A method for determining contents of multiple components in red peony root based on MRM mode UHPLC-QQQ-MS / MS

Abstract

The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a UHPLC-QQQ-MS / MS multi-component content determination method of Radix Paeoniae Rubra based on an MRM mode. Radix Paeoniae Rubra powder and methanol are mixed to perform ultrasonic extraction to obtain an extraction liquid; the extraction liquid is detected by ultrahigh performance liquid chromatography-tandem mass spectrometry in a multiple reaction monitoring mode to obtain a to-be-detected spectrum; according to the to-be-detected spectrum and a predetermined standard curve, the content of each component in Radix Paeoniae Rubra is obtained, and the standard curve is linearly fitted by taking the peak area ratio of each to-be-detected component to an internal standard as the concentration. The method can simultaneously complete quantitative detection of 54 components in Radix Paeoniae Rubra within 15 min, has the characteristics of simple operation, strong specificity, good precision and accuracy, and solves the technical problems of few detected components, long analysis time, great interference and insufficient accuracy of the existing Radix Paeoniae Rubra quantitative method.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for determining the content of multiple components of Paeonia lactiflora based on MRM mode using UHPLC-QQQ-MS / MS. Background Technology

[0002] Red peony is the herb Paeonia lactiflora, a plant belonging to the Ranunculaceae family. Paeonia lactiflora Pall. or Paeonia lactiflora Paeonia veitchii The dried root of *Lychium lychnophorum*. The *Fifty-Two Prescriptions* is the oldest extant document recording the medicinal use of peony. The *Shennong's Classic of Materia Medica* states: "Peony, bitter and neutral in nature, treats abdominal pain caused by pathogenic factors, eliminates blood stasis, breaks up hard masses, treats chills and fever, hernia, relieves pain, promotes urination, and invigorates qi." Modern research shows that red peony contains monoterpenes and their glycosides, flavonoids and their glycosides, phenolic acids, and other components, possessing various pharmacological effects such as neuroprotection, improving microcirculation, regulating immunity, anti-arthritis, anti-tumor, and hypoglycemic effects. Currently, red peony circulating in the market comes from a wide range of sources with inconsistent production areas, resulting in varying quality. However, the quality of red peony directly affects its efficacy; therefore, quality control is crucial. Quantitative analysis of active ingredients is indispensable in perfecting the quality evaluation system for red peony.

[0003] Currently, the main quantitative methods for the traditional Chinese medicine Paeonia lactiflora (red peony root) are high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC). While these methods are simple to operate, they suffer from drawbacks such as long analysis times, high solvent consumption, severe inter-compound interference, high detection limits, and a limited number of detectable components. Liquid chromatography-mass spectrometry (LC-MS) is a powerful analytical technique that combines the high separation efficiency of HPLC with the high sensitivity of mass spectrometry, and is widely used for the qualitative and quantitative analysis of traditional Chinese medicine. Some studies have also used UHPLC-MS / MS for the quantification of multiple components in Paeonia lactiflora, but this still suffers from a limited number of compounds and narrow coverage, failing to meet the quality control requirements of traditional Chinese medicine for "multiple components and multiple targets." Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a UHPLC-QQQ-MS / MS method for determining the content of multiple components in Paeonia lactiflora based on MRM mode. This method can simultaneously complete the quantitative detection of 54 components in Paeonia lactiflora within 15 minutes. It has the characteristics of simple operation, strong specificity, good precision and accuracy, and solves the technical problems of existing quantitative methods for Paeonia lactiflora, such as detecting few components, long analysis time, large interference, and insufficient accuracy.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for determining the content of multiple components of Paeonia lactiflora using UHPLC-QQQ-MS / MS based on MRM mode, comprising the following steps: Red peony powder and methanol were mixed and subjected to ultrasonic extraction to obtain an extract. The extract was detected by ultra-high performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode to obtain the spectrum to be measured. The content of each component in Paeonia lactiflora is obtained based on the spectrum to be tested and the predetermined standard curve. The standard curve is obtained by linearly fitting the concentration with the peak area ratio of each component to be tested to the internal standard. The components of Paeonia lactiflora include ethyl 4-hydroxybenzoate, 2,4-dihydroxyacetophenone, 2,5-dihydroxy-4-methylacetophenone, 4-hydroxyacetophenone, salicyl alcohol, vanillic acid, gallic acid, acetoxypaeoniflorin, paeoniflorin oxidase, benzoylpaeoniflorin, 4-O-methylpaeoniflorin, 3β,4β,23-Trihydroxy-24,30-Dinorolean-12,20(29)-dien-28-oicacid, 7-O-galloylcatechin, 3-O-galloylepicatechin, Bis-8,8′-catechinylmethane, ethyl gallate, 1-ethylDL-malate, methyl gallate, (1 S , 2 S , 5 R , 6 R ) -1,8-Dihydroxypin-4-one, Taxonitrile, 7 S 8 R -Dihydrodehydrobispineol, hederonium saponin, paeoniflorin ketone, naringenin, hesperidin, sennaol, butyl gallate, paeoniflorin, Tortoside A, 9-epi-paeonidanin, 6-O-β-D-Glucopyranosyl-8-O-benzoylpaeonisuffrone, (1 S 5 R 6 R The following are one or more of the following: 8-O-Benzoylmudanpioside F, Paeonidanin, Paeoniflorin, Catechin, Protocatechuic acid, 9-O-Butylpaeoniflorin, 4-O-n-Butyloxypaeoniflorin, 3,3′,4-Trimethylellagic acid-4′-O-β-D-glucoside, Lycium barbarum, Galloylpaeoniflorin, Euphorbin C, Paeonidanin E, 9-O-Butyloxypaeonidanin, 3-Methoxy-4-hydroxyphenylacetone, Vanillin, Stigmaster-4-en-3-one, Betulinic acid, Quercetin, Spinogenin C6, 3,3',4'-Tri-O-methylellagic acid, 3-O-methylellagic acid, methyl 4-hydroxybenzoate, and 3-hydroxy-2,4-dimethoxybenzoic acid.

