Method for detecting free carotenoid in pepper

Abstract

The invention relates to the technical field of chemical analysis, in particular to a method for detecting free carotenoid in chili. The detection method comprises the following steps: detecting the capsicum extract by using ultra-high performance liquid chromatography-tandem mass spectrometry, and the liquid chromatography in the ultra-high performance liquid chromatography-tandem mass spectrometry comprises the following mobile phase components: a mobile phase A: methanol or acetonitrile, formic acid and ammonium formate, the volume percentage of formic acid in the mobile phase A is 0.05-0.2%, the volume percentage of ammonium formate in the mobile phase A is 0.05-0.2%, and the volume percentage of ammonium formate in the mobile phase A is 0.05-0.2%; the concentration of ammonium formate in the mobile phase A is 4.5-5.5 mM; and the mobile phase B is an MTBE (Methyl Tert-Butyl Ether) solution containing 0.005 to 0.02 g / 100mL of antioxidant. The detection method can accurately detect the free carotenoid in the pepper and perform quantitative analysis, and meanwhile, the detection method is simple and rapid to operate, high in sensitivity and good in repeatability.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for detecting free carotenoids in peppers. Background Art

[0002] Chili pepper and its products contain a variety of bioactive compounds, among which carotenoids have good antioxidant and anticancer activities, playing an important role in preventing human chronic diseases such as cancer, cardiovascular disease, osteoporosis and diabetes. Carotenoids are also a high-quality natural pigment, widely used in commercial applications as food colorants and feed additives.

[0003] Carotenoids in peppers and their products include capsanthin, capsorubin, β-carotene, β-cryptoxanthin, zeaxanthin, violaxanthin, antheraxanthin and neoxanthin, etc., which mainly exist in free and esterified forms. In the past, the detection of carotenoids was mostly carried out by saponification to convert carotenoid esters into free carotenoids, and the total amount of free carotenoids and carotenoid esters was detected. However, the saponification step is very time-consuming and will cause the degradation and structural transformation of heat-sensitive carotenoids, resulting in the loss of the real carotenoid structural information in the sample. In recent years, studies have found that free carotenoids and carotenoid esters each have different biological activities and play different physiological functions during the growth and processing of peppers. Therefore, it is necessary to detect free carotenoids and carotenoid esters in peppers and their products separately.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting free carotenoids in peppers. The method can accurately detect free carotenoids in peppers and perform quantitative analysis, and the method is simple to operate, rapid, highly sensitive and reproducible.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a method for detecting free carotenoids in pepper, comprising detecting a pepper extract by ultra-high performance liquid chromatography-tandem mass spectrometry, wherein the mobile phase components of the liquid chromatography in the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows:

[0008] Mobile phase A: methanol or acetonitrile, formic acid and ammonium formate, wherein the volume percentage of the formic acid in the mobile phase A is 0.05%-0.2%, and the concentration of the ammonium formate in the mobile phase A is 4.5-5.5 mM;

[0009] Mobile phase B: MTBE solution containing 0.005-0.02 g / 100 mL antioxidant;

[0010] The mobile phase elution mode was gradient elution.

[0011] In an optional embodiment, the gradient elution process is as follows: 0 min, 3% mobile phase B; 0-3 min, 3%-15% mobile phase B; 3-5 min, 15%-80% mobile phase B; 5-8 min, 80%-100% mobile phase B; 8-10 min, 100% mobile phase B; 10.1 min, 3% mobile phase B; 10.1-15 min, 3% mobile phase B.

[0012] In an optional embodiment, the liquid phase conditions include: a C30 chromatographic column; a column temperature of 30° C.-40° C., and a flow rate of 0.2-0.4 mL / min.

[0013] In an optional embodiment, the mass spectrometry conditions in ultra-performance liquid chromatography-tandem mass spectrometry include: spray voltage: positive ion mode 3200-3800V; mass spectrometry ion source temperature: 300°C-330°C; sheath gas: 20-40Arb.

[0014] In an alternative embodiment, the antioxidant is selected from BHT.

[0015] In an optional embodiment, the preparation step of the chili extract is as follows: chili powder, an extraction solvent and an antioxidant are mixed and extracted under ultrasonic conditions.

[0016] In an optional embodiment, the extraction solvent is a mixed solution of an alcohol solvent and an ether solvent, wherein the volume ratio of the alcohol solvent to the ether solvent is 5:5-8:2.

