A method for separating and determining geraniol glycosides based on high performance liquid chromatography-mass spectrometry

Abstract

This invention discloses a method for separating and determining geraniol glycosides based on high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), relating to the field of analytical chemistry. HPLC-MS / MS is used to detect geraniol glycosides in standard solutions and pretreated test samples. First, geraniol glycosides are separated from the test samples by optimizing the elution conditions using HPLC-MS / MS. Then, a standard curve is established with the standard solution concentration as the X-axis and the peak area as the Y-axis to calculate the geraniol glycoside content of the test sample. Beneficial effects: This invention, by optimizing the elution conditions, can effectively distinguish and separate geraniol glycosides from nerol glycosides, improving the accuracy of geraniol glycoside content measurement. This method has good application prospects.

Description

Technical Field

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

[0002] Glycosides are aroma precursors widely found in plants and play an indispensable role in plant growth and development. When plants are under stress or under specific conditions, glycosides are broken down by glycoside hydrolases, releasing aroma molecules. These aroma molecules can enhance the plant's resistance to pests and diseases and effectively repel insects. Furthermore, glycosides are widely involved in plant secondary metabolism, generating various metabolites beneficial to plant physiological activities and stress resistance. Although glycosides are widely distributed in nature, their content is relatively low; therefore, enzymatic synthesis is the primary method for their synthesis.

[0003] Geraniol is one of the main components of terpenoids in plants, possessing a typical rose fragrance and widely found in over 250 plant species, including tea trees, geraniums, and roses. Furthermore, geraniol is primarily stored in plant cells as glucosinolates or primrose glycosides. Nerol is the cis isomer of geraniol, and both have the same molecular formula C10H18O. Nerol glycosides are aroma precursors and have no fragrance themselves. This substance is widely found in plants such as tea and fresh flowers. During plant maturation, nerol glycosides are broken down by β-glycoside hydrolases, releasing nerol and thus producing aroma.

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

[0005] The technical problem to be solved by this invention is how to solve the current difficulty in separating and accurately determining the content of geraniol glycosides.

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

[0007] The first aspect of this invention proposes a method for separating and determining geraniol glycosides based on high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). HPLC-MS / MS is used to detect geraniol glycosides in a standard solution and a pretreated sample. First, geraniol glycosides are separated from the sample using HPLC-MS / MS by optimizing the elution conditions. Then, a standard curve is established with the standard solution concentration as the X-axis and the peak area as the Y-axis to calculate the geraniol glycoside content of the sample. Specific chromatographic and mass spectrometric conditions are as follows:

[0008] (1) Mass spectrometry conditions: Electrospray ionization (ESI) source was used, the detection mode was negative ion mode (ESI-), the extraction cone voltage was 5.0V, the mass spectrometry detection mode was multiple reaction monitoring (MRM), and the ion source temperature was 350℃.

[0009] (2) High performance liquid chromatography 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℃; Mobile phase A: 0.1% (v / v) formic acid water, B: 100% acetonitrile; Gradient elution was used with an elution gradient of 28 min. The elution parameters were as follows: B phase from 0% to 10% in the first 3 min, B phase from 10% to 25% in the first 3 min, B phase from 25% to 40% in the first 15 min, B phase from 40% to 90% in the first 23 min, B phase maintained at 90% in the first 24 min, and B phase from 90% to 10% in the first 24.1 min, with ΔRt = 0.348 min.

[0010] Preferably, the elution gradient is 36 min, and the elution parameters are as follows: phase B increases from 0% to 10% within 0–3 min, phase B increases from 10% to 25% within 3–18 min, phase B increases from 25% to 40% within 18–30 min, phase B increases from 40% to 90% within 30–31 min, phase B remains at 90% within 31–31.1 min, phase B decreases from 90% to 10% within 31.1–36 min, and ΔRt = 0.446 min.

[0011] Preferably, regression analysis was performed on the peak area and concentration to obtain the standard curve equation for concentration-peak area as Y = 96615X - 50546, R0 2 =0.998, where Y represents the peak area, X represents the solution concentration, and R 2 This 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, or Zhongcha 108.

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

[0015] Preferably, the pretreatment method for the sample to be tested is as follows: weigh the sample, extract it with methanol, sonicate it in an ice bath, centrifuge it after sonication, transfer the supernatant to a new centrifuge tube, repeat the extraction once, collect the supernatant twice, and filter it with a filter membrane.

[0016] Preferred method for pretreatment of the sample to be tested is as follows: weigh 50 mg of tea sample, extract with 90% (v / v) methanol (4 mL), sonicate in an ice bath for 30 min, and mix by turning over every 5 min; after sonication, centrifuge at 4°C and 13400×g for 10 min using a low-temperature centrifuge, aspirate the supernatant into a new centrifuge tube, repeat the extraction once, and finally collect the supernatant twice and filter it through a 0.22 μm 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 Jin Mudan, the content of geraniol glycosides is 295.42±1.47μg / g; when the sample to be tested is Jin Guanyin, the content of geraniol glycosides is 227.99±2.90μg / g; and when the sample to be tested is Zhongcha 108, the content of geraniol glycosides is 57.94±0.78μg / g.