[0006] Preferably, the chromatographic column used in the ultra-high performance liquid chromatography-tandem mass spectrometry detection is an Agilent ZORBAX Eclipse XDB C18, 2.1 × 150 mm, 1.8 μm.

[0007] Preferably, in the ultra-high performance liquid chromatography-tandem mass spectrometry detection, mobile phase A is 0.1~0.2% formic acid water, and mobile phase B is methanol.

[0008] Preferably, the elution program in the ultra-high performance liquid chromatography-tandem mass spectrometry detection is: 0 min: mobile phase A 95 vol%, mobile phase B 5 vol%. 1 min: Mobile phase A 55 vol%, Mobile phase B 45 vol%. 10 min: Mobile phase A 5 vol%, Mobile phase B 95 vol%. 15 min: Mobile phase A 2 vol%, Mobile phase B 98 vol%.

[0009] Preferably, the column temperature in the ultra-high performance liquid chromatography-tandem mass spectrometry detection is 30~35℃.

[0010] Preferably, the elution time in the ultra-high performance liquid chromatography-tandem mass spectrometry detection is 15 min.

[0011] Preferably, the mass spectrometry parameters detected by ultra-high performance liquid chromatography-tandem mass spectrometry include: Scanning mode: Negative ion mode; Ion source temperature: 450~550℃; Ion spray voltage: 3500~4500V.

[0012] Preferably, the mass ratio of the peony root powder to the volume ratio of methanol is (0.1~0.5)g:(10.0~50.0)mL.

[0013] Preferably, the ultrasonic extraction power is 200~300W, the frequency is 30~50kHz, and the time is 30~60min.

[0014] Preferably, the ultrasonic extraction is performed at room temperature.