[0017] In an optional embodiment, the alcohol solvent is selected from C1-C5 solvents, preferably C1-C3 solvents, more preferably methanol or ethanol;

[0018] The ether solvent is selected from diethyl ether.

[0019] In an optional embodiment, the ultrasound time is 25-35 minutes;

[0020] The number of extractions is 2 or more.

[0021] In an optional embodiment, the ratio of the chili powder to the extraction solvent is 1:145-1:155 g / mL;

[0022] The added amount of antioxidant is 0.025-0.035g / 100mL.

[0023] The present invention has the following beneficial effects: the detection method provided by the embodiment of the present invention can efficiently, quickly and accurately qualitatively and quantitatively detect free carotenoids in peppers, and the detection method is simple to operate, has good repeatability and high sensitivity, and provides strong technical support for the quality evaluation and control of peppers and their products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a test result diagram provided in Example 1 of the present invention;

[0026] Figure 2 This is a test result diagram provided by Example 2 of the present invention;

[0027] Figure 3 This is a graph of the test results of the chromatographic column optimization in the present invention;

[0028] Figure 4 This is a test result diagram of the optimization of the mobile phase system in the present invention;

[0029] Figure 5 This is a graph showing the detection results of the optimization of the acid and salt in the mobile phase A of the present invention;

[0030] Figure 6 This is a graph showing the detection results of gradient elution optimization in the present invention;

[0031] Figure 7 This is a test result diagram of the optimization of the extraction solvent in the present invention;

[0032] Figure 8 This is a test result diagram of the optimization of the ratio of the extraction solvent in the present invention;

[0033] Fig. 9 This is a test result diagram of the optimization of the extraction solvent volume ratio in the present invention;

[0034] Fig.10 This is a test result diagram of the ultrasound time optimization in the present invention;

[0035] Fig.11 This is a graph showing the test results of optimizing the number of repeated extractions in the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0037] Liquid chromatography (LC) combined with a photodiode array or a UV-visible detector has always been the main method for separating and identifying carotenoids. However, the isomers of free carotenoids and carotenoid esters have the same UV-visible spectrum, which hinders the use of LC alone for analysis and identification. The inventors have found that liquid chromatography-tandem mass spectrometry (LC-MS / MS) integrates the chromatographic capacity of LC and the high resolution and high accuracy provided by MS, and can perform structural identification and high-sensitivity and accurate quantification of free carotenoids in peppers and their products. Therefore, the purpose of an embodiment of the present invention is to establish a detection method for free carotenoids in peppers based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), aiming to solve the problem that free carotenoids cannot be detected quickly and accurately in the prior art, and to provide strong technical support for the quality evaluation and control of peppers and their products.

[0038] First, pretreatment is performed. Specifically, if the peppers used are fresh peppers, gently scrub the peppers in clean water with a soft brush to remove surface dirt, take a clean and intact pepper sample and homogenize it in a wall breaking machine, and use a freeze dryer to freeze-dry the homogenized sample at -50°C, then grind the freeze-dried pepper sample into powder, pass it through a 0.18μm fine sieve, and place it in a -20°C low-temperature refrigerator for testing.

[0039] If dried chili pepper is used, grind the sample into powder, pass through a 0.18 μm fine sieve, and store it in a -20°C refrigerator for testing. If chili powder is used, pass through a 0.18 μm fine sieve, and store it in a -20°C refrigerator for testing.

[0040] It should be noted that carotenoids are easily degraded when exposed to oxygen and light, so all operations should be performed under dim light to prevent isomerization and photodegradation of carotenoids. Following "Good Laboratory Practice" procedures, all operations involving solvents were performed in a fume hood.

[0041] Secondly, free carotenoids were extracted by mixing chili powder, extraction solvent and antioxidant under ultrasonic conditions.

[0042] The ratio of chili powder to the extraction solvent is 1:145-1:155; that is, each gram (or kilogram) of freeze-dried chili powder corresponds to 145-155 milliliters (or liters) of extraction solvent, and the extraction solvent here refers to the amount of extraction solvent used in the total extraction process. For example, the ratio is 1::145, 1::146, 1:147, 1:148, 1:149, 1:150, 1:151, 1:152, 1:153, 1:154, 1:155, etc., any value between 1:145-1:155 or a range value between any two values.

