Nuclear magnetic method for simultaneously quantifying four types of monophthalide components in ligusticum wallichii based on HSQC spectrogram technology

Abstract

The invention provides a nuclear magnetic method for simultaneously quantifying four types of monophthalide components in ligusticum wallichii based on an HSQC spectrogram technology. The nuclear magnetic method comprises the following steps: 1) preparing an internal standard solution; (2) preparing a test solution; 3) detecting; 4) quantifying; the method disclosed by the invention has the advantages of high sampling efficiency, high accuracy and good repeatability, breaks through the limitation that one type of component is quantified by one method, realizes leap-type upgrading that four types of components are quantified simultaneously by one internal standard by one method, greatly improves the component quantification efficiency, and has good application prospects. Meanwhile, the method also has relatively good quantitative accuracy and repeatability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to an NMR method for simultaneously quantifying four monophthalide components in Ligusticum chuanxiong based on HSQC spectroscopy. Background Technology Chuanxiong is a plant of the Apiaceae family, namely Chuanxiong (Ligusticum chuanxiong). Ligusticum chuanxiong The dried rhizome of *Ligusticum chuanxiong* (Hort.) contains abundant phthalide compounds. Monophthalides, the most abundant type, constitute the important active substance basis of *Ligusticum chuanxiong*, with representative compounds including Z-ligustilide, 3-butenylphthalide, ligustilide A, and butylphthalide. Modern pharmacological studies have confirmed that phthalide compounds possess a wide range of biological activities, not only dilating blood vessels and improving microcirculation, but also exhibiting anti-inflammatory, antioxidant, neuroprotective, and tumor cell proliferation-inhibiting effects, demonstrating important value in the prevention and treatment of cardiovascular and cerebrovascular diseases and nervous system disorders.

[0002] The total content of phthalide compounds in Ligusticum chuanxiong is usually above 1%, and more than 70 such compounds have been reported so far. However, for a long time, the overall control of phthalide compounds in the quality standards of Ligusticum chuanxiong has been relatively weak. It was not until the 2025 edition of the Chinese Pharmacopoeia that the content determination of ligustilide was introduced for the first time, stipulating that its content should not be less than 0.80%. The efficacy of traditional Chinese medicine stems from the synergistic effect of multiple components and multiple targets. Measuring only one or two individual components is insufficient to fully reflect the overall quality of the medicinal material. Therefore, establishing a component analysis method that can rapidly quantify phthalide compounds in Ligusticum chuanxiong has become an urgent need to improve its quality control level and ensure clinical efficacy.

[0003] Quantitative nuclear magnetic resonance (qNMR) is a general quantitative analysis method based on the NMR signals of organic molecular structures, and it has developed rapidly in the past 20 years. Compared with traditional techniques such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), qNMR not only provides rich structural information, but also has advantages such as not requiring specific reference standards, non-destructive analysis, and absolute quantification of a class of components. Currently, the most widely used qNMR methods are mainly... 1 1H NMR quantitative nuclear magnetic resonance and 1 H- 13Two-dimensional HSQC quantitative NMR, with its wide spectral range and high signal resolution, offers significant advantages in the quantitative analysis of mixtures of traditional Chinese medicine (TCM). However, it still faces a series of technical challenges in practical applications, especially in the analysis of complex TCM systems. Firstly, the quantitative accuracy of qNMR is affected by multiple factors. TCM components are complex, and signals from different types of components may overlap, especially proton signals in the high-field region. Even using two-dimensional HSQC, signal interference cannot be completely avoided, affecting integration accuracy. Secondly, a suitable internal standard is also crucial to the accuracy of two-dimensional NMR quantitative analysis. Given the complexity of qNMR quantification, and considering the important pharmacological activity of monophthalic acid components in Ligusticum chuanxiong (Chuanxiong), representing a significant class of pharmacodynamic active components, and the fact that a dedicated and reliable qNMR quantitative method for these components has not yet been established, further challenges remain. Summary of the Invention

