A method for determining the content of 17 effective components in pericarpium citri reticulatae by UPLC-MS / MS

Abstract

The present application relates to the technical field of biological analysis, and more particularly to a method for determining the content of 17 effective components in pericarpium citri reticulatae by UPLC-MS / MS. The method comprises the following steps: (1) preparing a control solution; (2) preparing a test solution; (3) determining the content by using the multiple reaction monitoring (MRM) quantitative mode through the ultra-high performance liquid chromatography tandem mass spectrometry technology; (4) establishing the standard curve of 17 components by using a series of standard solution with different concentrations, and calculating the content of the 17 components in pericarpium citri reticulatae by the standard curve and the peak area of the sample. The determination method has the advantages of short analysis time, low detection limit and quantitative limit, and high sensitivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological analysis, and particularly relates to a method for determining the content of 17 effective components in pericarpium citri reticulatae by using UPLC-MS / MS. BACKGROUND

[0002] Pericarpium citri reticulatae is the dried mature fruit pericarp of Citrus reticulata Blanco and its cultivated varieties. The medicinal materials are divided into "pericarpium citri reticulatae" and "pericarpium citri reticulatae viride". The mature fruits are picked, the pericarp is peeled off, and then dried or low-temperature dried. The dried pericarp of Dahonggan produced in Xinhui District of Jiangmen City, Guangdong Province is a high-value medicinal material, and is also a traditional spice and seasoning delicacy. Pericarpium citri reticulatae has a long history of medicinal use, and has the functions of regulating qi, invigorating the spleen, drying dampness and reducing sputum.

[0003] The 1990 edition of Chinese Pharmacopoeia added the thin layer chromatography identification of reference materials, which greatly improved the identification of traditional Chinese medicines and Chinese patent medicines. At present, traditional Chinese medicines and foreign herbs pay more and more attention to the detection of multiple components or multiple components, such as the quality control of German ginkgo leaf extract. At present, there are two kinds of "reference materials" in the control of the quality of traditional Chinese medicines, chemical reference materials and traditional Chinese medicine reference materials. The chemical reference materials can be used for qualitative identification and quantitative analysis of medicinal materials, and the traditional Chinese medicine reference materials are used for microscopic identification and thin layer identification. However, both the chemical reference materials and the traditional Chinese medicine reference materials have their limitations in the quality control of traditional Chinese medicines. First, the chemical components of traditional Chinese medicines are diversified, and a single or several compounds cannot reflect the whole picture of the medicinal materials, and the existing standards often have many loopholes, and the phenomenon of using inferior products as good products occurs from time to time. The traditional Chinese medicine reference materials are affected by the production place and growth environment, and it is difficult to ensure the consistency of the quality of each batch, and can only be used for qualitative identification, and cannot reflect the high and low of the content of the components of the medicinal materials.

[0004] In the prior art, the HPLC method is generally used to determine the content of the main active components (hesperidin, citropten, sinensin and synephrine) in pericarpium citri reticulatae. Therefore, the present application provides a method for determining the content of 17 effective components in pericarpium citri reticulatae by using UPLC-MS / MS, so as to solve the problem of low sensitivity of the method for determining the effective components of pericarpium citri reticulatae in the prior art. SUMMARY

[0005] The purpose of the present application is to provide a method for determining the content of 17 effective components in pericarpium citri reticulatae by using UPLC-MS / MS, so as to more comprehensively determine the effective components of pericarpium citri reticulatae.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:

[0007] The present application provides a method for determining the content of 17 effective components in pericarpium citri reticulatae by using UPLC-MS / MS, which comprises the following steps:

[0008] (1) 17 kinds of control mixed with methanol to prepare a control stock solution;

[0009] (2) Chenpi mixed with methanol after ultrasonic treatment, cooling, centrifugal to take supernatant, filtration, to obtain test solution;

[0010] (3) UPLC-MS / MS method for detection.

[0011] Preferably, step (1) said 17 kinds of control are respectively octopine, N-methyl tyramine, vitexin-Ⅱ, rutin, eriodictyol, p-coumaric acid, naringin, hesperidin, melilotoside, naringenin, hesperetin, iso-scutellarein, sweet orange flavone, citropten, 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone, citranone, norcitropten.

[0012] Preferably, step (1) said control mixed weight is 0.50-1.50 mg; the concentration of the control stock solution is 0.5-1.5 mg / mL.

[0013] Preferably, step (2) said Chenpi dosage is 7-13 mg.

[0014] Preferably, step (2) said ultrasonic treatment is specifically as follows: extraction power is 200-300 W, frequency is 40-60 KHz, extraction time is 40-50 min.

