A method for simultaneous determination of 10 furanocoumarins in citrus by UPLC

Abstract

The present invention discloses a method for simultaneously detecting 10 kinds of furanocoumarins in citrus by UPLC, and relates to the field of food technology. The test conditions of the ultra-high performance liquid chromatograph used in the method include: chromatographic column: Waters CORTECS C18+ column, 3.0mm×150mm, 1.6μm; column temperature: 30°C; flow rate: 0.3mL / min; injection volume: 5μL; detection wavelength: 310nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; gradient elution. The method of the present invention can simultaneously determine the presence of 10 kinds of furanocoumarins in citrus samples, with simple steps, high accuracy, good reproducibility, and the characteristics of rapid and high-throughput detection.

Description

Technical Field

[0001] The invention relates to the field of food technology, and in particular to a method for simultaneously detecting 10 furanocoumarins in citrus by using UPLC. Background Art

[0002] Furanocoumarins are secondary metabolites widely found in nature. They are composed of a series of compounds consisting of furan rings connected to coumarins. They are a general term for a class of compounds and their derivatives formed by the condensation of the 7-hydroxyl group of the coumarin parent nucleus with the 6- or 8-substituted isopentenyl group to form a furan ring. Furanocoumarins are usually found in higher plants, mainly distributed in four higher plant families: Rutaceae, Moraceae, Leguminosae and Apiaceae.

[0003] At present, the main methods for detecting furanocoumarins include ultraviolet spectrophotometry, high performance liquid chromatography, liquid chromatography-mass spectrometry, etc. Ultraviolet spectrophotometry is easy to operate, but it measures the total amount of furanocoumarins in the sample, and the measurement results are easily interfered by other substances with strong ultraviolet absorption in the sample. HPLC is a commonly used method for analyzing furanocoumarin components in complex samples, but because the matrix of such samples is complex and there are many interferences, the separation time of conventional HPLC is long and the separation capacity is limited, which can easily lead to misjudgment. Liquid chromatography-mass spectrometry instruments are relatively expensive and complicated to operate and maintain.

[0004] Ultra-high performance liquid chromatography (UPLC) is one of the new development directions of liquid chromatography in recent years. It uses sub-2 μm fillers to greatly improve separation efficiency, peak capacity and analysis speed. Li Guijie et al. (Li Guijie, Tan Xiang, Wang Hua, et al. Simultaneous determination of 12 polymethoxyflavonoids and coumarins in citrus juice by HPLC-DAD-FLD [J]. Food Science, 2017, 38(20):7.) established a method for HPLC-DAD-FLD to simultaneously detect four furanocoumarins in citrus juice, namely bergamotrin, bergamot lactone, epoxybergamotrin and bergamotol. The column used was Thermo Scientific HyPURITYC18 (4.6×150 mm, 3 μm). However, there are many types of furanocoumarins in citrus and their structures are complex, far more than the above four. Zhao Xijuan et al. (Zhao XJ, GuoPM, Pang WH, et al. A Rapid UHPLC-QqQ-MS / MS methodfor the Simultaneous Qualitation and Quantitation of Coumarins,Furocoumarins, Flavonoids, PhenolicAcids in Pummelo Fruits[J]. FoodChemistry, 2020, 325:126835.) established a UHPLC-QqQ-MS / MS method for the simultaneous determination of eight furanocoumarins in pomelo, including zanthoxylum toxicol, psoralen, imperatorin, and isoimperatorin. The column used was Waters AcquityUPLC HSS T3 (2.1 × 100 mm, 1.8 µm), but it still could not achieve the effect of separating 10 furanocoumarins. At the same time, the pretreatment was relatively complicated, the LC-MS instrument was relatively expensive, and the operation and maintenance were complicated.