[0043] The amount of antioxidant added is 0.025-0.035 g / 100 mL, that is, 0.025-0.035 g (or kg) of antioxidant is added to every 100 mL (or liter) of extraction solvent. For example, any value between 0.025 and 0.035 or a range between any two values ​​such as 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034 and 0.035.

[0044] The extraction solvent is a mixed solution of an alcohol solvent and an ether solvent, wherein the volume ratio of the alcohol solvent to the ether solvent is 5:5-8:2, for example, any value between 5:5-8:2 or a range value between any two values ​​such as 5:5, 6:4, 7:3 and 8:2.

[0045] The alcohol solvent is selected from C1-C5 solvents, preferably C1-C3 solvents, more preferably methanol or ethanol; the ether solvent is selected from diethyl ether. The antioxidant is selected from BHT.

[0046] The number of extractions is 2 or more, preferably 2. The ultrasonic time is 25-35 minutes.

[0047] Next, conduct testing.

[0048] Preparation of standard solution: Use acetone as solvent to prepare carotenoid standard stock solution. The 8 carotenoid standards are β-carotene, neoxanthin, capsanthin, capsanthin, zeaxanthin, violaxanthin, antheraxanthin, and β-cryptoxanthin. Dissolve the standard with acetone solution and dilute to 5mL volumetric flask. Store the prepared standard stock solution in a refrigerator. Based on the standard stock solution, accurately prepare a mixed standard intermediate solution with a mass concentration of 20mg / L, and also store it in a -20℃ refrigerator for standby use. Prepare the mixed standard intermediate solution containing 8 carotenoids with methanol into a mixed standard working solution with a mass concentration of 0.1-2000μg / L. Each standard solution is prepared on demand.

[0049] The mixed standard working solution and the pepper extract were detected by ultra-high performance liquid chromatography-tandem mass spectrometry, wherein the liquid phase conditions include: C30 chromatographic column; column temperature of 30°C-40°C, flow rate of 0.2-0.4mL / min. The mobile phase includes mobile phase A and mobile phase B, the components of mobile phase A are methanol or acetonitrile, formic acid and ammonium formate, the volume percentage of formic acid in the mobile phase A is 0.05%-0.2%, and the concentration of ammonium formate in the mobile phase A is 4.5-5.5mM; mobile phase B is an MTBE solution containing 0.005-0.02g / 100mL antioxidant. Wherein, the antioxidant includes but is not limited to BHT.

[0050] It should be noted that the mobile phase A is a mixed solution formed by mixing one of methanol and acetonitrile with formic acid and ammonium formate.

[0051] Use mobile phase A and mobile phase B for gradient elution. The specific elution process is as follows:

[0052] 0min, 3% mobile phase B; 0-3min, 3%-15% mobile phase B; 3-5min, 15%-80% mobile phase B; 5-8min, 80%-100% mobile phase B; 8-10min, 100% mobile phase B; 10.1min, 3% mobile phase B; 10.1-15min, 3% mobile phase B.

[0053] The mass spectrometry conditions in ultra-high performance liquid chromatography-tandem mass spectrometry include: spray voltage: positive ion mode 3200-3800V; mass spectrometry ion source temperature: 300°C-330°C; sheath gas: 20-40Arb.

[0054] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0055] Example 1

[0056] The embodiment of the present invention provides a method for detecting free carotenoids in peppers, comprising:

[0057] Weigh 0.2g of chilli freeze-dried powder (accurate to 0.001g) accurately into a 50mL centrifuge tube, add 15mL of solvent accurately, the extraction solvent is methanol: ether (6:4 (v / v)), add 0.0045g of BHT. Vortex for 1min in a parallel vortexer, sonicate for 30min at room temperature, centrifuge the extracted mixed solution at 4℃ and 10000rpm for 5min, repeat the above steps, combine the two supernatants, dilute 3 times, filter through a 0.22μm filter membrane into a brown injection vial.

[0058] Dissolve the standard with acetone solution and dilute to 5mL volumetric flask, and store the prepared standard stock solution in a -20℃ refrigerator. Among them, the standards of 8 carotenoids are: β-carotene, neoxanthin, capsanthin, capsanthin, zeaxanthin, violaxanthin, antheraxanthin and β-cryptoxanthin. Take the above standard stock solutions to accurately prepare 20mg / L mixed standard intermediate solutions, which are also stored in a -20℃ refrigerator for standby use. The mixed solutions of 8 carotenoids are prepared with methanol into mixed standard intermediate solutions with mass concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000μg / L, and each standard solution is prepared on demand.