[0004] To address the above problems, this invention provides an NMR method for simultaneously quantifying four monophthalide components in Ligusticum chuanxiong based on HSQC spectroscopy, comprising the following steps: 1) Preparation of internal standard solution: Take magnolol and dissolve it in DMSO- d Add a relaxation reagent to step 6 and mix well to prepare an internal standard solution. 2) Preparation of the test solution: Take Ligusticum chuanxiong, extract with methanol, filter and dry the extract, dissolve the residue in the internal standard solution obtained in step 1), and the test solution is obtained. 3) Detection: The test solution was detected using a nuclear magnetic resonance (NMR) spectrometer. The NMR parameters included: relaxation delay time: 1.8 s; 4) Quantitative analysis: Acquire HSQC spectra, integrate the quantitative signal region, and calculate the content of various monophthalide components based on the integrated value; The four types of monophthalide components are BT-I to BT-IV type monophthalides: BT-I type monophthalides are a class of compounds having the structure shown in general formula I: Formula I, where R is each independently selected from hydrogen, hydroxyl or C1–C6 hydrocarbon group, m is 0~4, and R1 is C1–C6 alkyl; BT-II type monophthalides are a class of compounds having the structure shown in general formula II: Formula II, wherein R5 are each independently selected from hydrogen or hydroxyl, m is 0~4; R2 is C1–C6 alkyl; BT-III type monophthalides are a class of compounds having the structure shown in general formula III: Formula III, wherein R is each independently selected from hydrogen, hydroxyl or C1–C6 hydrocarbon group, m is 0 to 4, and R3 is C1–C6 alkyl; BT-IV type monophthalides are a class of compounds having the structure shown in general formula IV: Formula IV, wherein R5 are each independently selected from hydrogen or hydroxyl, m is 0 to 4; R4 is C1–C6 alkyl.

[0005] Further, in step 1), the concentration of magnolol in the internal standard solution is 1.50 mM, and the concentration of the relaxation reagent is 3.00 mM.

[0006] Furthermore, the relaxant is chromium acetylacetone.

[0007] Further, the extraction in step 2) is ultrasonic extraction, with a temperature not exceeding 25 ℃ and a power of 300 W.

[0008] Further, in step 2), the mass-to-volume ratio of Ligusticum chuanxiong, methanol, filtrate, and internal standard solution is 1-5 g: 25 ml: 2 mL: 400 g. µ L.

[0009] Further, the NMR parameters mentioned in step 3) also include: sampling mode 25% NUS, pulse sequence: hsqcetgpsisp2.2, temperature: 298 K, number of scans: 16; number of blank scans: 32, coupling constant: 156 Hz, gain: 101, spectral width: F2: 16.0 ppm, F1: 200.0 ppm, number of sampling points: F2: 2048, F1: 128; center frequency: O1P: 5.3 ppm, O2P: 115.0 ppm.

[0010] Further, the HSQC spectrum described in step 4) is subjected to phase correction and baseline correction using Topspin 4.1.4 software.

[0011] Further, in step 4), the quantitative signal region of BT-I type monophthalide is... d H 5.3 ~ 5.6 ppm, d C The quantitative signal region for BT-II type monophthalamide is 109.2 ~ 114.0 ppm. d H 5.9 ~ 6.0 ppm, d C The quantitative signal region for BT-III type monophthalamide is 107.8 ~ 110.8 ppm. d H 5.0 ~ 5.2 ppm, d C The quantitative signal range for BT-IV class monophthalamide is 79.3 ~ 83.7 ppm. dH 5.6 ~ 5.7 ppm, d C The quantitative signal region of magnolol is 79.4 ~ 83.6 ppm. d H 5.8 ~ 5.9 ppm, d C 136.9 ~ 140.2 ppm.

[0012] Further, the content mentioned in step 4) is calculated using the following formula: C S = N R / N S × C R × A S / A R In the formula: C S The molar concentration (mM) of the Ligusticum chuanxiong sample to be tested; N R The number of protons in the internal standard quantitative signal is 2; N S The number of protons in the quantitative signal of monophthalamides is 1; C R A is the molar concentration (mM) of the internal standard. S A represents the integrated peak volume of any one of the monophthalic acid quantitative signals from BT-I to BT-IV in the Ligusticum chuanxiong sample to be tested; R This is the integrated peak volume of the quantitative signal of the internal standard.