[0005] Due to the large number of furanocoumarins, there are few reports on the UPLC method for simultaneously detecting multiple furanocoumarins, and there is no report on the UPLC method for simultaneously detecting 10 furanocoumarins. Summary of the invention

[0006] In view of the deficiencies in the background technology, the present invention provides a method for simultaneously detecting 10 kinds of furanocoumarins in citrus by UPLC. The method can simultaneously determine whether the citrus samples contain zanthoxylum lactone, bergamotol, isoanisole lactone, epoxybergamotrin, bergamotrin, imperatorin, isoimperatorin, psoralen, bergamot lactone and isopsoralen components, and the method has simple steps, high accuracy, good reproducibility and the characteristics of rapid and high-throughput detection.

[0007] To achieve the above purpose, the technical solution of the present invention is as follows:

[0008] A method for simultaneously detecting 10 furanocoumarins in citrus by UPLC, the simultaneous detection method comprising the following steps:

[0009] 1) Preparation of standard solution: Weigh 10 kinds of furanocoumarin standards, place them in volumetric flasks, add methanol solution to dissolve and dilute to volume, and obtain a single standard stock solution;

[0010] 2) Prepare standard solution: pipette the single standard stock solution in step 1) respectively, add methanol solution to dilute and prepare mixed standard solution;

[0011] 3) Sample pretreatment: Weigh the citrus sample and place it in a centrifuge tube, add methanol solution, soak and extract, shake in a water bath to assist extraction, collect the supernatant by centrifugation, rotary evaporate until there is no alcohol, add methanol solution to re-dissolve, filter through a filter membrane to obtain the sample to be tested;

[0012] 4) Detection and analysis: The mixed standard solution of step 2) and the sample to be tested of step 3) are respectively subjected to ultra-high performance liquid chromatography for qualitative and quantitative analysis to obtain a standard working curve of the mixed standard solution and the type and content of furanocoumarins in the citrus sample.

[0013] Furthermore, the weight of the standard products of the 10 furanocoumarin components in step 1) is 5 mg; and the volume of the volumetric flask is 10 mL.

[0014] Furthermore, the concentration of the mixed standard solution in step 2) is 50 μg / ml.

[0015] Furthermore, 2 g of the citrus sample in step 3) is accurately weighed and placed in a 50 mL centrifuge tube, 15 mL of methanol is added, and the mixture is immersed and extracted for 30 min, and extracted by shaking in a water bath for 30 min (100 rpm, 30° C.), and centrifuged at 4000 rpm for 10 min, and the supernatant is collected. 10 mL of methanol is added to the residue, and the mixture is extracted by shaking in a water bath for 30 min, and centrifuged at 4000 rpm for 10 min, and the supernatant is collected. This process is repeated twice, and the supernatants are combined.

[0016] Furthermore, the combined supernatant in step 3) is rotary evaporated on a vacuum rotary evaporator at 35°C until there is no alcohol, and the volume is fixed to 5 mL with chromatographic grade methanol, and filtered through a 0.22 μm nylon filter membrane before detection.

[0017] Furthermore, the standard working curve of step 4) is obtained by taking 5 μL, 10 μL, 25 μL, 50 μL, 75 μL and 100 μL of the mixed standard solution of step 2) and placing them in 50 mL centrifuge tubes respectively, without adding samples and measuring them on a machine after processing according to step 3).

[0018] Furthermore, the test conditions of the ultra-high performance liquid chromatography in step 4) are as follows:

[0019] 1) Liquid chromatography conditions

[0020] Chromatographic column: Waters CORTECS C18+ column, 3.0mm×150mm, 1.6μm; column temperature: 30°C; flow rate: 0.3mL / min; injection volume: 5μL; detection wavelength: 310nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; gradient elution was used.