[0015] Preferably, step (2) said cooling to 22-28 ℃; centrifugal is 11800-12200 g centrifugal for 5-15 min.

[0016] Preferably, step (2) said filter membrane pore size is 0.22 μm.

[0017] Preferably, step (3) said detection uses the following ultra-high liquid chromatography conditions:

[0018] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm); column temperature: 22-28 ℃; flow rate: 0.1-0.5 mL·min -1 ; injection volume: 2-8 μL; gradient elution conditions are: 0-1 min, 13%-32% B; 1-3 min, 32%-42% B; 3-4 min, 42%-52% B; 4-8 min, 52%-58% B; mobile phase is A phase 0.1% formic acid water, B phase acetonitrile.

[0019] Preferably, step (3) said detection uses the following mass spectrometry conditions:

[0020] Ion source: electrospray ion source (ESI); capillary voltage: 3500V in positive and negative ion mode; capillary temperature: 300℃; drying gas flow rate: 7L·min-1; nebulizer pressure: 35psi; EMV voltage: 400V; detection mode: multiple reaction monitoring mode (MRM) as shown in Table 2 below: -1

[0021] Table 2

[0022]

[0023] Preferably, the volume fraction of the methanol solution is 70-90%.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] The determination method of the present application has short analysis time, low detection limit and quantitative limit, high sensitivity and high accuracy. The present application provides a method for detecting 17 kinds of effective components in pericarpium citri reticulatae based on UPLC-MS / MS, and the accuracy of the present application is verified by the sample recovery rate. The present application establishes an analysis method for simultaneously determining 17 kinds of effective components in pericarpium citri reticulatae by using UPLC-MS / MS, and the results show that the method has the characteristics of strong specificity, high sensitivity and solvent saving, and can provide reliable reference for the quality control of pericarpium citri reticulatae. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0027] Figure 1 A control sample MRM chart; B sample MRM chart;

[0028] (1. Octopine; 2. N-methyl tyramine; 3. Vicenin-Ⅱ; 4. Rutin; 5. Eriodictyol-7-rutinoside; 6. p-coumaric acid; 7. Nobiletin; 8. Hesperidin; 9. Melilotoside; 10. Naringenin; 11. Hesperetin; 12. Isohesperetin; 13. Sweet orange flavone; 14. Citrocutin; 15. 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone; 16. Citranone; 17. Norcitrocutin). DETAILED DESCRIPTION

[0029] The present application provides a method for determining the content of 17 kinds of effective components in pericarpium citri reticulatae by using UPLC-MS / MS, which comprises the following steps:

[0030] ​(1) 17 kinds of control samples are mixed with methanol to prepare a control sample stock solution;

[0031] (2) The pericarpium citri reticulatae is mixed with methanol, ultrasonic treatment, cooling, centrifugal to take supernatant, filtration to obtain a test sample solution;

[0032] (3) UPLC-MS / MS method is used for detection.

[0033] In the present application, the 17 kinds of control samples in step (1) are respectively synephrine, N-methyl tyramine, vitexin-Ⅱ, rutin, eriodictyol, p-coumaric acid, narirutin, hesperidin, melilotoside, naringenin, hesperetin, isohyperoside, sweet orange flavone, citropten, 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone, citranone, demethylcitropten.

[0034] In the present application, the weight of the control sample mixed in step (1) is 0.50-1.50 mg; preferably 0.70-1.30 mg; further preferably 0.90-1.10 mg; more preferably 1.0 mg.

[0035] In the present application, the concentration of the control sample stock solution in step (1) is 0.5-1.5 mg / mL; preferably 0.70-1.30 mg / mL; further preferably 0.90-1.10 mg / mL; more preferably 1.0 mg / mL.

[0036] In the present application, the amount of pericarpium citri reticulatae in step (2) is 7-13 mg; preferably 8-12 mg; further preferably 9-11 mg; more preferably 10 mg.

[0037] In the present application, the ultrasonic treatment in step (2) is specifically as follows: the extraction power is 200-300 W, the frequency is 40-60 KHz, and the extraction time is 40-50 min; preferably the extraction power is 210-290 W, the frequency is 43-57 KHz, and the extraction time is 41-49 min; further preferably the extraction power is 230-270 W, the frequency is 47-53 KHz, and the extraction time is 43-47 min; more preferably the extraction power is 250 W, the frequency is 50 KHz, and the extraction time is 45 min.

[0038] In the present application, the cooling in step (2) is to 22-28℃; preferably 23-27℃; further preferably 24-26℃; more preferably 25℃.