[0059] Then, UHPLC-MS / MS was used for detection and analysis, wherein the mass spectrometry conditions were as follows: ion source type: electrospray ion source (H-ESI); scanning mode: multiple reaction monitoring (MRM); spray voltage (Ion Spray Voltage): positive ion mode 3600V; mass spectrometry ion source temperature (Temperature): 325℃; sheath gas (Sheath Gas): 30Arb. The ion pairs of carotenoids and the corresponding collision energy (CE), injection voltage (RF Lens) and other plasma characteristic mass spectrometry conditions are shown in Table 1.

[0060] Table 1 Names, retention times and mass spectrometry detection parameters of 8 carotenoids

[0061]

[0062]

[0063] Chromatographic column: YMC Carotenoid C30 column (YMC, Japan), column length 100 mm, inner diameter 2.0 mm, particle size 3 μm; column oven temperature: 35°C; mobile phase: (phase A) methanol solution containing 0.1% formic acid and 5 mM ammonium formate (that is, the volume fraction of formic acid in mobile phase A is 0.1%, and the concentration of ammonium formate in mobile phase A is 5 mM), (phase B) MTBE solution containing 0.01 g / 100 mL BHT;

[0064] Method flow rate: 0.3 mL / min; mobile phase gradient elution: 0 min, 3% mobile phase B; 0-3 min, 3%-15% mobile phase B; 3-5 min, 15%-80% mobile phase B; 5-8 min, 80%-100% mobile phase B; 8-10 min, 100% mobile phase B; 10.1 min, 3% mobile phase B; 10.1-15 min, 3% mobile phase B. Sample injection volume: 5 μL.

[0065] Test results see Figure 1 .

[0066] Example 2

[0067] The present invention provides a method for detecting free carotenoids in peppers. The detection method is basically the same as that in Example 1, except that methanol in mobile phase A is replaced with acetonitrile. Figure 2 ,in, Figure 2 Among them, 1 represents neoxanthin, 2 represents violaxanthin, 3 represents antheraxanthin, 4 represents capsanthin, 5 represents capsanthin, 6 represents zeaxanthin, 7 represents β-cryptoxanthin, and 8 represents β-carotene.

[0068] Optimization of detection conditions

[0069] (1) Column optimization

[0070] The detection was performed according to the detection method provided in Example 1, except that the C30 chromatographic column was replaced with C18 (purchased from Waters), and the other operations and conditions were consistent with those in Example 1. Figure 3 ,in, Figure 3 Among them, 1 represents neoxanthin, 2 represents violaxanthin, 3 represents antheraxanthin, 4 represents capsanthin, 5 represents capsanthin, 6 represents zeaxanthin, 7 represents β-cryptoxanthin, and 8 represents β-carotene.

[0071] according to Figure 3 It can be seen that only 5 carotenoids can be detected using the C18 chromatographic column, while antheraxanthin, capsanthin and zeaxanthin were not detected, and carotenoids that are isomers of each other (such as violaxanthin and neoxanthin, capsanthin and antheraxanthin) cannot be effectively separated. The chromatographic column has very poor separation of carotenoids, and some peaks are not completely separated, making it impossible to detect the results.

[0072] (2) Optimization of mobile phase system

[0073] Mobile phases A and B were prepared according to the ratio in Table 2, and then the detection method provided in Example 1 was referred to for detection. The elution method, mass spectrometry conditions, etc. were consistent with those in Example 1, except for the mobile phase. Figure 4 .in, Figure 4 1 represents neoxanthin, 2 represents violaxanthin, 3 represents antheraxanthin, 4 represents capsanthin, 5 represents capsanthin, 6 represents zeaxanthin, 7 represents β-cryptoxanthin, and 8 represents β-carotene

[0074] Table 2 Mobile phase ratio

[0075]

[0076] according to Figure 4It can be seen that when the mobile phase ratio is a, b and c, the retention time of most compounds is in the first 5 minutes, the peaks of the compounds in Figures d and e show front peaks, and the peaks of the compounds in Figure f are all symmetrical sharp peaks. Based on the peak shape and response intensity, methanol (phase A) and MTBE (phase B) were selected as the mobile phase system.