[0021] Further, the procedure of the gradient elution is:

[0022] 0-3.00 min, volume fraction of mobile phase B 5% to 15%;

[0023] 3.00~8.00min, the volume fraction of mobile phase B increased from 15% to 21%;

[0024] 8.00-16.00 min, the volume fraction of mobile phase B was 21%;

[0025] 16.00-29.00 min, the volume fraction of mobile phase B increased from 21% to 30%;

[0026] 29.00-42.00 min, the volume fraction of mobile phase B increased from 30% to 42%;

[0027] 42.00-50.00 min, the volume fraction of mobile phase B increased from 42% to 60%;

[0028] 50.00-59.00 min, the volume fraction of mobile phase B increased from 60% to 95%;

[0029] 59.00-59.10min, the volume fraction of mobile phase B decreased from 95% to 5%;

[0030] 59.10~62.00min, the volume fraction of mobile phase B is 5%.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1) This invention reports for the first time a method for simultaneously detecting 10 furanocoumarins (xanthoxylum sutchulenol, bergamotol, isoanisole, epoxybergamotrin, bergamotrin, imperatorin, isoimperatorin, psoralen, bergamotol, and isopsoralen) in citrus, and the number of species measured is higher than that reported in existing literature.

[0033] 2) The chromatographic column used in the present invention is a Waters CORTECS C18+ column, 3.0 mm × 150 mm, 1.6 μm, which has the characteristics of smaller filler particle size and longer column than the columns in the reported methods, and thus has the advantage of high separation, solving the problem that some furanocoumarin monomers cannot be separated in the existing methods. At the same time, the column has high separation efficiency and the time is much shorter than other columns.

[0034] 3) A UPLC method for simultaneously detecting 10 furanocoumarins in citrus is provided. The method is simple to operate, has strong specificity, high accuracy, and wide applicability. It can better control the quality of citrus processed products and provide guarantee for food safety. The present invention provides a reliable detection method for quality control of citrus products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The standard working curve of the 10 furanocoumarin standards in Example 1;

[0036] Figure 2 is the chromatogram of 10 furanocoumarin standards (50 μg / ml) in Example 1;

[0037] Figure 3 is the chromatogram of 10 furanocoumarin standards (50 μg / ml) in Comparative Example 1;

[0038] Figure 4 is the chromatogram of 10 furanocoumarin standards (50 μg / ml) in Comparative Example 2;

[0039] Figure 5 is the chromatogram of 10 furanocoumarin standards (50 μg / ml) in Comparative Example 3;

[0040] Figure 6 is the content of furanocoumarins in the pulp of 9 kinds of citrus fruits in Example 2;

[0041] Figure 7 is the content of furanocoumarins in the peels of 8 kinds of citrus fruits in Example 2;

[0042] Figure 8 is the content of furanocoumarins in the pulp of 9 kinds of citrus fruits in Comparative Example 4;

[0043] Fig. 9 The furanocoumarin contents in the peels of 8 kinds of citrus fruits in Comparative Example 4 are shown in Table 1. DETAILED DESCRIPTION

[0044] The following combination Figures 1 to 9The specific embodiments of the present invention are described in detail so that those skilled in the art can more clearly understand how to practice the present invention. Although the present invention is described in conjunction with its preferred specific embodiments, these embodiments are only illustrative, rather than limiting, the scope of the present invention.

[0045] Example 1

[0046] The invention discloses a method for simultaneously detecting 10 kinds of furanocoumarins in citrus by UPLC, which are zanthoxylum sutchulenol, bergamotol, isoanisole, epoxybergamotol, bergamotol, imperatorin, isoimperatorin, psoralen, bergamotol, and isopsoralen. The simultaneous detection method comprises the following steps:

[0047] 1) Preparation of standard products: Weigh 5 mg of 10 kinds of furanocoumarin standards and place them in 10 mL volumetric flasks respectively. Add methanol solution to dissolve and dilute to volume to obtain a single standard stock solution.

[0048] 2) Prepare standard solution: pipette the single standard stock solution in step 1) and dilute it with methanol solution to prepare a mixed standard solution with a concentration of 50 μg / ml.