[0015] This invention provides a method for determining the content of multiple components of Paeonia lactiflora using UHPLC-QQQ-MS / MS based on MRM mode, comprising the following steps: Red peony powder and methanol were mixed and subjected to ultrasonic extraction to obtain an extract. The extract was detected by ultra-high performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode to obtain the spectrum to be measured. The content of each component in Paeonia lactiflora is obtained based on the spectrum to be tested and the predetermined standard curve. The standard curve is obtained by linearly fitting the concentration with the peak area ratio of each component to be tested to the internal standard. The components of Paeonia lactiflora include ethyl 4-hydroxybenzoate, 2,4-dihydroxyacetophenone, 2,5-dihydroxy-4-methylacetophenone, 4-hydroxyacetophenone, salicyl alcohol, vanillic acid, gallic acid, acetoxypaeoniflorin, paeoniflorin oxidase, benzoylpaeoniflorin, 4-O-methylpaeoniflorin, 3β,4β,23-Trihydroxy-24,30-Dinorolean-12,20(29)-dien-28-oicacid, 7-O-galloylcatechin, 3-O-galloylepicatechin, Bis-8,8′-catechinylmethane, ethyl gallate, 1-ethylDL-malate, methyl gallate, (1 S , 2 S , 5 R , 6 R ) -1,8-Dihydroxypin-4-one, Taxonitrile, 7 S 8 R -Dihydrodehydrobispineol, hederonium saponin, paeoniflorin ketone, naringenin, hesperidin, sennaol, butyl gallate, paeoniflorin, Tortoside A, 9-epi-paeonidanin, 6-O-β-D-Glucopyranosyl-8-O-benzoylpaeonisuffrone, (1 S 5 R 6 R The following are one or more of the following: 8-O-Benzoylmudanpioside F, Paeonidanin, Paeoniflorin, Catechin, Protocatechuic acid, 9-O-Butylpaeoniflorin, 4-O-n-Butyloxypaeoniflorin, 3,3′,4-Trimethylellagic acid-4′-O-β-D-glucoside, Lycium barbarum, Galloylpaeoniflorin, Euphorbin C, Paeonidanin E, 9-O-Butyloxypaeonidanin, 3-Methoxy-4-hydroxyphenylacetone, Vanillin, Stigmaster-4-en-3-one, Betulinic acid, Quercetin, Spinogenin C6, 3,3',4'-Tri-O-methylellagic acid, 3-O-methylellagic acid, methyl 4-hydroxybenzoate, and 3-hydroxy-2,4-dimethoxybenzoic acid.

[0016] This invention provides a highly efficient and comprehensive method for the quantitative detection of multiple components in Paeonia lactiflora. Multiple reaction monitoring (MRM), an important data acquisition mode of UHPLC-MS / MS, effectively eliminates interference from co-eluted components during analysis by selectively monitoring specific precursor and daughter ions, thus improving the accuracy and reliability of quantification. Based on this mode, this invention establishes a UHPLC-MS / MS detection method that can simultaneously complete the quantitative analysis of 54 active components in Paeonia lactiflora within 15 minutes. Compared with existing technologies, this invention has advantages such as high detection efficiency, low solvent consumption, and minimal interference. The detected components cover dozens of monoterpenoid glycosides and phenolic acids, enabling a more comprehensive and integrated evaluation of the quality of Paeonia lactiflora and providing strong technical support for its quality control. Attached Figure Description

[0017] Figure 1 UHPLC-MRM chromatograms of blank solvent (A), analyte solution (B), and peony sample solution (C). Detailed Implementation

[0018] This invention provides a method for determining the content of multiple components of Paeonia lactiflora using UHPLC-QQQ-MS / MS based on MRM mode, comprising the following steps: Red peony powder and methanol were mixed and subjected to ultrasonic extraction to obtain an extract. The extract was subjected to ultra-high performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring (MRM) mode to obtain the spectral data. The content of each component in Paeonia lactiflora is obtained based on the spectrum to be tested and the predetermined standard curve. The standard curve is obtained by linearly fitting the concentration to the peak area ratio of each component to be tested and the internal standard.

[0019] The components of Paeonia lactiflora include ethyl 4-hydroxybenzoate, 2,4-dihydroxyacetophenone, 2,5-dihydroxy-4-methylacetophenone, 4-hydroxyacetophenone, salicyl alcohol, vanillic acid, gallic acid, acetoxypaeoniflorin, paeoniflorin oxidase, benzoylpaeoniflorin, 4-O-methylpaeoniflorin, 3β,4β,23-Trihydroxy-24,30-Dinorolean-12,20(29)-dien-28-oicacid, 7-O-galloylcatechin, 3-O-galloylepicatechin, Bis-8,8′-catechinylmethane, ethyl gallate, 1-ethylDL-malate, methyl gallate, (1 S , 2 S , 5 R , 6 R) -1,8-Dihydroxypin-4-one, Taxonitrile, 7 S 8 R -Dihydrodehydrobispineol, hederonium saponin, paeoniflorin ketone, naringenin, hesperidin, sennaol, butyl gallate, paeoniflorin, Tortoside A, 9-epi-paeonidanin, 6-O-β-D-Glucopyranosyl-8-O-benzoylpaeonisuffrone, (1 S 5 R 6 R The following are one or more of the following: 8-O-Benzoylmudanpioside F, Paeonidanin, Paeoniflorin, Catechin, Protocatechuic acid, 9-O-Butylpaeoniflorin, 4-O-n-Butyloxypaeoniflorin, 3,3′,4-Trimethylellagic acid-4′-O-β-D-glucoside, Lycium barbarum, Galloylpaeoniflorin, Euphorbin C, Paeonidanin E, 9-O-Butyloxypaeonidanin, 3-Methoxy-4-hydroxyphenylacetone, Vanillin, Stigmaster-4-en-3-one, Betulinic acid, Quercetin, Spinogenin C6, 3,3',4'-Tri-O-methylellagic acid, 3-O-methylellagic acid, methyl 4-hydroxybenzoate, and 3-hydroxy-2,4-dimethoxybenzoic acid.