[0077] (3) Optimization of salt and acid in mobile phase A

[0078] Adding salts and acids to the mobile phase can increase the sensitivity of target analytes or improve the shape of chromatographic peaks. For example, adding formic acid can increase the intensity of [M+H]+ ions, and adding ammonium formate can improve the shape of chromatographic peaks, especially for target analytes eluting at longer retention times.

[0079] In the methanol / MTBE system, the concentrations of formic acid and ammonium formate were optimized, and ammonium formate and formic acid were added according to Table 3. Then, the detection was performed with reference to the detection method provided in Example 1. The elution mode and mass spectrometry conditions were the same as those in Example 1, except that the amount of salt and acid added to the mobile phase was different. Figure 5 .in, Figure 5 Among them, 1 represents neoxanthin, 2 represents violaxanthin, 3 represents antheraxanthin, 4 represents capsanthin, 5 represents capsanthin, 6 represents zeaxanthin, 7 represents β-cryptoxanthin, and 8 represents β-carotene.

[0080] Table 3 Addition amount of formic acid and ammonium formate in the mobile phase

[0081]

[0082] according to Figure 5 It can be seen that Figure 5 After adding ammonium formate to a, b and c, the peak shape of the target compound was significantly improved, and the peak shape, response intensity and separation were good at a concentration of 5 mM. Figure 5 The addition of formic acid to d, e, and f significantly increased [M+H] + The intensity of the ions was highest when the concentration was 0.1%. Therefore, a mobile phase consisting of methanol and MTBE containing 0.1% (v / v) formic acid and 5 mM ammonium formate was selected.

[0083] (4) Gradient elution optimization

[0084] The detection was performed according to the detection method provided in Example 1. The composition of mobile phase A and B, mass spectrometry conditions, etc. were the same as those in Example 1, and only the elution process was different. See Table 4 for the specific elution process.

[0085] Table 4 Elution process

[0086]

[0087]

[0088] Results Figure 6 .in, Figure 6 1 represents neoxanthin, 2 represents violaxanthin, 3 represents antheraxanthin, 4 represents capsanthin, 5 represents capsanthin, 6 represents zeaxanthin, 7 represents β-cryptoxanthin, and 8 represents β-carotene. When gradients 1 to 4 were selected, the peak time was too early, and a front peak appeared from gradient 1 to gradient 3; gradients 5 and 6 had better separation effects, but gradient 5 had more impurity peaks and a lower response intensity, so gradient 6 was finally selected.

[0089] (5) Extraction process optimization

[0090] (5.1) Extraction solvent optimization

[0091] The same amount of pepper was extracted and tested according to the detection method of Example 1, except that the extraction solvent was changed to acetone, dichloromethane, methanol, chloroform, petroleum ether, ethanol, ether, ethyl acetate and n-hexane. The rest of the operations were consistent with those of Example 1. The results are shown in Figure 7 .

[0092] according to Figure 7 It can be seen that methanol is the best solvent for extracting capsanthin, capsanthin, zeaxanthin, antheraxanthin and β-cryptoxanthin, but ether has the best extraction effect on β-carotene. The possible reason is that β-carotene is a highly lipophilic non-polar carotenoid, and its conjugated hydrocarbon structure lacks polar functional groups. Methanol is a polar solution, while ether is a medium-polar solution, so it has a better extraction effect on β-carotene. Compared with β-carotene, the addition of polar functional groups in the other seven carotenoids, such as epoxy (violaceousin and neoxanthin) and hydroxyl (lutein and zeaxanthin), increases the polarity of the compounds. Therefore, a mixed solvent of methanol and ether was selected as the extraction solvent.

[0093] (5.2) Optimization of extraction solvent ratio

[0094] The same amount of pepper was extracted and tested according to the detection method of Example 1, except that the ratio of methanol and ether in the extraction solvent was different. Specifically, the ratio of methanol to ether was 9:1, 8:2, 7:3, 6:4, 5:5 and 10:0 (v / v). The results are shown in Figure 8 .

[0095] according to Figure 8It can be seen that the contents of capsanthin, β-carotene, zeaxanthin, β-cryptoxanthin and neoxanthin increase with the increase of ether ratio, reaching the highest when methanol: ether is 6:4 (v / v), and violaxanthin reaches the highest when methanol: ether is 7:3 (v / v). There is no significant difference in antheraxanthin and capsanthin at different ratios. Considering the extraction effect of 8 carotenoids, methanol: ether 6:4 (v / v) was selected as the optimal extraction solvent.

