Method for separating and determining geraniol glycoside content based on high performance liquid chromatography-mass spectrometry

Abstract

The invention discloses a method for separating and determining the content of geraniol glycoside based on a high performance liquid chromatography-mass spectrometry technology, relates to the field of analytical chemistry, and adopts the high performance liquid chromatography-mass spectrometry technology to detect a standard substance solution and geraniol glycoside in a pretreated sample to be detected. The method comprises the following steps: separating geraniol glycoside from a to-be-detected sample by using a high performance liquid chromatography-mass spectrometry technology and optimizing elution conditions, establishing a concentration-peak area standard curve by taking the concentration of a standard solution as an X axis and the peak area as a Y axis, and calculating the content of geraniol glycoside in the to-be-detected sample. The method has the beneficial effects that by optimizing the elution conditions, geraniol glycoside and nerol glycoside can be well distinguished and separated, the accuracy of content measurement of geraniol glycoside is improved, and the method has a relatively good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for separating and determining the content of geraniol glycosides based on high performance liquid chromatography-mass spectrometry. Background Art

[0002] Glycosides are aroma precursors widely found in plants and play an indispensable role in the growth and development of plants. When plants are under stress or under certain conditions, glycosides are decomposed under the catalysis of glycoside hydrolases to release aroma molecules, which can improve the ability of plants to resist pests and diseases and effectively repel insects. In addition, glycosides are also widely involved in the secondary metabolism of plants, generating a variety of metabolites that are beneficial to plant physiological activities and stress resistance. Although glycosides are widely distributed in nature, their content is relatively low, so the bioenzymatic method is the main method for their synthesis.

[0003] Geraniol is one of the main components of terpene alcohol compounds in plants. It has a typical rose fragrance and is widely found in more than 250 plants such as tea trees, geraniums, and roses. In addition, geraniol is mainly stored in cells in plants in the form of glucoside or primulosidoside. Nerol is the cis isomer of geraniol. Both have the same molecular formula C10H18O. Nerol glycoside is a flavor precursor substance that has no aroma itself. This substance is widely found in plants such as tea and flowers. During the maturation of the plant, nerol glycoside will decompose and release nerol through the action of β-glycoside hydrolase, thereby producing aroma.

[0004] Since the two are isomers, the current separation and determination methods make it difficult to separate geraniol glycosides alone, resulting in the inability to accurately quantify geraniol glycosides. Therefore, optimizing the separation method of geraniol glycosides can provide a scientific basis for its development and utilization. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to solve the problem that it is currently difficult to separate and accurately determine the content of geraniol glycoside.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The first aspect of the present invention proposes a method for separating and determining the content of geraniol glycosides based on high performance liquid chromatography-mass spectrometry technology, wherein high performance liquid chromatography-mass spectrometry technology is used to detect geraniol glycosides in a standard solution and a pretreated sample to be tested, and high performance liquid chromatography-mass spectrometry technology is first used to separate geraniol glycosides from the sample to be tested by optimizing the elution conditions, and then a concentration-peak area standard curve is established with the concentration of the standard solution as the X-axis and the peak area as the Y-axis, and the geraniol glycoside content of the sample to be tested is calculated. The specific chromatography-mass spectrometry conditions are:

[0008] (1) Mass spectrometry conditions: an electrospray ion source (ESI) was used, the detection mode was negative ion mode (ESI-), the extraction cone voltage was 5.0 V, the mass spectrometry detection was in multiple reaction monitoring mode (MRM), and the ion source temperature was 350 °C;

[0009] (2) HPLC conditions: Column: Hypersil GOLD (100 mm × 2.1 mm 1.9 μm); Flow rate: 0.25 mL min -1 ; Injection volume: 5μL; Column temperature: 40℃; Mobile phase A: 0.1% (v / v) formic acid water, B: 100% acetonitrile; Gradient elution method was adopted, the elution gradient was 28min, and the elution parameters were: B phase from 0% to 10% in 0-3min, B phase from 10% to 25% in 3-15min, B phase from 25% to 40% in 15-23min, B phase from 40% to 90% in 23-24min, 90% B phase was maintained in 24-24.1min, B phase from 90% to 10% in 24.1-28min, △Rt=0.348min.

[0010] Preferably, the elution gradient is 36 min, and the elution parameters are: phase B from 0% to 10% in 0-3 min, phase B from 10% to 25% in 3-18 min, phase B from 25% to 40% in 18-30 min, phase B from 40% to 90% in 30-31 min, 90% phase B is maintained in 31-31.1 min, phase B from 90% to 10% in 31.1-36 min, △Rt=0.446 min.