[0049] 3) Sample pretreatment: Accurately weigh 2 g of citrus freeze-dried powder, add 15 mL of methanol, soak and extract for 30 min, extract in a water bath with shaking for 30 min (100 rpm, 30 °C), centrifuge at 4000 rpm for 10 min, collect the supernatant, add 10 mL of methanol to the residue, extract in a water bath with shaking for 30 min, centrifuge at 4000 rpm for 10 min, collect the supernatant, repeat twice, combine the supernatants, evaporate the combined supernatants on a vacuum rotary evaporator at 35 °C until there is no alcohol, make up to 5 mL with chromatographic grade methanol, filter through a 0.22 μm nylon filter membrane to obtain the sample to be tested.

[0050] 4) Detection and analysis: Pipette 5 μL, 10 μL, 25 μL, 50 μL, 75 μL and 100 μL of the mixed standard solution in step 2) into 50 mL centrifuge tubes respectively, without adding samples and process according to step 3) and then measure on the machine to obtain the standard working curve of the mixed standard solution, such as Figure 1 As shown, the 10 furanocoumarin standards showed a good linear relationship, indicating that the instrument has good precision. The samples to be tested in step 3) were respectively subjected to ultra-high performance liquid chromatography for qualitative and quantitative analysis to obtain the types and contents of furanocoumarins in the samples.

[0051] The test conditions of the ultra-high performance liquid chromatography are as follows:

[0052] 1) Ultra-high performance liquid chromatography (UPLC) conditions

[0053] Chromatographic column: Waters CORTECS C18+ column, 3.0 mm × 150 mm, 1.6 μm; column temperature: 30 ° C; flow rate: 0.3 mL / min; injection volume: 5 μ L; detection wavelength: 310 nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; running time: 62 min;

[0054] The gradient elution program was:

[0055] 0-3.00 min, volume fraction of mobile phase B 5% to 15%;

[0056] 3.00~8.00min, the volume fraction of mobile phase B increased from 15% to 21%;

[0057] 8.00-16.00 min, the volume fraction of mobile phase B was 21%;

[0058] 16.00-29.00 min, the volume fraction of mobile phase B increased from 21% to 30%;

[0059] 29.00-42.00 min, the volume fraction of mobile phase B increased from 30% to 42%;

[0060] 42.00-50.00 min, the volume fraction of mobile phase B increased from 42% to 60%;

[0061] 50.00-59.00 min, the volume fraction of mobile phase B increased from 60% to 95%;

[0062] 59.00-59.10min, the volume fraction of mobile phase B decreased from 95% to 5%;

[0063] 59.10~62.00min, the volume fraction of mobile phase B is 5%.

[0064] Chromatogram Figure 2 It can be seen that the 10 furanocoumarin standards, including zanthoxylum lactone, bergamotol, isopsoralen, epoxybergamotin, bergamotin, imperatorin, isopsoralen, psoralen, bergamot lactone and isopsoralen, can be well separated. Except for the resolution (R) of 1.42 between peak 3 (isopsoralen) and peak 4 (zanthoxylum lactone), the resolutions of other chromatographic peaks were all greater than 1.5, which can achieve complete separation. The peaks were sharp and symmetrical, which can meet the requirements of simultaneous separation of multiple furanocoumarins.

[0065] Comparative Example 1

[0066] The preparation of standard products, standard solutions and sample pretreatment processes were the same as in Example 1, and the HPLC conditions were:

[0067] Chromatographic column: Agilent Zorbax SB-C18 (250×4.6 mm, 5μm), pre-column C18 (30×4.6 mm, 5μm); column temperature: 30°C; flow rate: 1mL / min; injection volume: 20μL; detection wavelength: 310nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; running time: 55 min;

[0068] The gradient elution program was:

[0069] 0-15.00 min, volume fraction of mobile phase B 5% to 20%;

[0070] 15.00~20.00min, the volume fraction of mobile phase B increased from 20% to 30%;

[0071] 20.00-35.00 min, the volume fraction of mobile phase B increased from 30% to 100%;

[0072] 35.00~40.00min, the volume fraction of mobile phase B is 100%.