[0013] This invention is based on 1 H- 13 C HSQC (heteronuclear single quantum correlation) spectroscopy, through systematic organization and summarization of the NMR signals of monophthalide components in Ligusticum chuanxiong, combined with the structural framework of monophthalides, revealed that monophthalide components can be identified by the presence or absence of double bonds at the 3,8-position (…). △ 3,8 Based on whether ring A is a benzene ring, phthalides were classified into four categories: ligustilide (BT-I), butenylphthalide (BT-II), butylligustilide (BT-III), and butylphthalide (BT-IV). BT-I and BT-II can be collectively referred to as 3-enylphthalides, while BT-III and BT-IV can be collectively referred to as 3-methylenephthalides. Representative components from each of the four categories—Z-ligustilide (BT-I), 3-butenylphthalide (BT-II), ligustilide A (BT-III), and butylphthalide (BT-IV)—were selected for further study.

[0014] During the research, by screening the key parameters of HSQC qNMR, a new NMR method for simultaneously quantifying four types of monophthalic components in Ligusticum chuanxiong based on HSQC spectral technology was successfully established. This method has the advantages of high sampling efficiency, high accuracy and good repeatability. It breaks the limitation of "one method to quantify one type of component" and achieves a leapfrog upgrade of "one method and one internal standard to simultaneously quantify four types of components", which greatly improves the efficiency of component quantification, while also having good quantitative accuracy and repeatability.

[0015] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0016] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0017] Figure 1 The chemical structural formulas of magnolol and four types of monophthalide skeletons; Figure 2 Key quantitative NMR signal maps of mixed and single standards (containing magnolol and HPF) of representative components of four types of monophthalide fractions (BT-I~IV) in Ligusticum chuanxiong (left: mixed reference standard of the four types of monophthalide representative components containing internal standard; right: single standard of the four types of monophthalide reference standard and magnolol). Figure 3 For Class 4 monophthalide reference standards and Ligusticum chuanxiong samples 1 H- 13 C HSQC spectrum overlay (left image shows four types of monophthalide and chuanxiong samples) 1 H- 13 The C HSQC spectrum overlay image shows the full image on the right, which includes four types of monophthalides and chuanxiong samples. 1 H- 13 C HSQC quantitative signal region overlay map, DMSO- d 6); Figure 4 For magnolol (internal standard) and chuanxiong samples 1 H- 13 C HSQC spectrum overlay (left image shows magnolol and chuanxiong samples) 1 H- 13 The C HSQC spectrum overlay image shows the whole image, with the right image showing the magnolol and chuanxiong samples. 1 H- 13 C HSQC quantitative signal region overlay map, DMSO- d 6); Figure 5 The key quantitative signal peak of magnolol (internal standard) at different concentrations of chromium acetylacetone 1 Partial 1H NMR spectra (left image shows magnolol at different concentrations of CA) 1 ¹H NMR spectra; (a-e) represent different concentrations of CA (0, 2, 3, 4, 5 mM), respectively. 1 1H NMR spectrum; the right figure shows the QSIS of magnolol magnified to the same height and superimposed on... 1 H NMR d H Within the spectral range of 5.80 ~ 6.00 ppm, variations in the full width at half maximum (FWHM) are observed. Figure 6 Key quantitative signal peaks of four types of monophthalide reference standards at different concentrations of chromium acetylacetone 1 Partial 1H NMR spectra (A1 ~ A4 show BT-I, BT-II, BT-III, and BT-IV spectra at different concentrations of CA). 1 ¹H NMR spectra; (a-e) represent different concentrations of CA (0, 2, 3, 4, 5 mM), respectively. 1 1H NMR spectra; Figures B1 to B4 show the QSIS of BT-I, BT-II, BT-III, and BT-IV reference standards magnified to the same height and superimposed on... 1 Within the quantitative signal spectral range of the four types of monophthalides in H NMR, variations in the full width at half maximum (FWHM) are observed. Figure 7 Results of the linear study. Detailed Implementation

[0018] Example 1: NMR quantification method for four types of monophthalide components in Ligusticum chuanxiong according to the present invention. 1. Solution preparation 1.1 Chromium acetylacetone stock solution Accurately weigh chromium acetylacetone and dissolve it in DMSO- d In step 6, the solution is prepared by thorough shaking and ultrasonic dissolution to obtain a 0.1 M stock solution.