[0011] Preferably, the peak area and concentration are subjected to regression analysis to obtain a concentration-peak area standard curve equation of Y=96615X-50546, R 2 =0.998, where Y represents the peak area, X represents the solution concentration, and R 2 Represents the correlation coefficient.

[0012] Preferably, the sample to be tested includes a tea sample.

[0013] Preferably, the tea sample is any one of Golden Peony, Golden Guanyin, and Zhongcha 108.

[0014] Preferably, the characteristic ion peak of geraniol glycoside is 361.19.

[0015] Preferably, the pretreatment method of the sample to be tested is: weigh the sample, extract it with methanol, perform ultrasonic treatment in an ice bath, centrifuge after the ultrasonic treatment, draw the supernatant into a new centrifuge tube, repeat the extraction once, and finally collect the supernatant twice and filter it with a filter membrane.

[0016] Preferably, the pretreatment method of the sample to be tested is: weigh 50 mg of tea sample, extract it with 90% (v / v) methanol (4 mL), ultrasonicate in an ice bath for 30 minutes, and mix it by turning it upside down every 5 minutes; after the ultrasonication is completed, use a low-temperature centrifuge, 4°C, 13400×g centrifuge for 10 minutes, aspirate the supernatant into a new centrifuge tube, repeat the extraction once, and finally collect the supernatant twice, and filter it with a 0.22μm filter membrane.

[0017] Preferably, the peak area of ​​the sample to be tested is substituted into the standard curve to calculate the geraniol glycoside content of the sample to be tested.

[0018] Preferably, when the sample to be tested is Golden Peony, the content of geraniol glycoside is 295.42±1.47 μg / g; when the sample to be tested is Golden Guanyin, the content of geraniol glycoside is 227.99±2.90 μg / g; when the sample to be tested is Zhongcha 108, the content of geraniol glycoside is 57.94±0.78 μg / g.

[0019] The second aspect of the present invention proposes the use of CsUGT73D protein in catalyzing the production of glycosides from geraniol and nerol. The amino acid sequence of the CsUGT73D protein is shown in SEQ ID NO: 1, and the CDS sequence of the gene encoding the CsUGT73D protein is shown in SEQ ID NO: 2.

[0020] Beneficial effects of the present invention:

[0021] The invention is based on high performance liquid chromatography-mass spectrometry technology and can well distinguish and separate geraniol glycoside and nerol glycoside by optimizing elution conditions, thereby improving the accuracy of measuring the content of geraniol glycoside. The method has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is the SDS-PAGE gel image of CsUGT73D protein stained with Coomassie Brilliant Blue in Example 1 of the present invention, wherein: 1, 120kDa protein Marker; 2, before CsUGT73D induction; 3, after CsUGT73D induction; 4, supernatant after crushing; 5, precipitate after crushing; 6, flow-through; 7, CsUGT73D purified protein; the blue box marks the target protein.

[0023] Figure 2 This is the mass spectrum of the geraniol glucoside standard product in Example 1 of the present invention.

[0024] Figure 3 This is a chromatogram of detecting geraniol and nerol glycosides under a 16 min elution gradient in Example 1 of the present invention.

[0025] Figure 4 The chromatograms are for detecting geraniol and nerol glycosides under the 28 min elution gradient in Example 1 of the present invention, wherein Figure A is the chromatogram of geraniol and nerol glycosides, Figure B is the mass spectrum of nerol glycosides, and Figure C is the mass spectrum of geraniol glycosides.

[0026] Figure 5 The chromatograms are for detecting geraniol and nerol glycosides under the 36 min elution gradient in Example 1 of the present invention, wherein Figure A is the chromatogram of geraniol and nerol glycosides, Figure B is the mass spectrum of nerol glycosides, and Figure C is the mass spectrum of geraniol glycosides.

[0027] Figure 6 This is a standard curve diagram of geraniol glycosides in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0030] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0031] The main reagents and formulas used in the embodiments of the present invention are:

[0032] (1) Geraniol Glucoside

[0033] (2) Formic acid (LC / MS grade), pure acetonitrile (LC / MS grade), ultrapure water

[0034] Embodiment 1:

[0035] 1. Glycosyltransferase CsUGT73D protein purification

[0036] The prokaryotic expression recombinant vector pMAL-c5x-CsUGT73D was constructed and heterologously expressed in Escherichia coli Rosetta. The CsUGT73D recombinant protein was purified by affinity chromatography and the purification results of the recombinant protein were detected by SDS-PAGE protein gel.