[0073] 40.00-45.00 min, the volume fraction of mobile phase B decreased from 100% to 5%;

[0074] 45.00~55.00min, the volume fraction of mobile phase B is 5%.

[0075] Chromatogram Figure 3 It can be seen that within 55 min, except for peak 1 (bergamotol), peak 2 (psoralen), peak 6 (bergamot lactone) and peak 7 (epoxybergamotone), which can be completely separated; peak 3 (isopsoralen) and peak 4 (zanthoxylum lactone) overlap in peak shape, peak 5 (isoanisole lactone) and peak 6 (bergamot lactone) overlap in peak shape, peak 7 (epoxybergamotone) and peak 8 (imperatorin) overlap in peak shape. All 6 substances cannot be separated and their contents cannot be determined.

[0076] Comparative Example 2

[0077] The preparation of standard products, standard solutions and sample pretreatment processes were the same as in Example 1, and the HPLC conditions were:

[0078] Chromatographic column: Waters XBridge C18 (250×4.6 mm, 5μm); column temperature: 30°C; flow rate: 1mL / min; injection volume: 20μL; detection wavelength: 310nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; running time: 100 min;

[0079] The gradient elution program was:

[0080] 0-5.00min, volume fraction of mobile phase B 5% to 15%;

[0081] 5.00~15.00min, the volume fraction of mobile phase B increased from 15% to 25%;

[0082] 15.00-30.00 min, the volume fraction of mobile phase B was 25%;

[0083] 30.00-45.00 min, the volume fraction of mobile phase B increased from 25% to 42%;

[0084] 45.00-65.00 min, the volume fraction of mobile phase B increased from 42% to 60%;

[0085] 65.00-95.00 min, the volume fraction of mobile phase B increased from 60% to 100%;

[0086] 95.00-95.10min, the volume fraction of mobile phase B decreased from 100% to 5%;

[0087] 95.10~100.00min, the volume fraction of mobile phase B is 5%.

[0088] Chromatogram Figure 4 It can be seen that within 100 min, eight substances including zanthoxylum lactone, bergamotol, epoxybergamotin, bergamotin, imperatorin, isoimperatorin, psoralen and isopsoralen can be well separated, but the peaks 5 (isoanisole lactone) and 6 (bergamot lactone) overlap, the two substances cannot be separated, their contents cannot be determined, and the separation time is too long, resulting in a large amount of consumables in actual application.

[0089] Comparative Example 3

[0090] The preparation of standard products, standard solutions and sample pretreatment processes were the same as in Example 1, and the ultra-high performance liquid chromatography (UPLC) conditions were:

[0091] Chromatographic column: Agilent ZORBAX Extend-C18 (100×2.1 mm, 1.8μm); column temperature: 30°C; flow rate: 0.3mL / min; injection volume: 5μL; detection wavelength: 310nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; running time: 30 min;

[0092] The gradient elution program was:

[0093] 0-3.00 min, volume fraction of mobile phase B 5% to 15%;

[0094] 3.00~7.00min, the volume fraction of mobile phase B increased from 15% to 22%;

[0095] 7.00-10.00 min, the volume fraction of mobile phase B was 22%;

[0096] 10.00-15.00 min, the volume fraction of mobile phase B increased from 22% to 42%;

[0097] 15.00~20.00min, the volume fraction of mobile phase B increased from 42% to 60%;

[0098] 20.00~25.00min, the volume fraction of mobile phase B increased from 60% to 95%;

[0099] 25.10~30.00min, the volume fraction of mobile phase B is 5%.

[0100] Chromatogram Figure 5 It can be seen that within 30 minutes, the six substances of bergamotol, epoxybergamotin, bergamotin, imperatorin, isopsoralen and psoralen can be well separated, but the peaks 3 (isopsoralen) and 4 (zanthoxylum lactone) overlap, and the peaks 5 (isoanisole lactone) and 6 (bergamot lactone) overlap. The four substances cannot be separated and their contents cannot be determined.

