Multi-component quantitative analysis method for anti-aging tablets

Abstract

The application provides a multi-component quantitative analysis method of anti-aging tablets, acetonitrile-0.1% formic acid aqueous solution is used as a mobile phase, an HSS T3 chromatographic column is used for target compound separation, and 13 active components in the anti-aging tablets are simultaneously determined in an MRM mode by using an electrospray ionization triple quadrupole mass spectrometer, the linear range of the 13 active components is 0.4-100 ng / mL, the linear range of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 is 4-1000 ng / mL, the method has high sensitivity, strong specificity and good reproducibility, and realizes simultaneous quantitative analysis of the 13 active components in the anti-aging tablets.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a multi-component quantitative analysis method for anti-aging tablets. Background Technology

[0002] Aging has always been a perplexing problem in the medical field, a multifactorial process leading to a gradual decline in function at the cellular, tissue, and organismal levels. Traditional Chinese medicine (TCM) has a long history of anti-aging treatments and significant advantages, making it a current research hotspot in the field. This anti-aging powder is a compound TCM preparation developed based on the "Yishou Yongzhen Gao" formula from the Ming Dynasty Yongle Imperial Hospital. It possesses the effects of invigorating qi and nourishing yin, replenishing blood and promoting body fluids, and calming the mind and spirit. It is prepared from six medicinal herbs: Rehmannia glutinosa, red ginseng, Ophiopogon japonicus, Asparagus cochinchinensis, Lycium chinense root bark, and Poria cocos. The anti-aging powder contains a large amount of active small molecule components such as ginsenosides and polysaccharide components, making quality control a necessary step. Given the lack of comprehensive and rapid multi-component detection methods for anti-aging powders to date, establishing a sensitive, rapid, simple, and comprehensive quantitative analysis method that directly reflects the effective components in anti-aging powders is a trend in quality standard research and effective component control for anti-aging powders. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a multi-component quantitative analysis method for anti-aging tablets.

[0004] The technical solution adopted in this invention is:

[0005] A multi-component quantitative analysis method for anti-aging tablets is disclosed. Using an aqueous solution containing acetonitrile-formic acid as the mobile phase, target compounds are separated using a chromatographic column. Electrospray ionization triple quadrupole mass spectrometry (MRM mode) is employed to simultaneously determine the content of 13 active ingredients in the anti-aging tablets. These 13 active ingredients are verbascoside, isoverascoside, echinacoside, rehmannia glutinosa phenylethanol glycoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3.

[0006] Preferably, in the multi-component quantitative analysis method of the above-mentioned anti-aging tablets, the mobile phase is an aqueous solution containing acetonitrile-0.1% formic acid.

[0007] Preferably, in the multi-component quantitative analysis method of the above-mentioned anti-aging tablets, the chromatographic column is an HSS T3 chromatographic column.

[0008] Preferably, in the above-mentioned multi-component quantitative analysis method for anti-aging tablets, the anti-aging tablets are anti-aging tablet extract powders. The anti-aging tablet extract powders are ultrasonically extracted with 50% ethanol for 90 minutes, and the extract is centrifuged and filtered through a microporous membrane as the test solution.

[0009] Preferably, the chromatographic conditions for the multi-component quantitative analysis method of the above-mentioned anti-aging tablets using an HSS T3 column are as follows:

[0010] Chromatographic column: Waters UPLC ACQUITY HSS T3 (1.8 μm, 2.1 mm × 100 mm); mobile phase: acetonitrile (A) – 0.1% formic acid aqueous solution (B), gradient elution program, column temperature 40 °C, flow rate 0.3 mL / min, injection volume 4 μL. The gradient elution program was as follows: 0–2.5 min, 20–30% (A); 2.5–10 min, 30–60% (A); 10–10.5 min, 60–98% (A); 10.5–13 min, 98–98% (A); 13–13.5 min, 98–20% (A); 13.5–15 min, 20–20% (A).