[0020] Unless otherwise specified, the present invention does not have special requirements on the source of raw materials used, and commercially available products well known to those skilled in the art can be used.

[0021] This invention involves mixing red peony powder and methanol, followed by ultrasonic extraction to obtain an extract.

[0022] In one embodiment, the peony root powder is obtained by pulverizing and sieving peony root; the sieve used for sieving has an aperture of 80-100 mesh, specifically 100 mesh in this embodiment; the methanol is pure methanol; the mass ratio of the peony root powder to the volume of methanol is (0.1-0.5) g : (10.0-50.0) mL, specifically 0.1 g : 10.0 mL in this embodiment; the ultrasonic extraction power is 200-300 W, specifically 250 W in this embodiment, the frequency is 30-50 kHz, specifically 40 kHz in this embodiment, and the time is 30-60 min, specifically 30 min in this embodiment; the ultrasonic extraction is performed at room temperature.

[0023] In one embodiment, after ultrasonic extraction, the method further includes: separating the solid and liquid components of the ultrasonically extracted liquid, filtering it, and obtaining the extract; the solid-liquid separation is performed by centrifugation; the centrifugation speed is 8000~14000 rpm, specifically 14000 rpm, and the time is 10~15 min, specifically 10 min; the filter membrane used for filtration has a pore size of 0.22~0.45µm, specifically 0.22µm, and is made of nylon.

[0024] As one implementation method, before performing ultra-high performance liquid chromatography-tandem mass spectrometry detection, the method further includes: diluting the extract with an internal standard solution and storing it in a refrigerator at 4°C for later use; the internal standard solution is a mixture of gentic acid and 6'-O-β-D-glucosylgentiopicrin; the extract is diluted by a factor of 2 to 5 times, and in a specific embodiment, it is diluted by a factor of 2 times.

[0025] After obtaining the extract, the present invention performs ultra-high performance liquid chromatography-tandem mass spectrometry on the extract in multiple reaction monitoring mode to obtain the spectrum to be tested; The content of each component in Paeonia lactiflora is obtained based on the spectrum to be tested and the predetermined standard curve. The standard curve is obtained by linearly fitting the concentration to the peak area ratio of each component to be tested and the internal standard.

[0026] As one implementation method, the liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection conditions include: Column: Agilent ZORBAX Eclipse XDB C 18 (2.1×150 mm, 1.8 μm); Mobile phase A: 0.1~0.2% (v / v) formic acid water, specifically 0.1% (v / v) formic acid water in the embodiment; Mobile phase B: Methanol; Column temperature: 30~35℃, 30℃ in the specific embodiment; Flow rate: 0.2~0.4 mL / min, 0.3 mL / min in the specific embodiment; Injection volume: 2~4µL, 2µL in the specific example; Washing time: 15 min; Balancing time: 5~10 min, 5 min in the specific embodiment; Elution program: 0 min: Mobile phase A 95 vol%, Mobile phase B 5 vol%. 1 min: Mobile phase A 55 vol%, Mobile phase B 45 vol%. 10 min: Mobile phase A 5 vol%, Mobile phase B 95 vol%. 15 min: Mobile phase A 2 vol%, Mobile phase B 98 vol%.

[0027] The elution gradient is shown in Table 1.

[0028] Table 1 UHPLC elution procedure

[0029] As one implementation method, the mass spectrometry parameters detected by the ultra-high performance liquid chromatography-tandem mass spectrometry include: Scanning mode: Negative ion mode; Instrument parameters: Source temperature (TEM): 450~550℃, 450℃ in the specific embodiment; Ion spray voltage (ISV): 3500~4500V, 4500V in the specific embodiment; Nebulizer gas (GS1): 40~45psi, 45psi in the specific embodiment; Heater gas (GS2): 40~45psi, 45psi in the specific embodiment; Curtain gas (CUR): 10~20psi, 10psi in the specific embodiment; Collision gas (CAD): 5~10psi, 5psi in the specific embodiment; The MRM parameters of the compounds are shown in Table 2.