[0101] Example 2

[0102] A method for simultaneously detecting 10 furanocoumarins (xanthoxylum sutchulenol, bergamotol, isoanisole, epoxybergamotol, bergamotol, imperatorin, isoimperatorin, psoralen, bergamotol, and isopsoralen) in citrus by UPLC, the detection method comprising the following steps:

[0103] 1) Sampling: The experimental materials used in the research were 9 different varieties of mature citrus fruits (Taiwan Juicy Golden Grapefruit, Yunnan Pomelo, Changshan Pomelo, Pinghe White Pomelo, Guanxi Red Pomelo, Yongquan Mandarin Orange, Yongchun Lugan, Gannan Navel Orange, Yangshuo Kumquat). The fruits were picked at the commercial maturity stage. The fruits were selected with relatively uniform size, uniform color, and no mechanical damage or pests. The fruits were washed with clean water, divided into two parts: pulp and peel, and quickly frozen at -80°C. They were freeze-dried in a vacuum freeze dryer for 72 h to constant weight. The obtained dry samples were ground with a grinder and stored at -40°C for later use.

[0104] 2) Sample pretreatment: Accurately weigh 2 g of citrus freeze-dried powder, add 15 mL of methanol, soak and extract for 30 min, extract in a water bath with shaking for 30 min (100 rpm, 30 °C), centrifuge at 4000 rpm for 10 min, collect the supernatant, add 10 mL of methanol to the residue, extract in a water bath with shaking for 30 min, centrifuge at 4000 rpm for 10 min, collect the supernatant, repeat twice, combine the supernatants, evaporate the combined supernatants on a vacuum rotary evaporator at 35 °C until there is no alcohol, make up to 5 mL with chromatographic grade methanol, filter through a 0.22 μm nylon filter membrane to obtain the sample to be tested.

[0105] 3) Detection and analysis: The sample to be tested in step 2) is subjected to qualitative and quantitative analysis by ultra-high performance liquid chromatography to obtain the type and content of furanocoumarins in the sample.

[0106] The test conditions of the ultra-high performance liquid chromatography are as follows:

[0107] 1) Ultra-high performance liquid chromatography (UPLC) conditions

[0108] Chromatographic column: Waters CORTECS C18+ column, 3.0 mm × 150 mm, 1.6 μm; column temperature: 30 ° C; flow rate: 0.3 mL / min; injection volume: 5 μ L; detection wavelength: 310 nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; running time: 62 min;

[0109] The gradient elution program was:

[0110] 0-3.00 min, volume fraction of mobile phase B 5% to 15%;

[0111] 3.00~8.00min, the volume fraction of mobile phase B increased from 15% to 21%;

[0112] 8.00-16.00 min, the volume fraction of mobile phase B was 21%;

[0113] 16.00-29.00 min, the volume fraction of mobile phase B increased from 21% to 30%;

[0114] 29.00-42.00 min, the volume fraction of mobile phase B increased from 30% to 42%;

[0115] 42.00-50.00 min, the volume fraction of mobile phase B increased from 42% to 60%;

[0116] 50.00-59.00 min, the volume fraction of mobile phase B increased from 60% to 95%;

[0117] 59.00-59.10min, the volume fraction of mobile phase B decreased from 95% to 5%;

[0118] 59.10~62.00min, the volume fraction of mobile phase B is 5%.