[0039] In the present application, the centrifugal in step (2) is 11800-12200 g; preferably 11850-12150 g; further preferably 11950-12050 g; more preferably 12000 g.

[0040] In the present application, the centrifugation time in step (2) is 5-15 min; preferably 7-13 min; further preferably 9-11 min; more preferably 10 min.

[0041] In the present application, the pore size of the filter membrane in step (2) is preferably 0.22 μm.

[0042] In the present application, the ultra-high liquid chromatography conditions used in step (3) are as follows:

[0043] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm); column temperature: 22-28 °C; flow rate: 0.1-0.5 mL·min -1 ; injection volume: 2-8 μL; gradient elution conditions: 0-1 min, 13%-32% B; 1-3 min, 32%-42% B; 3-4 min, 42%-52% B; 4-8 min, 52%-58% B; mobile phase: A phase 0.1% formic acid water, B phase acetonitrile; preferably chromatographic column: Waters ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm); column temperature: 23-27 °C; flow rate: 0.1-0.4 mL·min -1 ; injection volume: 3-7 μL; gradient elution conditions: 0-1 min, 13%-32% B; 1-3 min, 32%-42% B; 3-4 min, 42%-52% B; 4-8 min, 52%-58% B; mobile phase: A phase 0.1% formic acid water, B phase acetonitrile; further preferably chromatographic column: Waters ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm); column temperature: 24-26 °C; flow rate: 0.2-0.4 mL·min -1 ; injection volume: 4-6 μL; gradient elution conditions: 0-1 min, 13%-32% B; 1-3 min, 32%-42% B; 3-4 min, 42%-52% B; 4-8 min, 52%-58% B; mobile phase: A phase 0.1% formic acid water, B phase acetonitrile; more preferably chromatographic column: Waters ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm); column temperature: 25 °C; flow rate: 0.3 mL·min -1 ; injection volume: 5 μL; gradient elution conditions: 0-1 min, 13%-32% B; 1-3 min, 32%-42% B; 3-4 min, 42%-52% B; 4-8 min, 52%-58% B; mobile phase: A phase 0.1% formic acid water, B phase acetonitrile.

[0044] In the present application, the mass spectrometry conditions used for the detection in step (3) are as follows:

[0045] Ion source: electrospray ion source (ESI); capillary voltage: 3500 V in positive and negative ion mode; capillary temperature: 300℃; drying gas flow rate: 7 L·min -1 ; atomizer pressure: 35 psi; EMV voltage: 400 V; detection mode: multiple reaction detection mode (MRM) as shown in Table 3 below:

[0046] Table 3

[0047]

[0048] In the present application, the volume fraction of the methanol solution is 70-90%; preferably 73-87%; further preferably 77-83%; and more preferably 80%.

[0049] The technical solutions provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0050] The experimental methods used in the following examples are conventional methods unless otherwise specified; and the reagents, materials, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0051] 1. Instruments

[0052] Agilent 1290 ultra-high performance liquid chromatograph (Agilent, USA); Agilent 6470 triple quadrupole mass spectrometer (Agilent, USA); Agilent Mass Hunter analysis software (Agilent, USA); AS60 / 220.R2 type hundredth balance (RADWAG, Poland); Milli-Q ultrapure water preparation instrument (Millipore); G3KT18273 vortex mixer (Thermo Fisher Scientific); 5430R centrifuge (Eppendorf); ZZ-L6DT ultrasonic cleaner.

[0053] 2. Reagents

[0054] The origin and number of pericarpium citri reticulatae are shown in Table 4. Methanol is chromatographically pure and purchased from Merk Company; formic acid is chromatographically pure and purchased from ROE Company. The control 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone (DST201227-104), hesperidin (DST180619-038), hesperetin (DST191024-054), nobiletin (DSTDC003701), p-coumaric acid (DST210508-057), citranone (DSTDJ000401), rutin (DST210520-017), nor-nobiletin (DST220222-094), rutinoside (DST211225-014), sinensetin (DST201009-005), vicenin-Ⅱ (DST201104-023), jaceosidin (DST200815-048), synephrine (DSTDX002001), iso-hesperidin (DST220217-065), naringenin (DST211116-100), aurantiamarin (DST211102-098) are purchased from Chengdu Desite Biotechnology Co., Ltd.; N-methyltyramine (RH351718) is purchased from R & N Reagent Co., Ltd.