[0030] Table 2. MRM parameters for target compound and internal standard

[0031] Compound 19 is described in Chinese Patent CN 105481670 A, a pinane-type monoterpene compound and its preparation method and application; Compound 42 is found in the literature Wang, An Hua, et al. "Phenolic glycosides and monoterpenoids from the roots of Euphorbia ebracteolata and their bioactivities." Fitoterapia 121(2017):175-182; Compound 44 can be found in the literature Ding, et al. "New monoterpene glycosides from Paeoniasuffruticosa Andrews and their inhibition on NO production in LPS-induced RAW264.7 cells." Bioorganic and Medicinal Chemistry Letters (2012):7243-7247; Compound 50 is found in the literature Miyakoshi, and M. "3α-hydroxy-oleanene type triterpeneglycosyl esters from leaves of Acanthopanax spinosus." Phytochemistry, Vol.46, No.7, PP.1255-1259 (1997).

[0032] As one implementation method, the preparation method of the standard curve includes the following steps: (1) Preparation of standard solutions; (2) Preparation of mixed standard solutions; (3) Preparation of standard curve solution; (4) The standard curve solution is detected by ultra-high performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode to obtain the spectrum to be tested; based on the peak area and corresponding concentration of each standard in the spectrum to be tested, a linear relationship curve is plotted to obtain the standard curve.

[0033] As one implementation method, the specific process for preparing the standard solution is as follows: accurately weigh appropriate amounts of each standard, add pure methanol to prepare standard stock solutions with a concentration of 1.00 mg / mL.

[0034] As one implementation method, the specific process for preparing the mixed standard solution is as follows: Accurately transfer appropriate amounts of each standard stock solution and prepare solutions with methanol as the solvent at concentrations of 1 μg / mL (2, 4, 11, 15, 27, 51), 0.1 μg / mL (16, 29), 0.125 μg / mL (31), 0.5 μg / mL (3, 33, 46, 53), 2.5 μg / mL (47, 48), 5 μg / mL (1, 5, 6, 8, 10, 12, 17, 20, 21, 24, 25, 26, 28, 30, 32, 36, 37, 38, 40, 42, 44, 45, 49), and 10 μg / mL (13, 23, 39, 43, 52). Mixed standard solutions of 12.5 μg / mL (54), 25 μg / mL (14, 19, 35, 50), 50 μg / mL (18, 22), 100 μg / mL (7, 9, 41), and 1 mg / mL (34).

[0035] As one implementation method, the specific process for preparing the standard curve solution is as follows: the mixed standard solution is gradually diluted with methanol as a solvent to obtain the standard curve solution.

[0036] The preparation process for the mixed standard solutions of low, medium, and high concentrations used for method validation is the same as above and will not be repeated here. The quantitative method used in this experiment is the internal standard method, which requires the addition of an internal standard compound to the Paeonia lactiflora extract for quantification. The internal standard compounds are gentic acid and 6'-O-β-D-glucosylgentiopicrin, with concentrations of 2 µg / mL and 5 µg / mL in the sample, respectively.

[0037] This invention, based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) and employing multiple reaction monitoring (MRM) mode, aims to determine the content of multiple components in Paeonia lactiflora, addressing the technical problems of existing quantitative methods for Paeonia lactiflora, such as limited component detection, long analysis time, significant interference, and insufficient accuracy. The detection method provided by this invention, through optimized gradient elution conditions, can simultaneously complete the quantitative detection of 54 components in Paeonia lactiflora within 15 minutes. It boasts advantages such as simple operation, high specificity, and good precision and accuracy. While ensuring the chromatographic peak shape and resolution of the target components, it significantly shortens the detection time, improves analytical efficiency, and provides a significantly wider coverage of detected components than existing technologies, offering a reliable technical means for the quality evaluation of Paeonia lactiflora.

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Instruments and reagents used in the embodiments of this invention: The liquid chromatography-mass spectrometry (LC-MS) system was purchased from Agilent Technologies, Inc. (USA) and Applied Biosystems Sciex, Inc. (Canada), specifically an Agilent 1290 ultra-high performance liquid chromatograph tandem with an Applied Biosystems Sciex API 3200 triple quadrupole mass spectrometer. The high-speed refrigerated centrifuge was purchased from Eppendorf GmbH in Germany, model 5430R; The ultrasonic cleaner was purchased from Xiaomei Ultrasonic Instruments (Kunshan) Co., Ltd., model XM-P240H. The 1 / 100,000 balance was purchased from Sartorius GmbH, Germany, model MSA225P-0CE-DU; Mass spectrometry grade methanol and formic acid were purchased from Honeywell Pharmaceuticals, USA. The ultrapure water was prepared using the Milli-Q ultrapure water purification system.

