After crushing and homogenizing the fruit and vegetable samples, they were extracted in the dark using a mixed organic solvent containing 0.01 wt% 2,6-di-tert-butyl-p-cresol in hexane, acetone and anhydrous ethanol. The extract was centrifuged and filtered, and then redissolved in acetonitrile to obtain the test solution. S2: Liquid chromatography separation: A C18 column with a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm was used to separate the test solution at a flow rate of 0.3 mL / min, a column temperature of 40℃, and an injection volume of 2 μL. Mobile phase A is ultrapure water containing 0.1% formic acid and 5 mmol / L ammonium acetate by volume, and mobile phase B is a mixed solution of methanol and acetonitrile by volume ratio of 4:

6. The following gradient elution program was used: 0–1 min: mobile phase A volume fraction 15%, mobile phase B volume fraction 85%; 1–8 min: mobile phase A was linearly gradiented from 15% to 0%, mobile phase B was linearly gradiented from 85% to 100%; 8–22 min: mobile phase A remained at 0%, mobile phase B remained at 100%. S3: Mass spectrometry detection: A triple quadrupole mass spectrometer was used with an electrospray ionization source in positive ion mode and multiple reaction monitoring mode to collect characteristic parent ion-daughter ion pairs of the above 13 carotenoid monomers: neoxanthin, azoxanthin, antheroxin, capsanthin, lutein, zeaxanthin, β-cryptoxanthin, lycopene, γ-carotene, α-carotene, β-carotene, (E / Z)-octahydrolycopene and β-apo-8'-carotene aldehyde; S4: Results Analysis: The content of 13 carotenoid monomers in the test solution was calculated based on the standard curves of each standard using the matrix-matched external standard method.

2. The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 1, characterized in that, In step S3, the mass spectrometry detection uses the following ion source parameters: nebulizer gas pressure is 45.0 psi, dryer gas flow rate is 8.0 L / min, dryer gas temperature is 250℃, sheath gas flow rate is 11.0 L / min, sheath gas temperature is 350℃, capillary voltage is 3000 V, and nozzle voltage is 500 V.

3. The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 1, characterized in that, In step S2, the total running time of the gradient elution procedure is 15–22 minutes.

4. The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 1, characterized in that, In step S1, the volume ratio of n-hexane, acetone and anhydrous ethanol is 2:1:

1.

5. The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 1, characterized in that, In step S3, the parent ion and daughter ion pairs of the 13 types of carotenoid monomers are as follows: Neoxanthin: parent ion m / z 601.4, daughter ions m / z 221.1 and 565.5; Violet-yellow ion: parent ion m / z 601.4, daughter ions m / z 221.0, 565.5 and 565.8; Anther xanthophyll: parent ion m / z 585.4, daughter ions m / z 175.1, 493.5, 505.4 and 568.6; Capsaicin: parent ion m / z 585.4, daughter ions m / z 109.1, 175.2 and 209.1; Lutein: parent ion m / z 569.4, daughter ions m / z 175.0 and 338.3; Zeaxanthin: parent ion m / z 569.4, daughter ions m / z 174.8 and 477.5; β-apo-8′-carotene aldehyde: parent ion m / z 417.3, daughter ions m / z 161.2 and 325.2; β-cryptoxanthin: parent ion m / z 553.5, daughter ions m / z 105.1, 119.1 and 461.2; Lycopene: parent ion m / z 537.4, daughter ions m / z 69.1, 144.9 and 445.4; γ-Carotene: parent ion m / z 537.4, daughter ions m / z 104.9, 118.9 and 176.9; α-Carotene: parent ion m / z 537.4, daughter ions m / z 145.3 and 444.5; β-Carotene: parent ion m / z 537.4, daughter ions m / z 119.0, 177.1 and 445.5; (E / Z)-Hydroxylycopene: parent ion m / z 545.4, daughter ions m / z 69.0, 81.0 and 339.

1.