[0019] 1.2 Internal standard solution Take chromium acetylacetone stock solution and dissolve the internal standard magnolol in DMSO- d 6. The final concentrations of magnolol and chromium acetylacetone after mixing were 1.50 mM and 3.00 mM, respectively.

[0020] 1.3 Test solution Accurately weigh approximately 1.0 g of Ligusticum chuanxiong powder (using a No. 2 sieve) and transfer it to a stoppered Erlenmeyer flask. Accurately add 25 mL of methanol, weigh the flask, and sonicate at 300 W power and a water temperature not exceeding 25°C for 30 min. Cool to 20°C, weigh the flask again, and replenish the lost weight with methanol. Shake well, filter, and accurately pipette 2 mL of the filtrate. Recover the solvent under reduced pressure at 30°C until dry. Accurately add 400 mL of methanol to the residue. µ Dissolve the L internal standard solution and transfer it to a 5 mm NMR tube to obtain the final product.

[0021] 2. Testing The test solution was analyzed using a nuclear magnetic resonance (NMR) spectrometer; the HSQC NMR quantitative analysis parameters were as follows: 25% NUS sampling mode; pulse sequence: hsqcetgpsisp2.2; temperature: 298 K; number of scans (NS): 16; number of blank scans (DS): 32; relaxation delay time (… D 1): 1.8 s; Coupling constant (CNST2): 156 Hz; Gain (RG): 101; Spectral width (SW): 16.0 ppm (F2), 200.0 ppm (F1); Number of sampling points (TD): 2048 (F2), 128 (F1); Center frequency: 5.3 ppm (O1P), 115.0 ppm (O2P).

[0022] 3. Calculate the content Topspin 4.1.4 software was used to perform phase correction and baseline correction on the measured HSQC spectrum. The quantitative signal regions of the four analytes and internal standards were selected for integration, and then the contents were calculated. The four types of analytes are as follows: BT-I type monophthalides are a class of compounds having the structure shown in general formula I: Formula I, where R is each independently selected from hydrogen, hydroxyl or C1–C6 hydrocarbon group, m is 0~4, and R1 is C1–C6 alkyl; BT-II type monophthalides are a class of compounds having the structure shown in general formula II: Formula II, wherein R5 are each independently selected from hydrogen or hydroxyl, m is 0~4; R2 is C1–C6 alkyl; BT-III type monophthalides are a class of compounds having the structure shown in general formula III: Formula III, wherein R is each independently selected from hydrogen, hydroxyl or C1–C6 hydrocarbon group, m is 0 to 4, and R3 is C1–C6 alkyl; BT-IV type monophthalides are a class of compounds having the structure shown in general formula IV: Formula IV, wherein R5 are each independently selected from hydrogen or hydroxyl, m is 0 to 4; R4 is C1–C6 alkyl.

[0023] The quantitative signal region of BT-I type monophthalide is d H 5.3 ~ 5.6, d C 109.2 ~ 114.0 ppm; the quantitative signal region for BT-II type monophthalide is d H 5.9 ~ 6.0 ppm, d C 107.8 ~ 110.8 ppm; Quantitative signal region of BT-III type monophthalide d H 5.0 ~ 5.2 ppm, d C 79.3 ~ 83.7 ppm; the quantitative signal region for BT-IV class monophthalamide is d H 5.6 ~ 5.7 ppm, d C 79.4 ~ 83.6 ppm; the quantitative signal region of the internal standard magnolol is d H 5.8~5.9 ppm, d C 136.9~140.2 ppm.

[0024] The calculation formula is: C S = N R / N S × C R × A S / A R In the formula: C S The molar concentration (mM) of the Ligusticum chuanxiong sample to be tested; N R The number of protons in the internal standard quantitative signal is 2; N S The number of protons in the quantitative signal of monophthalamides is 1; C R A is the molar concentration (mM) of the internal standard. S A represents the integrated peak volume of any one of the monophthalic acid quantitative signals from BT-I to BT-IV in the Ligusticum chuanxiong sample to be tested; R This is the integrated peak volume of the quantitative signal of the internal standard.