[0011] Preferably, the mass spectrometry conditions for the multi-component quantitative analysis method of the above-mentioned anti-aging tablets using electrospray ionization triple quadrupole mass spectrometry are as follows:

[0012] The ion source is an electrospray ionization (ESI) source, employing a negative ion scanning detection mode. In negative ion mode, the capillary voltage is 2.0 kV, the cone voltage is 37 V, and the desolventizing temperature is 350 °C. In positive ion mode, the capillary voltage is 3.0 kV, the cone voltage is 30 V, and the desolventizing temperature is 350 °C. Multiple reaction monitoring (MRM) mode is used.

[0013] The beneficial effects of this invention are:

[0014] The aforementioned method for quantitative analysis of multiple components in anti-aging tablets is the first UPLC / QQQ-MS method to simultaneously determine 13 chemical components in anti-aging tablets, including verbascoside, isoverascoside, echinacoside, rehmannia glutinosa phenylethanoid A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3, taking into account both the active and characteristic components. The method's specificity, linearity, accuracy, precision, stability, and recovery rate all meet the requirements, providing a new technical approach for the simultaneous determination of multiple components in anti-aging tablets.

[0015] The multi-component quantitative analysis method described above for anti-aging tablets can simultaneously quantify 13 components in the anti-aging tablets. Among the 13 active components, the linear range of verbascoside, isoverascoside, echinacoside, pyrethroid phenylethanol glycoside A1, ginsenoside Rg2, and ginsenoside Rg3 is 0.4-100 ng / mL, and the linear range of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1 is 4-1000 ng / mL. This method has high sensitivity, strong specificity, and good reproducibility, achieving simultaneous quantitative analysis of 13 active components in anti-aging tablets. Attached Figure Description

[0016] Figure 1 This is a mass spectrum of the mixed reference solution and the test solution.

[0017] Among them, A is blank solution; B is reference solution; and C is test solution.

[0018] In the diagram: Ⅰ Verbascoside; Ⅱ Isorbascoside; Ⅲ Echinacoside; Ⅳ Rehmannia glutinosa phenylethanol glycoside A1; Ⅴ Ginsenoside Rb1; Ⅵ Ginsenoside Rb2; Ⅶ Ginsenoside Rc; Ⅷ Ginsenoside Rd; Ⅸ Ginsenoside Re; Ⅹ Ginsenoside Rf; Ⅺ Ginsenoside Rg1; Ⅻ Ginsenoside Rg2; ⅩⅢ Ginsenoside Rg3. Detailed Implementation

[0019] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Example 1

[0021] 1. Experimental apparatus

[0022] ACQUITY UPLC (Ultra-High Performance Liquid Chromatograph, Waters Corporation, USA); Waters Xevo TQ-S Triple Quadrupole Mass Spectrometer (Waters Corporation, USA); Waters UPLC ACQUITY HSS T3 (1.8μm, 2.1mm×100mm); AX–205 0.0000g electronic balance (Mettler Toledo, Switzerland); SB–5200DTS ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); VX-200 vortex mixer (Labnet International); rotary evaporator (BUCHI, Switzerland, model: R-220SE); freeze dryer (EYELA Tokyo Rika K.K., model: FDU-2100); high-speed centrifuge (Eppendorf AG, Germany, model 22331Hamburg); Milli-Q pure water system (Millipore, USA).

[0023] 2. Preparation of standard stock solutions and establishment of multiple reaction detection (MRM) scanning methods

[0024] Accurately weigh 1 mg each of the following reference standards: verbascoside, isoverascoside, echinacoside, pyrodiol phenylethanoid A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3. Dissolve each reference standard in 50% ethanol to prepare a 1 mg / mL solution. Then, gradually dilute each solution to a concentration of 2 μg / mL for verbascoside, isoverascoside, echinacoside, pyrodiol phenylethanoid A1, ginsenoside Rg2, and ginsenoside Rg3, and 20 μg / mL for ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1. Take an appropriate amount of 20 μg / mL single standard stock solution, add 50% ethanol and dilute stepwise to a final concentration of 200 ng / mL solution, and set aside for use.