[0096] (5.3) Optimization of extraction solvent dosage

[0097] The amount of extraction solvent is also a key factor affecting the extraction efficiency. When the extraction solvent dosage is small, carotenoids cannot be completely extracted. The higher the extraction solvent dosage, the higher the sensitivity requirement of the instrument. Subsequently, nitrogen blowing or rotary evaporation and other concentration steps may be added, resulting in the loss of carotenoids. Therefore, the embodiment of the present invention optimizes the ratio of sample to extraction solvent.

[0098] Specifically, the same amount of pepper was extracted and tested according to the detection method of Example 1, except that the amount of extraction solvent was different, as follows: the ratio of sample to solvent was 1:25, 1:50, 1:100, 1:150, 1:200, 1:225 g / mL, respectively. Figure 8 .

[0099] according to Fig. 9 It can be seen that with the increase of solvent dosage, the content of carotenoids shows a trend of first increasing and then decreasing. Except for violaxanthin, the content of other carotenoids has the highest extraction efficiency when the extraction solvent dosage ratio is 1:150g / mL, and their contents are 208.109μg / g for β-carotene, 154.013μg / g for capsanthin, 82.657μg / g for zeaxanthin, 30.888μg / g for β-cryptoxanthin, 8.565μg / g for antheraxanthin, 3.969μg / g for capsanthin, and 0.260μg / g for neoxanthin. From a theoretical perspective, the results show that at first, with the increase of solvent dosage, the solubility of carotenoids increases, so the extraction effect increases. When the solvent dosage exceeds 1:150g / mL, the impurities are extracted more due to the excessive solvent dosage, which makes the extraction effect worse. Therefore, the final extraction solvent dosage is selected as 1:150g / mL.

[0100] (5.4) Ultrasound time optimization

[0101] The same amount of pepper was extracted and tested according to the detection method of Example 1, the only difference being the ultrasonic time, specifically 0-60 min. The results are shown in Fig.10 ,according to Fig.10It can be seen that ultrasound can significantly improve the extraction efficiency of carotenoids. As the ultrasound time increases, the extraction efficiency increases, but after 30 minutes of ultrasound, the extraction efficiency decreases significantly. The reason may be that under the condition of constant ultrasonic power, long-term ultrasound may generate a certain amount of heat, which affects the stability of carotenoids. Therefore, 30 minutes is selected as the optimal ultrasonic extraction time.

[0102] (5.5) Optimization of repeated extraction times

[0103] The same amount of pepper was extracted and tested according to the detection method of Example 1, the only difference being the number of extractions. The results are shown in Fig.11 .

[0104] according to Fig.11 It can be seen that when the extraction is repeated twice, the contents are 206.961μg / g of β-carotene, 157.463μg / g of capsanthin, 74.429μg / g of zeaxanthin, 24.691μg / g of β-cryptoxanthin, 8.238μg / g of anther xanthin, 5.080μg / g of capsanthin, and 0.182μg / g of neoxanthin, among which the contents of neoxanthin, violaxanthin and capsanthin increased by 12.8%, 41.0% and 11.5% respectively after two repeated extractions compared with the first extraction. Subsequent repeated extractions did not have a significant effect on the extraction of carotenoids in peppers. It was also found in the experiment that after the second extraction, the color of the extraction solution had changed to a transparent color. Therefore, it was chosen to repeat the extraction twice.

[0105] (6) Methodological evaluation

[0106] The established method was evaluated by linearity, limit of detection (LOD), limit of quantification (LOQ), intra-day precision, inter-day precision, recovery and matrix effect. The external standard method was used for accurate quantitative determination. The LOD and LOQ of carotenoids were determined with a signal-to-noise ratio (S / N) of 3 and 10, respectively. The precision of the established method was evaluated based on repeatability (intra-day precision) and reproducibility (inter-day precision), and the results were expressed as relative standard deviation (RSD) values. The recovery was used to evaluate the accuracy of the method. During the recovery evaluation process, three recovery spike levels of L (low), M (medium) and H (high) were designed, and the added compound content was approximately 50%, 100% and 200% of the original content of the compound in pepper; among them, the content of capsanthin, zeaxanthin, β-cryptoxanthin and β-carotene was relatively high, so 2, 5 and 10 times LOQ were selected for spike recovery. The matrix effect was evaluated by the slope method, and the calculation formula of the slope method was:

[0107] Slope = matrix standard curve slope / solvent standard curve slope (Formula 1)

[0108] The linear equation, linear range, and correlation coefficient (r 2 ), LOD and LOQ are shown in Table 5.