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

[0020] The beneficial effects of this invention are:

[0021] This invention is based on high performance liquid chromatography-mass spectrometry (HPLC-MS). By optimizing the elution conditions, geraniol glycosides and nerol glycosides can be effectively distinguished and separated, improving the accuracy of geraniol glycoside content measurement. This method has good application prospects. Attached Figure Description

[0022] Figure 1This is an 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, CsUGT73D before induction; 3, CsUGT73D after induction; 4, supernatant after disruption; 5, precipitate after disruption; 6, flow-through; 7, purified CsUGT73D protein; the target protein is marked by a blue box.

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

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

[0025] Figure 4 The images show the chromatograms of geraniol and nerol glycosides detected under a 28-minute elution gradient in Example 1 of this invention. Figure A shows the chromatogram of geraniol and nerol glycosides, Figure B shows the mass spectrum of nerol glycosides, and Figure C shows the mass spectrum of geraniol glycosides.

[0026] Figure 5 The images show the chromatograms of geraniol and nerol glycosides detected under a 36-minute elution gradient in Example 1 of this invention. Figure A shows the chromatogram of geraniol and nerol glycosides, Figure B shows the mass spectrum of nerol glycosides, and Figure C shows the mass spectrum of geraniol glycosides.

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

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0030] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0031] The main reagents and formulations used in the embodiments of this invention are as follows:

[0032] (1) Geraniol glucoside

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

[0034] Example 1:

[0035] 1. Purification of glycosyltransferase CsUGT73D protein

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

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

[0038] The purified CsUGT73D protein was used to catalyze the conversion of geraniol and nerol into glycosides. The enzyme activity reaction system (200 μL) consisted of: 50 mM Tris-HCl (pH = 7.5), 10 mM DTT, 5 mM geraniol (nerol), 10 mM UDP-Glucose, 50 μg CsUGT73D protein, and 10% glycerol. The mixture was incubated at 30 °C for 15 h. After incubation, the protein was extracted twice with an equal volume of ethyl acetate. The organic solvent was evaporated by rotary evaporation, and the extract was dissolved in 300 μL of 80% methanol. The mixture was centrifuged at 13400 × g for 10 min, and the supernatant was collected. After filtration through a 0.22 μm organic phase filter membrane, the supernatant was added to a sample vial for analysis.

[0039] Coomassie Brilliant Blue stained CsUGT73D protein SDS-PAGE gel image as shown below Figure 1 As 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). - The extraction cone voltage was 5.0V, and the mass spectrometry detection was performed in multiple reaction monitoring (MRM) mode with an ion source temperature of 350℃.

[0042] (2) High performance liquid chromatography 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℃. Mobile phase A: 0.1% (v / v) formic acid in water, B: 100% acetonitrile;

[0043] like Figure 3As shown, under a 16-minute elution gradient (phase B increases from 0% to 10% in the first 3 minutes, from 10% to 25% in the first 3 minutes, from 25% to 40% in the first 9 minutes, from 40% to 90% in the first 11 minutes, maintaining 90% phase B in the first 12 minutes, and from 90% to 10% in the first 12.1 minutes, with ΔRt = 0.057 min), isomers are difficult to separate. Therefore, the elution gradient needs to be optimized.

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

[0045] The elution gradient was optimized again (phase B increased from 0% to 10% in the first 3 min, from 10% to 25% in the first 3 min, from 25% to 40% in the first 18 min, from 40% to 90% in the first 30 min, maintained at 90% phase B in the first 31 min, and decreased from 90% to 10% in the first 31.1 min, with ΔRt = 0.446 min). The results are as follows: Figure 5 As shown, under a 36-minute elution gradient, the difference in glycoside retention time between geraniol and nerol further increases, making it easier to distinguish between the two.

[0046] 4. Construction of the standard curve for geraniol glycosides and determination of their content

[0047] Geraniol glycoside standards were diluted to eight concentration gradients: 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. A concentration-peak area standard curve was constructed based on the peak areas measured using the optimized elution gradients. Regression analysis was performed on the peak areas and concentrations, and the final standard curve equation was Y = 96615X - 50546, R0. 2 =0.998 (e.g.) Figure 6 As shown in the figure, where Y represents the peak area, X represents the solution concentration, and R... 2 This represents the correlation coefficient.