[0119] Depend on Figure 6 Figure 7 It can be seen that bergamotol was detected in the pulp of the 9 different citrus varieties tested. Among the varieties where bergamotol was detected, the content of bergamotol in grapefruit pulp was significantly higher than that in other citrus varieties, which was 17.12±0.18 ug / g DW; the lowest content was in navel orange, which was 0.30±0.04 ug / g DW; bergamotol, psoralen, isopsoralen and zanthoxylum lactone were in high content in citrus pulp and distributed in many varieties; isoanisole lactone, epoxybergamotin, bergamotin, imperatorin, isoimperatorin and bergamot lactone were in low content in citrus pulp and distributed in few varieties; the lowest epoxybergamotin was detected in mandarin orange, which was 0.17±0.02 ug / g DW. High levels of bergamotol and psoralen were detected in the peels of the eight different citrus varieties tested. The content of bergamotol in grapefruit peel was significantly higher than that in other citrus varieties, at 24.94±0.78 ug / g DW; the content of psoralen in mandarin orange peel was significantly higher than that in other citrus varieties, at 38.23±0.26 ug / g DW; the lowest content was in white grapefruit peel, at 8.22±0.41 ug / g DW; bergamotol, psoralen, isopsoralen, zanthoxylum lactone, imperatorin, and isoimperatorin were found in high levels in citrus peel and were distributed in many varieties; isoanisole, epoxybergamot, bergamotrin, and bergamot lactone were found in low levels in citrus peel and were distributed in few varieties; the lowest level of isoimperatorin was detected in mandarin orange peel, at 0.61±0.10 ug / g DW.

[0120] Comparative Example 4

[0121] The sampling and sample pretreatment process were the same as in Example 1, and the HPLC conditions were:

[0122] Chromatographic column: Waters XBridge C18 (250×4.6 mm, 5μm); column temperature: 30°C; flow rate: 1mL / min; injection volume: 20μL; detection wavelength: 310nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; running time: 100 min;

[0123] The gradient elution program was:

[0124] 0-5.00min, volume fraction of mobile phase B 5% to 15%;

[0125] 5.00~15.00min, the volume fraction of mobile phase B increased from 15% to 25%;

[0126] 15.00-30.00 min, the volume fraction of mobile phase B was 25%;

[0127] 30.00-45.00 min, the volume fraction of mobile phase B increased from 25% to 42%;

[0128] 45.00-65.00 min, the volume fraction of mobile phase B increased from 42% to 60%;

[0129] 65.00-95.00 min, the volume fraction of mobile phase B increased from 60% to 100%;

[0130] 95.00-95.10min, the volume fraction of mobile phase B decreased from 100% to 5%;

[0131] 95.10~100.00min, the volume fraction of mobile phase B is 5%.

[0132] Depend on Figure 8 Fig. 9 It can be seen that bergamot ol was detected in the pulp of the 9 different citrus varieties tested. Among the varieties where bergamot ol was detected, the content of bergamot ol in grapefruit pulp was significantly higher than that in other citrus varieties, which was 15.19±0.09 ug / g DW; the lowest content was in mandarin orange, which was 0.31±0.02 ug / g DW; bergamot ol and psoralen were found in high levels in citrus pulp and were distributed in many varieties; isopsoralen, zanthoxylum lactone, epoxybergamot, bergamotin, imperatorin and isoimperatorin were found in low levels in citrus pulp and were distributed in few varieties; the lowest bergamot ol was detected in mandarin orange, which was 0.31±0.01 ug / g DW. High levels of bergamotol and psoralen were detected in the peels of the eight different citrus varieties tested. The content of bergamotol in grapefruit peel was significantly higher than that in other citrus varieties, at 21.15±0.42 ug / g DW; the content of psoralen in mandarin orange peel was significantly higher than that in other citrus varieties, at 35.43±0.31 ug / g DW; the lowest content was in red grapefruit peel, at 7.19±0.34 ug / g DW; bergamotol, psoralen, zanthoxylide, and imperatorin were high in citrus peel and distributed in many varieties; isopsoralen, epoxybergamot, bergamotin, and isoimperatorin were low in citrus peel and distributed in few varieties; the lowest epoxybergamot was detected in mandarin orange peel, at 0.93±0.05 ug / g DW; and the retention times of isoanisole and bergamot lactone overlapped, so their contents in citrus samples could not be detected. Only the contents of 8 furanocoumarins in citrus samples could be detected.