[0037] 2. Preparation of geraniol glucoside and nerol glycoside mixture by enzymatic reaction

[0038] Purified CsUGT73D protein was used to catalyze geraniol and nerol to generate glycosides. The enzyme activity reaction system (200 μL) was as follows: 50 mM Tris-hcl (pH = 7.5), 10 mM DTT, 5 mM geraniol (nerol), 10 mM UDP-Glucose, 50 μg CsUGT73D protein, 10% glycerol, and incubated at 30°C for 15 h. After incubation, the mixture was extracted twice with an equal volume of ethyl acetate, the organic solvent was evaporated cleanly, and 300 μL of 80% methanol was added to dissolve the mixture, and the mixture was centrifuged at 13400 × g for 10 min. The supernatant was taken, filtered through an organic phase filter membrane with a pore size of 0.22 μm, and then added to a sample injection bottle for analysis.

[0039] The SDS-PAGE gel of CsUGT73D protein stained with Coomassie Brilliant Blue is shown in the figure below. Figure 1 shown.

[0040] 3. Liquid chromatography / quadrupole-time of flight mass spectrometry (LC / Q-TOF) detection:

[0041] (1) Mass spectrometry conditions: Electrospray ionization (ESI) source was used, and the detection mode was negative ion mode (ESI - ), extraction cone voltage was 5.0 V, mass spectrometry detection was in multiple reaction monitoring (MRM) mode, and the ion source temperature was 350 °C.

[0042] (2) HPLC conditions: Column: Hypersil GOLD (100 mm × 2.1 mm 1.9 μm); Flow rate: 0.25 mL min -1 ; Injection volume: 5 μL; Column temperature: 40°C. Mobile phase A: 0.1% (v / v) formic acid water, B: 100% acetonitrile;

[0043] like Figure 3As shown, under the 16 min elution gradient (B phase from 0% to 10% in 0-3 min, from 10% to 25% in 3-9 min, from 25% to 40% in 9-11 min, from 40% to 90% in 11-12 min, 90% B phase is maintained in 12-12.1 min, from 90% to 10% in 12.1-16 min, ΔRt=0.057 min), isomers are difficult to separate, so the elution gradient is optimized;

[0044] After adjusting the elution gradient to 28 min (Phase B from 0% to 10% in 0-3 min, from 10% to 25% in 3-15 min, from 25% to 40% in 15-23 min, from 40% to 90% in 23-24 min, from 90% to 10% in 24-24.1 min, from 90% to 10% in 24.1-28 min, ΔRt = 0.348 min), the results are as follows: Figure 4 As shown, geraniol and nerol glycosides can be separated under the 28-min elution gradient;

[0045] The elution gradient was optimized again (Phase B from 0% to 10% in 0-3 min, from 10% to 25% in 3-18 min, from 25% to 40% in 18-30 min, from 40% to 90% in 30-31 min, 90% of Phase B in 31-31.1 min, from 90% to 10% in 31.1-36 min, ΔRt = 0.446 min). The results are as follows: Figure 5 As shown in the figure, the difference in retention time of glycosides of geraniol and nerol increased further under the 36 min elution gradient, which could more clearly distinguish the two.

[0046] 4. Drawing of the standard curve of geraniol glycoside and its content determination

[0047] The geraniol glycoside standard was diluted to 8 concentration gradients of 0 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL. The concentration-peak area standard curve was constructed based on the peak area measured by the optimized elution gradient. The peak area and concentration were subjected to regression analysis, and the final standard curve equation was Y=96615X-50546, R 2 =0.998 (e.g. Figure 6 As shown), where Y represents the peak area, X represents the solution concentration, and R 2 Represents the correlation coefficient.

[0048] Sample preparation: Weigh 50 mg of sample, extract with 90% (v / v) methanol, add 4 mL of extract to each sample, ultrasonicate in an ice bath for 30 minutes, and mix by turning upside down every 5 minutes; after ultrasonication, centrifuge at 4°C, 13400×g for 10 minutes, aspirate the supernatant into a new centrifuge tube, repeat the extraction once, and finally collect the supernatant twice, filter with a 0.22μm filter membrane as the sample to be tested. According to the drawn standard curve, the content of geraniol glycoside in the sample is calculated, and the peak area of ​​the sample to be tested is substituted into the standard curve, and the content is shown in Table 1

[0049] Table 1

[0050]

[0051] from Figure 2 It can be seen that the characteristic ion peak of geraniol glycoside is 361.19.

[0052] from Figure 3 It can be seen that under the elution condition of 16 min, the peak time of geraniol glycoside overlaps with the mixture and cannot be separated.