[0025] The following experimental examples illustrate the beneficial effects of the present invention.

[0026] Experimental Example 1: Development and Application of NMR Quantification Method for Simultaneous Quantification of Four Monophthalide Components in Ligusticum chuanxiong 1.1 Instruments and Reagents Bruker Avance NEO 600 MHz NMR spectrometer (Bruker Corporation); BP221S electronic analytical balance (Sartorius GmbH, Germany); ultrapure water system (Milli-Q® reference, Millipore, USA); rotary evaporator (RE-2000A, Shanghai Xiande Experimental Instrument Co., Ltd.); multi-functional grinder (400A, Yongkang Hongtaiyang Electromechanical Co., Ltd.); NMR tube (5 mm, Shanghai Titan Technology Co., Ltd.); ultrasonic cleaner (DTC-10J, Hubei Dingtai High-Tech Co., Ltd.); Pharmacopoeia No. 2 sieve (Shaoxing Shangyu Daoxu Laboratory Instrument and Equipment Factory).

[0027] Detailed information on the samples of Ligusticum chuanxiong is shown in Table 1. Ligusticum chuanxiong lactone I (PS012579, purity: 99.56%), Ligusticum chuanxiong lactone A (PS013875, purity: 93.37%), n-butylphthalide (batch number PS020613, purity: 99.41%), and butenylphthalide (PS020962, purity: 99.00%) reference standards were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; magnolol (110729-202316, purity: 99.00%) was purchased from the National Institutes for Food and Drug Control. Chromium acetylacetone (Sigma, batch number: MKCN5034, purity: 97.00%), deuterated DMSO- d 6 (Shanghai Titan Technology Co., Ltd., batch number: P3045331); Methanol (analytical grade, Chengdu Kelon Chemical Co., Ltd.).

[0028] 1.2 Experimental solutions and conditions 1.2.1 Chromium acetylacetone stock solution Accurately weigh 72.00 mg of chromium acetylacetone reference standard and dissolve it in 2.0 mL of DMSO. d 6. Shake thoroughly and sonicate to dissolve and prepare a 0.1 M stock solution.

[0029] Table 1. Source and Origin Information of Ligusticum chuanxiong Samples 1.2.2 Internal standard solution Take an appropriate amount of chromium acetylacetone stock solution, and dissolve the internal standard magnolol in DMSO- d 6. The final concentrations of magnolol and chromium acetylacetone after mixing were approximately 1.50 mM and 3.00 mM, respectively.

[0030] 1.2.3 Standard Solution 1) Monophthalide standard solution Accurately weigh appropriate amounts of ligustilide I, ligustilide A, n-butylphthalide, and butenylphthalide, dissolve them in the prepared internal standard solution, and prepare solutions with mass concentrations of 8.400, 4.315, 2.112, and 2.045 mg·mL, respectively. -1 Prepare a mixed reference standard stock solution for later use. Take an appropriate amount of the mixed reference standard stock solution, dilute it to prepare a series of mixed standard solutions, and take 400 ml of each prepared solution. µ The L is contained within a 5 mm NMR tube and is used to investigate linearity, limit of quantitation, limit of detection, and precision.

[0031] 2) Magnolol standard relaxation solution and four types of monophthalide standard relaxation solutions Take appropriate amounts of chromium acetylacetone stock solution, magnolol, and four types of representative monophthalides to prepare magnolol and monophthalide standard relaxation solutions containing 0, 2, 3, 4, and 5 mM chromium acetylacetone, respectively. Investigate the effect of different doses of chromium acetylacetone on the quantitative signal. T The influence of 1 and half-width at half maximum (WHM).

[0032] 3) Four types of monophthalide standard sample solutions Prepare a mixed standard sample 1 (BTHB1) containing 3.00 mM chromium acetylacetone and 1.50 mM honokiol by taking an appropriate amount of internal standard solution and four types of monophthalide standard solution. D 1. Time and optimization 1 J C-H TDF1 and NS.