6. The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 5, characterized in that, In step S3, the fragmentation voltage and impact energy of each individual are as follows: Neoxanthine: fragmentation voltage 172 V, collision energy corresponding to daughter ion m / z 221.1 is 10 eV, collision energy corresponding to daughter ion m / z 565.5 is 18 eV; Violet-yellow matter: fragmentation voltage 142 V, collision energy corresponding to daughter ion m / z 221.0 is 15 eV, collision energy corresponding to daughter ion m / z 565.5 is 20 eV, and collision energy corresponding to daughter ion m / z 565.8 is 22 eV; Anther xanthophyll: Fragmentation voltage 130 V, collision energy corresponding to daughter ion m / z 175.1 is 18 eV, collision energy corresponding to daughter ion m / z 493.5 is 18 eV, collision energy corresponding to daughter ion m / z 505.4 is 20 eV, collision energy corresponding to daughter ion m / z 568.6 is 20 eV; Capsaicin: Fragmentation voltage 178 V, collision energy corresponding to daughter ion m / z 109.1 is 25 eV, collision energy corresponding to daughter ion m / z 175.2 is 20 eV, and collision energy corresponding to daughter ion m / z 209.1 is 20 eV; Lutein: Fragmentation voltage 168 V, collision energy corresponding to daughter ion m / z 175.0 is 22 eV, and collision energy corresponding to daughter ion m / z 338.3 is 18 eV; Zeaxanthin: Fragmentation voltage 138 V, collision energy corresponding to daughter ion m / z 174.8 is 22 eV, and collision energy corresponding to daughter ion m / z 477.5 is 15 eV; β-apo-8′-carotene aldehyde: fragmentation voltage 118 V, collision energy corresponding to daughter ion m / z 161.2 is 20 eV, and collision energy corresponding to daughter ion m / z 325.2 is 5 eV; β-cryptoxanthin: fragmentation voltage 175 V, collision energy corresponding to daughter ion m / z 105.1 is 75 eV, collision energy corresponding to daughter ion m / z 119.1 is 40 eV, and collision energy corresponding to daughter ion m / z 461.2 is 12 eV; Lycopene: Fragmentation voltage 152 V, collision energy corresponding to daughter ion m / z 69.1 is 48 eV, collision energy corresponding to daughter ion m / z 144.9 is 38 eV, and collision energy corresponding to daughter ion m / z 445.4 is 10 eV; γ-Carotene: Fragmentation voltage 165 V, collision energy corresponding to daughter ion m / z 104.9 is 65 eV, daughter ion m / z 118.9 is 40 eV, daughter ion m / z 176.9 is 18 eV; α-Carotene: Fragmentation voltage 175 V, collision energy corresponding to daughter ion m / z 145.3 is 42 eV, collision energy corresponding to daughter ion m / z 444.5 is 15 eV; β-Carotene: Fragmentation voltage 135 V, collision energy corresponding to daughter ion m / z 119.0 is 45 eV, collision energy corresponding to daughter ion m / z 177.1 is 22 eV, and collision energy corresponding to daughter ion m / z 445.5 is 15 eV; (E / Z)-Hydroxylycopene: Fragmentation voltage 142 V, collision energy corresponding to daughter ion m / z 69.0 is 62 eV, collision energy corresponding to daughter ion m / z 81.0 is 28 eV, and collision energy corresponding to daughter ion m / z 339.1 is 32 eV.

7. The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 1, characterized in that, In step S1, the sample pretreatment specifically includes: cutting the fruit and vegetable samples into small pieces, freezing them at -80℃ for 8 h, then placing them in a freeze dryer and freeze-drying them in the dark at -50℃ to -40℃ for 24 h, and then pulverizing them through a 60-mesh sieve to obtain sample powder; using a mixed organic solvent containing 0.01wt% 2,6-di-tert-butyl-p-cresol in hexane, acetone, and anhydrous ethanol for extraction in the dark; after extraction, adding 1 mL of ultrapure water and stirring magnetically for 10 min; repeating the extraction 3 times; centrifuging the extract at 4℃ and 12000 rpm for 10 min; drying with nitrogen and then redissolving in acetonitrile, vortexing for 3 min, and filtering through a 0.22 μm organic filter membrane.

8. The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 1, characterized in that, In step S1, a liquid-to-solid ratio of 10:1, mL / g, and anhydrous ethanol mixed organic solvent containing 0.01 wt% 2,6-di-tert-butyl-p-cresol were used for extraction in the dark, and the extraction time was 20 min.

9. The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 1, characterized in that, The limits of detection (LODs) for 13 carotenoid monomers were 0.09–1.5 μg / L, and the limits of quantitation (LOQs) were 0.2–4.5 μg / L. The precision RSDs were 1.2%–2.5%, and the recoveries were 85.2%–106.2%. Among them, the LOD for β-apo-8′-carotene aldehyde was 0.09 μg / L, and the LOD for quantitation was 0.2 μg / L; the LODs for antheroxin, capsanthin, lutein, and zeaxanthin were 0.2 μg / L; the LOD for β-cryptoxanthin was 0.3 μg / L; the LODs for neoxanthin and amethystoxanthin were 0.5 μg / L; the LOD for β-carotene was 0.8 μg / L; the LODs for γ-carotene, α-carotene, and (E / Z)-hydrolycopene were 1.0 μg / L; and the LOD for lycopene was 1.5 μg / L.

10. The high-performance liquid chromatography-mass spectrometry method for detecting 13 carotenoid monomers in fruits and vegetables as described in claim 1, characterized in that, Fruits and vegetables include yellow peaches, sweet potatoes, carrots, pumpkins, tomatoes, mangoes, and citrus fruits.

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