[0053] from Figure 4 It can be seen that under the elution condition of 28 minutes, geraniol glycoside can be preliminarily separated.

[0054] from Figure 5 It can be seen that under the elution condition of 36 minutes, the difference between the retention time of geraniol glycoside and the retention time of the mixture is further increased, and it can be better separated.

[0055] CDS sequence of glycosyltransferase CsUGT73D gene (SEQ ID NO.1)

[0056] ATGGCTTCCCAAAATAACCACCTTCACTTCGTTTTAGTACCTCTAATGGCCCCAGGC

[0057] CACTTCATACCCATGATTGACATGGCCAAATTATTGGCACAACACGGTGTGACCGT

[0058] CACCGTCGTCACCAGCCCTCTCATTGCTTCCAGATTCAACCCAATCATCAACCGTG

[0059] CAATTGAATCCGGACTCCACCTCCGCCTTCTTCCTTCCAGTCCCGTCCGTAGAG

[0060] GCTGGCCTGCCGGAGGGGTGCGAAACGGCAGACACTCTTCCCTCTATACACCTAAT

[0061] GAGAAATTTCTTCGTTGCCATCTGCCTGTTGCAAGAACCATTCGAGCAGTTGTTTG

[0062] AAACGATCGAGCCGTCTCCAATTAGTTGCATAATAGCTGACAAAAATATTCCTTGG

[0063] GTAGCCGACACTGCTCGAAAGTTTCAAATTCCCAGAATTATTTTTGATGGAATGAG

[0064] TTGCTTCACTCTTTTATCTACACACAATTTACATATTACCAAAGTTCATGAGAGTGT

[0065] GTCCGAGTCAGAACCGTTCGAGCTCCCGGGCTTGCCTGACAAAATTCAGCTAACA

[0066] AAATCTCAGTTGCCCCCATCAATCAATCCAGGTTCATTATTGGACGTGCAAGACTTC

[0067] CGGGTGAAAATACAAGCTGCGGAAGCCGAAGCATATGGGGTTGTGATTAACAGTT

[0068] TCGAAGAGCTGGAACCGAGATACGTGGACGGGTGTCGAAAGGTGAAACGAGATA

[0069] AAGTTTGGTGCGTTGGACCTCTATCGTTATGCAATAAAGACAACCTGGACAAGGCT

[0070] CAAAGAGGAAACAAACCTGCCATTGACGAAAACCAGTGTTTGAAGTGGCTCGATG

[0071] AACAAGAGCCAGGATCGGTGGTCTACGCATGCTTTGGAAGCACCGGTCACCTCTC

[0072] ACCTCTTCAATTAATCGAGCTGGGTTTAAGCTTGGAAGCATCAAAACGCCCATTTG

[0073] TGTGGGTCATAAGAGGCGGAGCCAAAGCAGCAGAGATAGAGAAGTGGGTGGAAG

[0074] AAGATGGATTCGAGGAAAGAGTAAAAGGGAAAGGCCTTGTGATCCGAGGGTGGG

[0075] CACCGCAAGTACTCATATTGTCTCACCCAGCTGTGGGGGCATTTTTGACACACTGT

[0076] GGGTGGAATTCGACATTGGAAGGGGTTTGTGCGGGTGTGCCAATGATAACTTGGC

[0077] CCATGTTCGCAGAGCAATTTTTTAATGAGAAGCTGATTGTACAGGTTTTGGAGACC

[0078] GGAGTGAGTGTTGGGGCTCAGGCAATTGGGCACATGTTTGGGGAAGACAAGTGTA

[0079] CGGTGCTGGTGAAGAAAGAGGTAGTTAGGGAAGCCGTGGAGGAAGTAATGGGTG

[0080] AAGGAATAGAAGGGGAAGAGAGAAGGAAAAGAGCGAGAGTGCTTGGGGAGATG

[0081] GCAAAAAAGGCAATGGAAGAAGGGGGTTCTTCGTACTTGAATGTGAGATTGTTGA

[0082] TTCAAGATGTCATCAAACAAGTTAACCACAAGGAATCAATTGAAGAAGACGGTGC

[0083] GTGTGGGTCTTAA

[0084] Amino acid sequence of glycosyltransferase CsUGT73D protein (SEQ ID NO.2)