[0040] Reference standards used in the embodiments of this invention: The reference standards used in the experiment were all isolated from Paeonia lactiflora by the research group in the previous stage, and were identified by infrared, mass spectrometry and nuclear magnetic resonance spectroscopy, and the purity was higher than 95%.

[0041] Gentianic acid and 6'-O-β-D-glucosylgentiopicrin were purchased from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China), both with a purity higher than 98%.

[0042] Example 1 Take an appropriate amount of each batch of Paeonia lactiflora sample, pulverize and pass through a 100-mesh sieve, accurately weigh 0.1g of each batch of sample powder, add 10.0mL of methanol, and extract by ultrasonication at 250W power and 40kHz frequency for 30min at room temperature. After centrifugation at 14000rpm for 10min, filter through a 0.22µm nylon membrane to obtain the filtrate. Then, dilute the filtrate by 2 times with an internal standard solution to obtain the test solution. All sample solutions are stored in a 4℃ refrigerator for later use before testing. The test solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) in multiple reaction monitoring mode (MRM) to determine the content of components in Paeonia lactiflora and obtain the chromatogram of the test sample. Based on the spectral data to be tested and the predetermined standard curve, the content of each component in Paeonia lactiflora was obtained. The standard curve was obtained by linearly fitting the concentration to the peak area ratio of each component to the internal standard. The HPLC conditions for ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) detection are as follows: Column type: Agilent ZORBAX Eclipse XDB C 18(2.1×150 mm, 1.8 μm); Mobile phase A: 0.1% formic acid water, Mobile phase B: methanol; Column temperature: 30℃; Flow rate: 0.3 mL / min; Injection volume: 2 µL; Elution time: 15 min; Equilibration time: 5 min; Specific elution gradient is shown in Table 1. Mass spectrometry parameters are as follows: Scan mode: negative ion mode; Instrument parameters: TEM (Source temperature), 450℃; ISV (Ionspray voltage), 4500V; GS1 (Nebulizer gas), 45psi; GS2 (Heater gas), 45psi; CUR (Curtain gas), 10psi; CAD (Collision gas), 5 psi; The MRM parameters of the compounds are shown in Table 2.

[0043] Preparation of standard solutions: Accurately weigh appropriate amounts of each standard and add methanol to prepare standard stock solutions with a concentration of 1.00 mg / mL. Accurately transfer appropriate amounts of each standard stock solution and prepare solutions with methanol as the solvent at concentrations of 1 μg / mL (2, 4, 11, 15, 27, 51), 0.1 μg / mL (16, 29), 0.125 μg / mL (31), 0.5 μg / mL (3, 33, 46, 53), 2.5 μg / mL (47, 48), 5 μg / mL (1, 5, 6, 8, 10, 12, 17, 20, 21, 24, 25, 26, 28, 30, 32, 36, 37, 38, 40, 42, 44, 45, 49), and 10 μg / mL (13, 23, 39, 43, 52). Mixed standard solutions of 12.5 μg / mL (54), 25 μg / mL (14, 19, 35, 50), 50 μg / mL (18, 22), 100 μg / mL (7, 9, 41), and 1 mg / mL (34); The standard curve solution was obtained by stepwise dilution of the above mixed standard solution with pure methanol as the solvent. The preparation process of the low, medium and high concentration mixed standard solutions used for method validation was the same as above. The internal standard compounds were gentic acid and 6'-O-β-D-glucosylgentiopicrin, and the concentrations of the two in the sample were 2 µg / mL and 5 µg / mL, respectively.

[0044] Performance testing Methodological investigation IS1 and IS2 are internal standard compounds. In the results of the spiking recovery test and sample detection, the samples were prepared using specific medicinal materials. Therefore, some compounds did not reach the detection limit and were not detected.

[0045] The established method for determining the content of multiple components in Paeonia lactiflora was subjected to methodological evaluation. The evaluation items included: specificity, linearity, limit of detection, limit of quantitation, precision, repeatability, stability, and recovery rate. All test results met the requirements.

[0046] (1) Statistical analysis All experimental data are presented as mean ± SD (Standard deviation).

[0047] (2) Exclusivity The specificity of the quantitative method was determined by comparing the chromatograms of the blank solvent, the mixed standard solution, and the peony sample solution.