[0033] 1.2.4 Test solution Accurately weigh approximately 1.0 g of Ligusticum chuanxiong powder (using a No. 2 sieve) and transfer it to a stoppered Erlenmeyer flask. Accurately add 25 mL of methanol, weigh the flask, and sonicate at 300 W power and a water temperature not exceeding 25 °C for 30 min. Cool to 20 °C, weigh the flask again, and replenish the lost weight with methanol. Shake well, filter, and accurately pipette 2 mL of the filtrate. Recover the solvent under reduced pressure at 30 °C until dry. Accurately add 400 mL of methanol to the residue. m Dissolve the L internal standard solution and transfer it to a 5 mm NMR tube to obtain the final product.

[0034] 1.2.5 Experimental Methods The experiment used 25% NUS sampling; pulse sequence: hsqcetgpsisp2.2; temperature: 298 K; number of scans (NS): 16; number of blank scans (DS): 32; relaxation delay time ( D1): 1.8 s; Coupling constant (CNST2): 156 Hz; Gain (RG): 101; Spectral width (SW): 16.0 ppm (F2), 200.0 ppm (F1); Number of sampling points (TD): 2048 (F2), 128 (F1); Center frequency: 5.3 ppm (O1P), 115.0 ppm (O2P).

[0035] 1.3 Determination of HSQC qNMR Method 1.3.1 Determination of characteristic NMR quantitative signals of phthalide components in Ligusticum chuanxiong A suitable quantitative signal is key to achieving HSQC qNMR. Through systematic organization and summarization of the NMR signals of monophthalic acid components in Ligusticum chuanxiong, it was found that the monophthalic acid components in Ligusticum chuanxiong can be identified by the presence or absence of a double bond at the 3,8-position (…). △ 3,8 Based on whether ring A is a benzene ring, phthalide components are classified into four categories: ligustilide (BT-I), butenylphthalide (BT-II), butylligustilide (BT-III), and butylphthalide (BT-IV); among which BT-I has... △ 3,8 Furthermore, ring A is a non-benzene ring; BT-II class has △ 3,8 However, ring A is a benzene ring; BT-III does not contain... △ 3,8 Furthermore, ring A is a non-benzene ring; BT-IV class does not contain... △ 3,8 Furthermore, ring A is a benzene ring; BT-I and BT-II can be collectively referred to as 3-alkenylphthalides, with the key quantitative signal being the 8-alkenyl group signal; BT-III and BT-IV can be collectively referred to as 3-methylenephthalides, with the key quantitative signal being the 3-oxymethylene group. For example... Figure 1 The quantitative signal regions of the four types of monophthalide components are shown to be... d H 5.4 ~ 5.6, d C 110.0 ~ 114.0 ppm (BT-I class); d H 5.9 ~ 6.0 ppm, d C 108.5 ~ 111.4 ppm (BT-II); d H 5.0 ~ 5.2 ppm, d C 80.7 ~ 84.0 ppm (BT-III); d H 5.6 ~ 5.7 ppm, d C 80.0 ~ 83.0 ppm (BT-IV) (see Figure 2 ).

[0036] The 8' and 8' alkenyl groups in magnolol are the key quantitative signals, and the quantitative signal region is... d H 5.8 ~ 5.9 ppm, d C 136.9 ~ 140.2 ppm (see) Figure 2 In the samples of Ligusticum chuanxiong 1 H- 13 In the C HSQC spectrum, it can be found that the key quantitative signals of the four types of monophthalide components are not affected by other signals (see...). Figure 3 (This can be used for NMR quantification of phthalide components in Ligusticum chuanxiong).

[0037] 1.3.2 Coupling constant 1 J C-H Optimization and selection of internal standards The HETLOC assay was used to determine the key quantitative signals of four types of monophthalide components and internal standards. 1 J C-H Experimental methods: Pulse sequence: dipsi2etgpjcsix1; 25% NUS; Temperature: 298 K; Number of scans (NS): 16; Number of blank scans (DS): 16; Relaxation delay time ( D 1): 2.0 s; Gain (RG): 101; Spectral width (SW): 16.0 ppm (F2), 16.0 ppm (F1); Number of sampling points (TD): 4096 (F2), 512 (F1); Center frequency: 5.5 ppm (O1P), 5.5 ppm (O2P).