[0085] MASQNNHLHFVLVPLMAPGHFIPMIDMAKLLAQHGVTVTVVTSPLIASRFNPIINRAIESGLHLRLLSLQFPSVEAGLPEGCETADTLPSIHLMRNFFVAICLLQEPFEQLFETIEPSPISCIIA DKNIPWVADTARKFQIPRIIFDGMSCFTLLSTHNLHITKVHESVSESEPFELPGLPDKIQLTKSQLPPSINPGSLLDVQDFRVKIQAAEEAYGVVINSFEELEPRYVDGCRKVKRDKVWCVGPLS LCNKDNLDKAQRGNKPAIDENQCLKWLDEQEPGSVVYACFGSTGHLSPLQLIELGLSLEASKRPFVWVIRGGAKAAEIEKWVEEDGFEERVKGKGLVIRGWAPQVLILSHPAVGAFLTHCGWNST LEGVCAGVPMITWPMFAEQFFNEKLIVQVLETGVSVGAQAIGHMFGEDKCTVLVKKEVVREAVEEVMGEGIEGEERKRARVLGEMAKKAMEEGGSSYLNVRLLIQDVIKQVNHKESIEEDGACGS

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for separating and determining the content of geraniol glycosides based on high performance liquid chromatography-mass spectrometry, characterized in that: High performance liquid chromatography-mass spectrometry was used to detect geraniol glycosides in the standard solution and the pretreated sample to be tested. First, high performance liquid chromatography-mass spectrometry was used to separate geraniol glycosides from the sample to be tested by optimizing the elution conditions. Then, a concentration-peak area standard curve was established with the concentration of the standard solution as the X-axis and the peak area as the Y-axis to calculate the geraniol glycoside content of the sample to be tested. The specific chromatography-mass spectrometry conditions were: (1) Mass spectrometry conditions: an electrospray ion source (ESI) was used, the detection mode was negative ion mode (ESI-), the extraction cone voltage was 5.0 V, the mass spectrometry detection was in multiple reaction monitoring mode (MRM), and the ion source temperature was 350 °C; (2) HPLC conditions: Column: Hypersil GOLD (100 mm × 2.1 mm 1.9 μm); Flow rate: 0.25 mL min -1 ; Injection volume: 5μL; Column temperature: 40℃; Mobile phase A: 0.1% (v / v) formic acid water, B: 100% acetonitrile; Gradient elution method was adopted, the elution gradient was 28min, and the elution parameters were: B phase from 0% to 10% in 0-3min, B phase from 10% to 25% in 3-15min, B phase from 25% to 40% in 15-23min, B phase from 40% to 90% in 23-24min, 90% B phase was maintained in 24-24.1min, B phase from 90% to 10% in 24.1-28min, △Rt=0.348min.

2. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that: The elution gradient is 36 minutes, and the elution parameters are: phase B from 0% to 10% in 0-3 minutes, phase B from 10% to 25% in 3-18 minutes, phase B from 25% to 40% in 18-30 minutes, phase B from 40% to 90% in 30-31 minutes, phase B maintains 90% in 31-31.1 minutes, phase B from 90% to 10% in 31.1-36 minutes, △Rt=0.446min.

3. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that: The peak area and concentration were subjected to regression analysis, and the standard curve equation of concentration-peak area was obtained as Y=96615X-50546, R 2 =0.998, where Y represents the peak area, X represents the solution concentration, and R 2 Represents the correlation coefficient.

4. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that: The sample to be tested includes a tea sample.

5. The method for separating and determining the content of geraniol glycosides according to claim 4, characterized in that: The tea sample is any one of Golden Peony, Golden Guanyin, and Zhongcha 108.

6. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that: The characteristic ion peak of geraniol glycoside is 361.

19.

7. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that: The pretreatment method of the sample to be tested is: weigh the sample, extract it with methanol, perform ultrasonic treatment in an ice bath, centrifuge after the ultrasonic treatment, draw the supernatant into a new centrifuge tube, repeat the extraction once, and finally collect the supernatant twice and filter it with a filter membrane.

8. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that: Substitute the peak area of ​​the sample to be tested into the standard curve to calculate the geraniol glycoside content of the sample to be tested.

9. The method for separating and determining the content of geraniol glycosides according to claim 5, characterized in that: When the sample to be tested is Golden Peony, the content of geraniol glycoside is 295.42±1.47μg / g; when the sample to be tested is Golden Guanyin, the content of geraniol glycoside is 227.99±2.90μg / g; when the sample to be tested is Zhongcha 108, the content of geraniol glycoside is 57.94±0.78μg / g.

10. The use of CsUGT73D protein in catalyzing the production of glycosides from geraniol and nerol, characterized in that: The amino acid sequence of the CsUGT73D protein is shown in SEQ ID NO:1, and the CDS sequence of the gene encoding the CsUGT73D protein is shown in SEQ ID NO:2.

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