[0025] The process for establishing the MRM scanning mode based on the QQQ mass spectrometer was as follows: Single standard solutions of 200 ng / mL each of verbascoside, isoverascoside, echinacoside, rehmannia glutinosa phenylethanol glycoside A1, ginsenoside Rg2, ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1 were inserted one by one into tuning port A, ensuring the syringe was as low as possible below the liquid surface. Infurion was then selected, and under ACQUITY UPLC SYSTEM, the XevoTQ-S MSDetector was used to set the molecular formula, positive and negative ion modes, file save path, injection volume, and other information. Ion pair information was then acquired. Detailed ion pair information is shown in Table 2.

[0026] 3. Preparation of working solutions for standard curves

[0027] A standard curve working solution with a linear range of 0.4-100 ng / mL was prepared by using 50% ethanol to prepare verbascoside, isoverascoside, echinacoside, pyrodiclofenac phenylethanol glycoside A1, ginsenoside Rg2, and ginsenoside Rg3. A standard curve working solution with a linear range of 4-1000 ng / mL was prepared by using ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1.

[0028] 4. Methods for extracting and preparing test samples

[0029] Accurately weigh 0.1 g of anti-aging tablet extract powder, add 10 mL of 50% ethanol, and extract ultrasonically for 90 min. Take an appropriate amount of the extract, centrifuge at 12000 g / min for 10 min, filter through a 0.22 μm microporous membrane, accurately measure 1 mL of the filtrate into a 100 mL volumetric flask, add 50% ethanol to the mark, mix well, and you will get a 100 μg / mL test solution. Take an appropriate amount of the test solution and put it into a vial for analysis.

[0030] 5. Content determination

[0031] The aforementioned detection system was used to detect the standards, and a standard curve was established for each analyte with the concentration of each analyte on the x-axis and the peak area of ​​the analyte on the y-axis.

[0032] The test solution was tested using the aforementioned detection system. Three samples were prepared in parallel for each condition and analyzed by LC-MS. The peak area of ​​each component in the test solution was determined and its content was calculated. The results are shown in Table 3.

[0033] Chromatographic conditions

[0034] Chromatographic column: Waters UPLC ACQUITY HSS T3 (1.8 μm, 2.1 mm × 100 mm); mobile phase: acetonitrile (A) – 0.1% formic acid aqueous solution (B), gradient elution program as shown in Table 1, column temperature 40℃, flow rate 0.3 mL / min, injection volume 4 μL. The gradient elution program is shown in Table 1.

[0035] Table 1 Gradient elution program

[0036]

[0037] Mass spectrometry conditions

[0038] The ion source was an electrospray ionization (ESI) source, using negative ion scanning detection mode. In negative ion mode, the capillary voltage was 2.0 kV, the cone voltage was 37 V, and the desolventizing temperature was 350 °C. In positive ion mode, the capillary voltage was 3.0 kV, the cone voltage was 30 V, and the desolventizing temperature was 350 °C. Multiple reaction monitoring (MRM) mode was used. The mass spectrometry analysis conditions for the 13 compounds are shown in Table 2.

[0039] Table 2 Mass Spectrometry Analysis Conditions

[0040]

[0041] Table 3. Content determination results

[0042]

[0043]

[0044] Example 2

[0045] Methodological Results Examination

[0046] 1. Exclusivity

[0047] Inject blank solution (50% ethanol), mixed reference solution, and test solution separately for analysis, and record their mass spectra. Figure 1 As shown.

[0048] 2. Linear relationship

[0049] Preparation of reference standard stock solution

[0050] Accurately weigh 1 mg each of the following reference standards: verbascoside, isoverascoside, echinacoside, pyrodiol phenylethanoid A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3. Dissolve each reference standard in 50% ethanol to prepare a 1 mg / mL solution. Then, gradually dilute each solution to a concentration of 2 μg / mL for verbascoside, isoverascoside, echinacoside, pyrodiol phenylethanoid A1, ginsenoside Rg2, and ginsenoside Rg3, and 20 μg / mL for ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1.