[0048] The MRM spectra of the blank solvent, analyte solution (i.e., the mixed standard solution, the target compound will also be described as the analyte in this article), and the Paeonia lactiflora sample solution are as follows: Figure 1 As shown, the results indicate that the analytes are well separated and the analytical method is highly specific.

[0049] (3) Linearity, limit of detection and limit of quantitation The standard curve solution was sequentially injected into the UHPLC-MS / MS system from low to high concentration. Sample concentration is plotted on the x-axis (X), and the ratio of analyte peak area to internal standard peak area is plotted on the y-axis (Y). The linear regression equation was fitted using AppliedBiosystems Analyst 1.6.2 software. LOD (Limit of detection) and LOQ (Limit of quantification) correspond to compound concentrations with S / N (Signal-to-noise ratio) of 3 and 10, respectively.

[0050] All standard curves exhibited good linearity over a wide concentration range, with correlation coefficients (r) all greater than 0.9952. The detection limit ranged from 0.2 to 200 ng / mL, and the quantitation limit ranged from 0.65 to 665 ng / mL. Specific parameters are shown in Table 3.

[0051] Table 3. Linear parameters of the target compounds in Paeonia lactiflora

[0052] (4) Precision, repeatability and stability Six parallel determinations were performed on mixed standard solutions of low, medium, and high concentrations within one day and six times on three consecutive days to assess intra-day and inter-day precision. Six parallel determinations of Paeonia lactiflora sample solutions were prepared and analyzed, and the peak areas of each compound were recorded. The relative standard deviation (RSD) was calculated to assess repeatability. The same Paeonia lactiflora sample solution was analyzed at room temperature at 0, 2, 4, 6, 8, 10, 12, and 24 hours, and the peak areas of the compounds were recorded. The RSD was calculated to assess stability.

[0053] The intra-day and inter-day precision RSD values ​​ranged from 0.2% to 4.9% and 0.1% to 4.6%, respectively, while the accuracy ranged from 90% to 113% (Table 4). The stability RSD value ranged from 0.2% to 4.9%, and the repeatability RSD value ranged from 0.1% to 4.3% (Table 5), indicating that the established method is accurate, reliable, and stable, and can be successfully applied to the quantitative analysis of Paeonia lactiflora samples.

[0054] Table 4. Intra-day and inter-day precision studies of the target compound and internal standard.

[0055] (5) Recovery rate Six parallel samples of Paeonia lactiflora (0.1 g) were weighed and mixed with 10 mL of pure methanol containing 80%, 100%, and 120% of the analyte, respectively. Samples were then prepared according to the established procedure. Considering the linear range of some compounds, the extract was diluted 2-fold with an internal standard solution, and the content of the target compound in the sample was determined to assess the recovery rate.

[0056] The average recoveries of the target compounds ranged from 87.1% to 112.3%, indicating that the background matrix had little impact on the accuracy of the determination results and that the sample preparation method was stable and reliable (Table 5).

[0057] Table 5. Methodological parameters for target compounds and internal standards

[0058] The UHPLC-MS / MS method established in this invention was used to determine the content of four different commercial grades of Paeonia lactiflora (Table 6). A total of 37 compounds were detected. The remaining undetected compounds may be due to their extremely low content in Paeonia lactiflora, which did not reach the mass spectrometry detection limit. Among the detected target compounds, the content of each component varied considerably. The contents of paeoniflorin, gallopaeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, paeoniflorin ketone, gallic acid, catechin, and 3-O-galloepicatechin were relatively high. Among them, paeoniflorin had the highest content, ranging from 39 to 51 mg / g. The total content of the four grades of Paeonia lactiflora was ordered as follows: rhizome > small strip > medium strip > large strip. This result provides a theoretical basis for the quality control of Paeonia lactiflora.

[0059] Table 6. Determination of Paeonia lactiflora content (μg / g) in different product specifications

[0060] This invention establishes a UHPLC-MS / MS detection method based on Multiple Reaction Monitoring (MRM) mode, which can simultaneously complete the quantitative analysis of 54 active components in Paeonia lactiflora within 15 minutes. Compared with existing technologies, it has advantages such as high detection efficiency, low solvent consumption, minimal interference, broad coverage of detected components, and accurate results. It can more comprehensively evaluate the quality of Paeonia lactiflora and provide strong technical support for the quality control of Paeonia lactiflora.