[0055] Table 4 Origin and number of 10 batches of pericarpium citri reticulatae

[0056]

[0057] Example 1

[0058] 1. Preparation of solution and chromatography-mass spectrometry conditions

[0059] Preparation of test solution

[0060] Precisely weigh 10.0 mg of pericarpium citri reticulatae powder into a 10 mL volumetric flask, add 80% methanol to the mark, shake well, and then ultrasonically treat for 45 min. Cool to room temperature, and then add methanol to the mark. Centrifuge the supernatant at 12000 g for 10 min. Filter the supernatant through a 0.22 μm filter membrane to obtain the test solution, which is stored in a 4 ℃ refrigerator for standby use.

[0061] 2. Preparation of control solution

[0062] Precisely weigh 1.00 mg of each of synephrine, N-methyltyramine, vicenin-Ⅱ, rutin, rutinoside, p-coumaric acid, aurantiamarin, hesperidin, jaceosidin, naringenin, hesperetin, iso-hesperidin, sinensetin, nobiletin, 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone, citranone, and nor-nobiletin control, and dissolve in methanol to prepare a control stock solution with a concentration of 1 mg / mL. Store in a 4 ℃ refrigerator for standby use.

[0063] 3. Chromatography conditions

[0064] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm); mobile phase A 0.1% formic acid water, B phase acetonitrile, gradient elution, elution program 0-1 min, 13%-32% B; 1-3 min, 32%-42% B; 3-4 min, 42%-52% B; 4-8 min, 52%-58% B; flow rate 0.3 mL·min -1 ; column temperature 25 °C; injection volume 5 μL.

[0065] 4. Mass spectrometry conditions

[0066] The ion source is electrospray ion source (ESI), the detection mode is multiple reaction monitoring mode (MRM), the capillary temperature is 300 °C; the capillary voltage is 3500 V in positive and negative ion mode; the dry gas flow rate is 7 L·min -1 ; the atomizer pressure is 35 psi; the EMV voltage is 400 V. The ion pairs and mass spectrometry parameters of 17 measured substances are shown in Table 5.

[0067] Table 5 Mass spectrometry parameters of 17 chemical components of Pericarpium Citri Reticulatae Viride

[0068]

[0069]

[0070] Example 2

[0071] Methodology investigation

[0072] 1. Linear range investigation

[0073] An appropriate amount of control sample stock solution was accurately taken, and the solution was diluted 2.5, 2, 2, 2, 2.5, 2, 2, 2 times in sequence with methanol to obtain 9 concentrations of control sample mixed solutions. 5 μL of each of the 9 concentrations of the above solutions was injected for analysis. With the concentration of the test substance (x) as the abscissa and the peak area of the test substance (y) as the ordinate, the regression calculation was performed by weighted least squares method, and the weight coefficient was set to 1 / X. The regression equation and correlation coefficient of each compound were calculated, and the results were recorded in Table 6, and the MRM diagram is shown in Figure 1 .

[0074] Table 6 Standard curve regression equation of 17 chemical components in Pericarpium Citri Reticulatae Viride

[0075]

[0076]

[0077] 2. Precision experiment

[0078] Intra-day precision: 10.0 mg of Pericarpium Citri Reticulatae (S10) powder was precisely weighed, and the test solution was prepared according to Step 1 of Example 1. The sample was injected 6 times under the chromatographic and mass spectrometric conditions described in Step 3 and Step 4 of Example 1, the peak areas of each compound were recorded, and the RSD value was calculated. The results in Table 7 show that the intra-day precision of the method is good.

[0079] Inter-day precision: 10.0 mg of Pericarpium Citri Reticulatae (S10) powder was precisely weighed, and the test solution was prepared according to Step 1 of Example 1. The same sample was continuously injected for three days, twice a day, and the peak areas of each compound were recorded. The RSD value was calculated. The results in Table 8 show that the inter-day precision of the method is good.

[0080] Table 7 Intra-day precision of 17 components in Pericarpium Citri Reticulatae (n = 6)

[0081]

[0082]

[0083] Table 8 Inter-day precision of 17 components in Pericarpium Citri Reticulatae (n = 6)

[0084]

[0085]

[0086] 3. Stability experiment

[0087] 10.0 mg of Pericarpium Citri Reticulatae (S10) powder was precisely weighed, and the test solution was prepared according to Step 1 of Example 1. The sample was injected at 0, 2, 4, 6, 8, and 12 h under the chromatographic and mass spectrometric conditions described in Step 3 and Step 4 of Example 1, the peak areas of each compound were recorded, and the RSD value was calculated. The results in Table 9 show that the 17 components in the sample are stable for 12 h in the autosampler.