[0133] Compared with Comparative Example 4, Example 2 shortens the time by 38 minutes, and has a sharp peak shape and good symmetry, and can achieve complete separation of 10 furanocoumarins; and the types of furanocoumarins detected are accurate, and the detection limit is lower, and a variety of furanocoumarins with low content in citrus samples can be detected, for example, a small amount of isopsoralen was detected in pomelo in Example 2, a small amount of bergamot was detected in navel orange, and a small amount of zanthoxylum toxicol was detected in white pomelo, while none of them were detected in Comparative Example 4; the furanocoumarin content of some furanocoumarins detected in Example 2 is slightly higher than that in Comparative Example 4, and it is more accurate and stable.

[0134] 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 simultaneously detecting 10 furanocoumarins in citrus by UPLC, characterized in that: The simultaneous detection method comprises the following steps: S1: Preparation of standard products: Weigh 10 kinds of furanocoumarin standards, place them in volumetric flasks respectively, add methanol solution to dissolve and dilute to volume, and obtain a single standard stock solution; the weight of the 10 kinds of furanocoumarin standards in S1 is 5 mg, and the volume of the volumetric flask is 10 mL; The 10 furanocoumarins are specifically zanthoxylum lactone, bergamotol, isoanisole, epoxybergamotol, bergamotol, imperatorin, isoimperatorin, psoralen, bergamotol, and isopsoralen; S2: prepare standard solution: take the single standard stock solution in S1 respectively, add methanol solution to dilute and prepare mixed standard solution; the concentration of the mixed standard solution of S2 is 50 μg / ml; S3: Sample pretreatment: accurately weigh 2g of citrus sample, place in a 50mL centrifuge tube, add 15mL of methanol, soak and extract for 30min, shake in a water bath at 100rpm and 30℃ for 30min, centrifuge at 4000rpm for 10min, collect the supernatant, add 10mL of methanol to the residue, shake in a water bath for 30min, centrifuge at 4000rpm for 10min, collect the supernatant, repeat twice, and combine the supernatants; the combined supernatant is rotary evaporated on a vacuum rotary evaporator at 35℃ until there is no alcohol, and the volume is fixed to 5mL with chromatographic grade methanol, and filtered through a 0.22μm nylon filter membrane for detection; S4: Detection and analysis: The mixed standard solution of step S2 and the sample to be tested of step S3 are respectively subjected to ultra-high performance liquid chromatography for qualitative and quantitative analysis to obtain a standard working curve of the mixed standard solution and the type and content of furanocoumarins in the citrus sample; The standard working curve of S4 is obtained by taking 5 μL, 10 μL, 25 μL, 50 μL, 75 μL and 100 μL of the mixed standard solution of step S2 and placing them in 50 mL centrifuge tubes respectively, without adding samples and measuring them on a machine after processing according to S3; The test conditions of the S4 ultra-high performance liquid chromatograph are as follows: chromatographic column: Waters CORTECS C18+ column, 3.0 mm×150 mm, 1.6 μm; column temperature: 30°C; flow rate: 0.3 mL / min; injection volume: 5 μL; detection wavelength: 310 nm; mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; gradient elution; The gradient elution procedure is: 0-3.00 min, volume fraction of mobile phase B 5% to 15%; 3.00~8.00min, the volume fraction of mobile phase B increased from 15% to 21%; 8.00-16.00 min, the volume fraction of mobile phase B was 21%; 16.00-29.00 min, the volume fraction of mobile phase B increased from 21% to 30%; 29.00-42.00 min, the volume fraction of mobile phase B increased from 30% to 42%; 42.00-50.00 min, the volume fraction of mobile phase B increased from 42% to 60%; 50.00-59.00 min, the volume fraction of mobile phase B increased from 60% to 95%; 59.00-59.10min, the volume fraction of mobile phase B decreased from 95% to 5%; 59.10~62.00min, the volume fraction of mobile phase B is 5%.

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