[0109] Table 5 Standard curves, detection limits and quantification limits of 8 carotenoids

[0110]

[0111] It can be seen from Table 5 that the linearity of the detection method is good, and the linear correlation coefficient r 2 >0.9982; the LOD of 8 carotenoids is between 0.5-2μg / L, and the LOQ is between 1-50μg / L, which can meet the detection requirements. Compared with traditional detection methods, this method adopts MRM scanning mode, which has high sensitivity and accurate quantitative results, and can provide technical support for the detection of free carotenoid content in peppers.

[0112] The recovery, matrix effect and precision results of 8 carotenoids in pepper are shown in Table 6.

[0113] Table 6 Recovery, matrix effect and precision of 8 carotenoids in pepper

[0114]

[0115] According to Table 6, the low-level recovery rate is between 69% and 110%, the intermediate-level recovery rate is between 72% and 118%, and the high-level addition recovery rate is between 73% and 116%. The intra-day and inter-day reproducibility RSD is <20%, indicating that this instrument method and pretreatment method meet the methodological evaluation and can be used to detect 8 free carotenoids in peppers. The matrix effect of the 8 compounds is between 0.8 and 1.2, indicating that the matrix effect of this method is not significant, and the conditions for qualitative and quantitative external standard method using solvent standard are met. Therefore, this method is suitable for the qualitative and quantitative detection of 8 free carotenoids in peppers.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting free carotenoids in pepper, characterized in that: include: The pepper extract was detected by ultra-high performance liquid chromatography-tandem mass spectrometry, wherein the mobile phase components of the liquid chromatography in the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows: Mobile phase A: methanol or acetonitrile, formic acid and ammonium formate, wherein the volume percentage of the formic acid in the mobile phase A is 0.05%-0.2%, and the concentration of the ammonium formate in the mobile phase A is 4.5-5.5 mM; Mobile phase B: MTBE solution containing 0.005-0.02 g / 100 mL antioxidant; The mobile phase elution mode was gradient elution.

2. The method for detecting free carotenoids in pepper according to claim 1, characterized in that: The process of gradient elution was as follows: 0 min, 3% mobile phase B; 0-3 min, 3%-15% mobile phase B; 3-5 min, 15%-80% mobile phase B; 5-8 min, 80%-100% mobile phase B; 8-10 min, 100% mobile phase B; 10.1min, 3% mobile phase B; 10.1-15min, 3% mobile phase B.

3. The method for detecting free carotenoids in pepper according to claim 1, characterized in that: The liquid phase conditions include: C30 chromatographic column; column temperature of 30°C-40°C, and flow rate of 0.2-0.4 mL / min.

4. The method for detecting free carotenoids in pepper according to claim 1, characterized in that: The mass spectrometry conditions in ultra-high performance liquid chromatography-tandem mass spectrometry include: spray voltage: positive ion mode 3200-3800V; mass spectrometry ion source temperature: 300°C-330°C; sheath gas: 20-40Arb.

5. The method for detecting free carotenoids in pepper according to claim 1, characterized in that: The antioxidant is selected from BHT.

6. The method for detecting free carotenoids in pepper according to any one of claims 1 to 5, characterized in that: The preparation steps of the pepper extract are as follows: pepper powder, extraction solvent and antioxidant are mixed and extracted under ultrasonic conditions.

7. The method for detecting free carotenoids in pepper according to claim 6, characterized in that: The extraction solvent is a mixed solution of an alcohol solvent and an ether solvent, wherein the volume ratio of the alcohol solvent to the ether solvent is 5:5-8:

2.

8. The method for detecting free carotenoids in pepper according to claim 7, characterized in that: The alcohol solvent is selected from C1-C5 solvents, preferably C1-C3 solvents, more preferably methanol or ethanol; The ether solvent is selected from diethyl ether.

9. The method for detecting free carotenoids in pepper according to claim 6, characterized in that: Ultrasound time is 25-35 minutes; The number of extractions is 2 or more.

10. The method for detecting free carotenoids in pepper according to claim 6, characterized in that: The ratio of the chili powder to the extraction solvent is 1:145-1:155 g / mL; The added amount of antioxidant is 0.025-0.035g / 100mL.

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