[0088] Table 9 Stability of 17 components in Pericarpium Citri Reticulatae (n = 6)

[0089]

[0090]

[0091] 4. Reproducibility experiment

[0092] 6 portions of Pericarpium Citri Reticulatae (S10), each 10.0 mg, were precisely weighed, and the test solution was prepared according to Step 1 of Example 1. The sample was injected under the chromatographic and mass spectrometric conditions described in Step 3 and Step 4 of Example 1, and the concentration and RSD value of each compound were calculated. The results in Table 10 show that the method has good reproducibility.

[0093] Table 10 Reproducibility of components in Pericarpium Citri Reticulatae (n = 6)

[0094]

[0095]

[0096] 5. Sample loading recovery test

[0097] Accurately weigh 6 portions of dried tangerine or orange peel (S10) powder, 5.0 mg each, and add synephrine 8200 ng, N-methyl tyramine 17 ng, vitexin-Ⅱ 2600 ng, rutin 1100 ng, eriodictyol 2400 ng, p-coumaric acid 25 ng, naringin 47500 ng, hesperidin 70000 ng, melilotoside 15500 ng, naringenin, hesperetin 110 ng, isohyperoside 140 ng, sweet orange flavonoid 160 ng, obacunone 3100 ng, 3,5,6,7,8,3',4'-heptamethoxy flavone 4500 ng, citranone 750 ng, and norobacunone 120 ng. Make up to 10 mL with 80% methanol. Prepare the test sample solution according to Step 1 of Example 1, and determine the sample according to the chromatographic and mass spectrometric conditions in Step 3 and Step 4 of Example 1. Calculate the recovery of each compound and the RSD value. The results are shown in Table 11.

[0098] Table 11. Recovery of 17 components in dried tangerine or orange peel (n = 6)

[0099]

[0100] Example 3

[0101] Sample determination

[0102] Accurately weigh 10 portions of dried tangerine or orange peel powder, 10.0 mg each, and prepare the test sample solution according to Step 1 of Example 1. Determine the sample according to the chromatographic and mass spectrometric conditions in Step 3 and Step 4 of Example 1, and calculate the content of 17 components. The results are recorded in Table 12.

[0103] Table 12. Determination results of dried tangerine or orange peel from different origins (ng / mL, n = 3)

[0104]

[0105]

[0106] The above only describes the preferred embodiments of the present application, and it should be noted that those of ordinary skill in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A method for determining the content of 17 active ingredients in dried tangerine peel using UPLC-MS / MS, characterized in that, Includes the following steps: (1) A reference standard stock solution was prepared by mixing 17 reference standards with methanol; (2) Mix dried tangerine peel with methanol, sonicate, cool, centrifuge, collect the supernatant, filter, and obtain the test solution; (3) Detection was performed using UPLC-MS / MS method; The 17 reference standards mentioned in step (1) are synephrine, N-methyltyramine, vezin-II, rutin, senna-2, p-coumaric acid, rutin, hesperidin, lemon balm glycoside, naringenin, hesperidin, isohesperidin, sweet orange flavonoids, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavonoids, tangerine, and demethylnobiletin; The ultra-high performance liquid chromatography (UHPLC) conditions used in step (3) are as follows: Chromatographic column: Waters ACQUITY UPLC BEH C18 column; column temperature: 22~28℃; flow rate: 0.1~0.5mL·min -1 Injection volume: 2~8μL; Gradient elution conditions: 0~1min, 13%~32%B; 1~3min, 32%~42%B; 3~4min, 42%~52%B; 4~8min, 52%~58%B; Mobile phase: Phase A 0.1% formic acid water, Phase B acetonitrile; The mass spectrometry conditions used for the detection are as follows: Ion source: Electrospray ion source; Capillary voltage: 3500V (positive and negative ion mode); Capillary temperature: 300℃; Drying gas flow rate: 7L·min -1 Atomizer pressure: 35 psi; EMV voltage: 400V; Detection method: Multiple reaction detection mode as shown in Table 1 below: Table 1 。 2. The method according to claim 1, characterized in that, The weight of the reference standard mixture in step (1) is 0.50~1.50 mg; the concentration of the reference standard stock solution is 0.5~1.5 mg / mL.

3. The method according to claim 1, characterized in that, The dosage of dried tangerine peel in step (2) is 7~13mg.

4. The method according to claim 1, characterized in that, The specific procedure for ultrasonic treatment in step (2) is as follows: the extraction power is 200~300 W, the frequency is 40~60 KHz, and the extraction time is 40~50 min.

5. The method according to claim 1, characterized in that, Step (2) involves cooling to 22-28℃ and centrifuging at 11800-12200g for 5-15 minutes.

6. The method according to claim 1, characterized in that, The volume fraction of the methanol solution is 70-90%.

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