[0048] Sample preparation: Weigh 50 mg of sample and extract with 90% (v / v) methanol. Add 4 mL of extract to each sample and sonicate on ice for 30 min, mixing by inverting the sample every 5 min. After sonication, centrifuge at 4°C and 13400 × g for 10 min. Transfer the supernatant to a new centrifuge tube and repeat the extraction once. Collect the supernatant from both extractions and filter it through a 0.22 μm filter membrane as the sample to be tested. Calculate the geraniol glycoside content in the sample based on the standard curve. Substitute the peak area of ​​the sample to be tested into the standard curve to obtain the content as shown in Table 1.

[0049] Table 1

[0050]

[0051] from Figure 2 As can be seen from the data, the characteristic ionic peak of geraniol glycoside is 361.19.

[0052] from Figure 3 As can be seen, under elution conditions of 16 min, the elution time of geraniol glycosides coincides with that of the mixture, making them impossible to separate.

[0053] from Figure 4 It can be seen that geraniol glycosides can be initially separated under elution conditions of 28 min.

[0054] from Figure 5 As can be seen, under elution conditions of 36 min, the difference between the retention time of geraniol glycoside and the retention time of the mixture further increases, indicating that they can be separated relatively well.

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

[0056] ATGGCTTCCCAAAATAACCACCTTCACTTCGTTTTAGTACCTCTAATGGCCCCAGGC

[0057] CACTTCATACCCATGATTGACATGGCCAAATTATTGGCACAACACGGTGTGACCGT

[0058] CACCGTCGTCACCAGCCCTCTCATTGCTTCCAGATTCAACCCAATCATCAACCGTG

[0059] CAATTGAATCCGGACTCCACCTCCGCCTTCTCTTCCTCCAGTTCCCGTCCGTAGAG

[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 LEGVCAGVPMITWPMFAEQFFNEKLIVQVLETGVSVGAQAIGHMFGEDKCTVLVKKEVVREAAVEEVMGEGIEGEERKRARVLGEMAKKAMEEGGSSYLNVRLLIQDVIKQVNHKESIEEDGACGS

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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, Geraniol glycosides in standard solutions and methanol-pretreated test samples were detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Geraniol glycosides and nerol glycosides were first separated using HPLC-MS / MS. A concentration-peak area standard curve was then established with the standard solution concentration as the X-axis and peak area as the Y-axis. The geraniol glycoside content of the test sample (tea sample) was calculated. Specific HPLC-MS / MS conditions were as follows: (1) Mass spectrometry conditions: an electrospray ion source was used, the detection mode was negative ion mode, the extraction cone voltage was 5.0 V, the mass spectrometry detection was performed in multiple reaction monitoring mode, and the ion source temperature was 350 ℃; (2) High performance liquid chromatography 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 ℃; Mobile phase A: 0.1% formic acid aqueous solution, B: 100% acetonitrile; A gradient elution method was used, with the following elution parameters: phase B increased from 0% to 10% within 0–3 min; phase B increased from 10% to 25% within 3–15 min; phase B increased from 25% to 40% within 15–23 min; phase B increased from 40% to 90% within 23–24 min; phase B remained at 90% within 24–24.1 min; and phase B decreased from 90% to 10% within 24.1–28 min, with ΔRt = 0.348 min. or The elution parameters were as follows: phase B increased from 0% to 10% within 0-3 min, phase B increased from 10% to 25% within 3-18 min, phase B increased from 25% to 40% within 18-30 min, phase B increased from 40% to 90% within 30-31 min, phase B remained at 90% within 31-31.1 min, and phase B decreased from 90% to 10% within 31.1-36 min, with ΔRt = 0.446 min.

2. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that, Regression analysis was performed on the peak area and concentration to obtain the standard curve equation for concentration-peak area as Y = 96615X - 50546, R0 2 =0.998, where Y represents the peak area, X represents the solution concentration, and R... 2 This represents the correlation coefficient.

3. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that, The tea sample is any one of Golden Peony, Golden Guanyin, or Zhongcha 108.

4. The method for separating and determining the content of geraniol glycosides according to claim 3, characterized in that, The tea sample was Golden Peony.

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

19.

6. The method for separating and determining the content of geraniol glycosides according to claim 1, characterized in that, The pretreatment method for the sample to be tested is as follows: weigh the sample, extract it with methanol, sonicate it in an ice bath, centrifuge it after sonication, transfer the supernatant to a new centrifuge tube, repeat the extraction once, collect the supernatant twice, and filter it with a filter membrane.

7. 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.

8. The method for separating and determining the content of geraniol glycosides according to claim 3, characterized in that, When the sample to be tested was *Peony peony*, the content of geraniol glycosides was 295.42 ± 1.47 μg / g.

9. The method for separating and determining the content of geraniol glycosides according to claim 3, characterized in that, When the sample to be tested was *Gynostemma pentaphyllum*, the content of geraniol glycosides was 227.99 ± 2.90 μg / g.

10. The method for separating and determining the content of geraniol glycosides according to claim 3, characterized in that, When the sample to be tested was Zhongcha 108, the content of geraniol glycosides was 57.94 ± 0.78 μg / g.

Images