[0051] Preparation of linear working fluid

[0052] Take 50 μL of each standard dilution and add 350 μL of 50% ethanol, vortex to mix, and obtain a solution containing verbascoside, isoverascoside, echinacoside, pyrethroid phenylethanol glycoside A1, ginsenoside Rg2, ginsenoside Rg3 100 ng / mL, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1. The 1000 ng / mL mixed standard stock solution was serially diluted with 50% ethanol to obtain eight concentration points for verbascoside, isoverascoside, echinacoside, pyrethroid phenylethanol glycoside A1, ginsenoside Rg2, and ginsenoside Rg3: 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 4 ng / mL, 2 ng / mL, 0.8 ng / mL, and 0.4 ng / mL. The eight concentration points for ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1 were 1000 ng / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 40 ng / mL, 20 ng / mL, 8 ng / mL, and 4 ng / mL.

[0053] A series of mixed linear working solutions were prepared, and a standard curve was plotted with the concentration of the reference standard x (ng / mL) as the abscissa and the corresponding peak area y of each reference standard compound as the ordinate. Linear regression was performed on the standard curve to obtain the linear regression equation and correlation coefficient. The results are shown in Table 4.

[0054] Table 4. Linear regression equations for 13 components of anti-aging tablets

[0055]

[0056] As shown in Table 4, verbascoside, isoverascoside, echinacoside, pyrethroid phenylethanol glycoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3 exhibit good linearity within their respective concentration ranges.

[0057] 3. Limit of Quantification and Limit of Detection

[0058] The mixed reference solution was further diluted and injected for analysis. The concentration of each component at a signal-to-noise ratio (SNR) of 10 was used as its limit of quantitation (LOQ); the concentration of each component at a SNR of 3 was used as its limit of detection (LOD). The results showed that the LOQs for verbascoside, isoverascoside, echinacoside, rehmannia glutinosa phenylethanoid A1, ginsenoside Rg2, ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, and ginsenoside Rg1 were 0.08 ng / mL, 0.08 ng / mL, 0.2 ng / mL, 0.2 ng / mL, 0.2 ng / mL, 0.2 ng / mL, 2 ng / mL, and 0.8 ng / mL, respectively. mL, 2ng / mL, 2ng / mL, 0.8ng / mL, 0.8ng / mL, 0.8ng / mL, the detection limits are 0.02ng / mL, 0.02ng / mL, 0.06ng / mL, 0.06ng / m respectively L, 0.06ng / mL, 0.06ng / mL, 0.6ng / mL, 0.24ng / mL, 0.6ng / mL, 0.6ng / mL, 0.24ng / mL, 0.24ng / mL, 0.24ng / mL.

[0059] 4. Accuracy and Precision

[0060] Take appropriate amounts of each linear working solution and dilute with 50% ethanol to prepare three quality control (QC) samples of mixed standard working solution at low, medium and high concentrations. Prepare three parallel samples and measure them continuously for three days. Analyze and determine according to the detection conditions. Calculate the measured concentration of each QC sample based on the peak area of ​​the sample using the accompanying standard curve. Calculate the intra-batch and inter-batch accuracy and precision. Accuracy is expressed as (RE%) and precision is expressed as relative standard deviation (RSD%). The results are shown in Table 5.

[0061] Table 5

[0062]

[0063]

[0064]

[0065] As shown in Table 5, the intra-day and inter-day accuracy (RE) values ​​of verbascoside, isoverascoside, echinacoside, pyrodiclofenac phenylethanol glycoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3 are all less than or equal to 4.78%, and the intra-day and inter-day precision (RSD) values ​​are all less than or equal to 7.16%, indicating that the method has good accuracy and the instrument has good precision.

[0066] 5. Stability

[0067] Take appropriate amounts of each linear working solution, dilute with 50% ethanol, and prepare three quality control (QC) samples of mixed standard working solution at low, medium and high concentrations. Place them in an autosampler at 10℃ and analyze them at 0, 2, 4, 8, 12 and 24 h according to the detection conditions. Calculate the measured concentration and RSD value of each QC sample based on the peak area of ​​the sample using the accompanying standard curve. The results are shown in Table 6.