[0061] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for determining the content of multiple components of Paeonia lactiflora using UHPLC-QQQ-MS / MS based on MRM mode, characterized in that, Includes the following steps: Red peony powder and methanol were mixed and subjected to ultrasonic extraction to obtain an extract. The extract was detected by ultra-high performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode to obtain the spectrum to be measured. The content of each component in Paeonia lactiflora is obtained based on the spectrum to be tested and the predetermined standard curve. The standard curve is obtained by linearly fitting the concentration with the peak area ratio of each component to be tested to the internal standard. The components of Paeonia lactiflora include ethyl 4-hydroxybenzoate, 2,4-dihydroxyacetophenone, 2,5-dihydroxy-4-methylacetophenone, 4-hydroxyacetophenone, salicyl alcohol, vanillic acid, gallic acid, acetoxypaeoniflorin, paeoniflorin oxidase, benzoylpaeoniflorin, 4-O-methylpaeoniflorin, 3β,4β,23-Trihydroxy-24,30-Dinorolean-12,20(29)-dien-28-oicacid, 7-O-galloylcatechin, 3-O-galloylepicatechin, Bis-8,8′-catechinylmethane, ethyl gallate, 1-ethylDL-malate, methyl gallate, (1 S , 2 S , 5 R , 6 R ) -1,8-Dihydroxypin-4-one, Taxonitrile, 7 S 8 R -Dihydrodehydrobispineol, hederonium saponin, paeoniflorin ketone, naringenin, hesperidin, sennaol, butyl gallate, paeoniflorin, Tortoside A, 9-epi-paeonidanin, 6-O-β-D-Glucopyranosyl-8-O-benzoylpaeonisuffrone, (1 S 5 R 6 R The following are one or more of the following: 8-O-Benzoylmudanpioside F, Paeonidanin, Paeoniflorin, Catechin, Protocatechuic acid, 9-O-Butylpaeoniflorin, 4-O-n-Butyloxypaeoniflorin, 3,3′,4-Trimethylellagic acid-4′-O-β-D-glucoside, Lycium barbarum, Galloylpaeoniflorin, Euphorbin C, Paeonidanin E, 9-O-Butyloxypaeonidanin, 3-Methoxy-4-hydroxyphenylacetone, Vanillin, Stigmaster-4-en-3-one, Betulinic acid, Quercetin, Spinogenin C6, 3,3',4'-Tri-O-methylellagic acid, 3-O-methylellagic acid, methyl 4-hydroxybenzoate, and 3-hydroxy-2,4-dimethoxybenzoic acid.

2. The determination method according to claim 1, characterized in that, The chromatographic column used in the ultra-high performance liquid chromatography-tandem mass spectrometry detection was an Agilent ZORBAX Eclipse XDB C18, 2.1 × 150 mm, 1.8 μm.

3. The determination method according to claim 1 or 2, characterized in that, In the ultra-high performance liquid chromatography-tandem mass spectrometry detection, mobile phase A is 0.1~0.2% formic acid in water, and mobile phase B is methanol.

4. The determination method according to claim 1 or 2, characterized in that, The elution program for ultra-high performance liquid chromatography-tandem mass spectrometry detection is as follows: 0 min: mobile phase A 95 vol%, mobile phase B 5 vol%. 1 min: Mobile phase A 55 vol%, Mobile phase B 45 vol%. 10 min: Mobile phase A 5 vol%, Mobile phase B 95 vol%. 15 min: Mobile phase A 2 vol%, Mobile phase B 98 vol%.

5. The determination method according to claim 1, characterized in that, The column temperature in the ultra-high performance liquid chromatography-tandem mass spectrometry detection is 30~35℃.

6. The determination method according to claim 1, characterized in that, The elution time in the ultra-high performance liquid chromatography-tandem mass spectrometry detection is 15 min.

7. The determination method according to claim 1, characterized in that, The mass spectrometry parameters detected by ultra-high performance liquid chromatography-tandem mass spectrometry include: Scanning mode: Negative ion mode; Ion source temperature: 450~550℃; Ion spray voltage: 3500~4500V.

8. The determination method according to claim 1, characterized in that, The mass ratio of the peony root powder to the volume ratio of methanol is (0.1~0.5)g:(10.0~50.0)mL.

9. The determination method according to claim 1, characterized in that, The ultrasonic extraction power is 200~300W, the frequency is 30~50kHz, and the time is 30~60min.

10. The determination method according to claim 1 or 9, characterized in that, The ultrasonic extraction was performed at room temperature.

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