[0068] Table 6

[0069]

[0070]

[0071]

[0072] As shown in Table 6, the RSDs of verbascoside, isoverascoside, echinacoside, pyrodiclofenac phenylethanol glycoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3 are all less than or equal to 4.75%, indicating that the stability of each test index is good within 24 hours at a sample pan temperature of 10℃.

[0073] 6. Recovery rate

[0074] Accurately weigh three portions of the anti-aging tablet extract powder with the same known content of the analyte, each approximately 50 mg. Add appropriate amounts of the reference standards of verbascoside, isoverascoside, echinacoside, pyrodiclofenac phenylethanol A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3 respectively. Prepare the test solution according to the method in section "2.1" and dilute it until each component is within the linear range. Calculate the recovery rate and its RSD value. The results are shown in Table 7 below.

[0075] Table 7

[0076]

[0077]

[0078]

[0079] As shown in Table 7, the average recoveries of verbascoside, isoverascoside, echinacoside, pyrethroid phenylethanol glycoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3 ranged from 87.12% to 97.35%, with RSD values ​​all less than or equal to 8.03%, indicating that the method has good recovery rates.

[0080] In summary, the multi-component quantitative analysis method for the aforementioned anti-aging tablets, based on UPLC / QQQ-MS technology, comprehensively considers the active and characteristic components contained in the anti-aging tablets. For the first time, a method has been established for the simultaneous detection of 13 chemical components in anti-aging tablets, including verbascoside, isoverascoside, echinacoside, rehmannia glutinosa phenylethanol glycoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2, and ginsenoside Rg3. This method enables comprehensive and rapid quality control of anti-aging tablets. Although the contents of these 13 components are different, rapid detection of these 13 components can be achieved by preparing test solutions with different dilution ratios. The methodological investigation and content determination of these 13 chemical components showed that the linearity, accuracy, precision, stability and recovery rate of each component all met the analytical method validation guidelines of the 2020 edition of the Chinese Pharmacopoeia 9101.

[0081] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for quantitative analysis of multiple components in an anti-aging tablet, characterized in that: The anti-aging tablets are prepared from six medicinal materials: Rehmannia glutinosa, red ginseng, Ophiopogon japonicus, Asparagus cochinchinensis, Lycium chinense root bark, and Poria cocos. The extract powder of the anti-aging tablets was extracted with 50% ethanol by ultrasonication, centrifuged, and filtered to obtain the test solution. The target compounds were separated using a Waters UPLC ACQUITY HSS T3 column with an acetonitrile-formic acid aqueous solution as the mobile phase. The chromatographic conditions were as follows: Column: Waters UPLC ACQUITY HSS T3 1.8 μm, 2.1 mm × 100 mm; Mobile phase A: acetonitrile, Mobile phase B: 0.1% formic acid aqueous solution, gradient elution program, column temperature 40℃, flow rate 0.3 mL / min, injection volume 4 μL; Gradient elution program: 0-2.5 min, 20-30% A; 2.5-10 min. The concentrations of 13 active ingredients in the anti-aging tablets were simultaneously determined using electrospray ionization triple quadrupole mass spectrometry in MRM mode. The mass spectrometry conditions were as follows: the ion source was an electrospray ionization source, and the positive and negative ion scanning detection modes were used. In the negative ion mode, the capillary voltage was 2. 0 kV, cone voltage 37 V, desolventizing temperature 350 °C; in positive ion mode: capillary voltage 3.0 kV, cone voltage 30 V, desolventizing temperature 350 °C, multiple reaction monitoring mode is adopted, the 13 active ingredients are verbascoside, isoverascoside, echinacoside, pyrethroid phenylethanol glycoside A1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Rg2 and ginsenoside Rg3.

2. The method for quantitative analysis of multiple components in anti-aging tablets according to claim 1, characterized in that: The anti-aging tablet is an anti-aging tablet extract powder. The anti-aging tablet extract powder is extracted with 50% ethanol by ultrasound for 90 minutes. The extract is then centrifuged and filtered through a microporous membrane as the test solution.

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