[0038] The results revealed the key quantitative signal of ligustilide-I (BT-I class). 1 J C-H The value is 158 Hz, and the key quantitative signal of butenylphthalide (BT-II) is... 1 J C-H For the key quantitative signal of 160 Hz ligustilide-A (BT-III class) 1 J C-H For the key quantitative signal of 154 Hz n-butylphthalide (BT-IV class) 1 J C-HThe Hz value is 154 Hz. For HSQC qNMR, when selecting an internal standard, besides needing structural stability, non-reaction with solvents and samples, and ensuring that the quantitative signal does not overlap with the quantitative signal of the analyte or other signal peaks, the most important point is that the hybridization type of the internal standard and the quantitative signal of the analyte should be consistent. After screening, magnolol was determined to be an ideal choice for HSQC qNMR internal standards of the four monophthalide components of Ligusticum chuanxiong. Figure 4 As shown, the key quantitative signal of magnolol has a signal range in the HSQC spectrum of [missing information]. d H 5.8 ~ 5.9 ppm, d C 136.9 ~ 140.2 ppm, this region is similar to four types of monophthalides (such as... Figure 2 (as shown) and Chuanxiong samples (such as) Figure 4 Other signals (shown) do not overlap; HETLOC experiments determined the signal at the 8-position alkenyl group of magnolol. 1 J C-H The value is 154 Hz, which is similar to that of the four types of phthalide components. 1 J C-H Since the values ​​are similar, magnolol was chosen as the internal standard.

[0039] In HSQC spectra, CNST2 (coupling constant) is crucial for quantitative accuracy. To determine appropriate CNST2 values, representative components from four classes of monophthalide fractions were used as samples to investigate different... 1 J C-H The quantitative accuracy of the HSQC experiment was assessed. Using the BTHB1 mixed standard sample as the research object, the hsqcetgpsisp2.2 pulse sequence was employed, with CNST2 set to 152, 154, 156, 158, and 160 Hz for measurement. The results are shown in Table 2.

[0040] Table 2. Differences 1 J C-H Effect of value on the accuracy of phthalide content of four types in BTHB1 sample As shown in Table 2, different CNST2 values ​​have a certain impact on the quantitative accuracy. When CNST2 is 156 Hz, the experimental values ​​of the content of four types of monophthalides in the BTHB1 mixed standard sample are closest to the true values. Therefore, the CNST2 parameter is determined to be 156 Hz.

[0041] 1.3.3 Relaxation delay time of HSQC qNMR method ( D 1) Determination Relaxation delay time (D 1) This is a key parameter affecting the accuracy of HSQC qNMR quantification; if it is too small... D If the maximum magnetic resonance signal cannot be detected, the quantitative error will increase, and if it is too large... D 1. This can lead to excessively long sampling times, affecting the efficiency of quantitative analysis. Furthermore, the slowest relaxing proton... T 1. The intensity of the ion will dynamically change with factors such as sample composition, ionic strength, temperature, and even NMR tube type. Therefore, for the Ligusticum chuanxiong sample system, this study used the following method to... D 1. Systematic customization and optimization were carried out: 1.3.3.1 Quantitative signals of monophthalide components and internal standards T 1. Measurement Measurement of longitudinal relaxation time T Method 1: Pulse sequence: t1ir; uniform sampling mode; temperature: 298 K; number of scans (NS): 16; number of blank scans (DS): 4; relaxation delay time ( D 1): 20 s; Gain: 33; Spectral width (SW): 16.0 ppm (F2), 2.0 ppm (F1); Number of sampling points (TD): 16384 (F2), 10 (F1); Center frequency (O1P): 5.5 ppm (F2), 5.5 ppm (F1).

[0042] Experimental results show that the key quantitative signals of four types of monophthalides, represented by ligustilide I (BT-I type), butylphthalide (BT-II type), ligustilide A (BT-III type), and n-butylphthalide (BT-IV type), are... T The values ​​were 1.411, 2.756, 2.649, 2.258, and 2.756 s, respectively; key quantitative signals of magnolol. d H At 5.8-5.9 ppm T The value is 3.353 s.

[0043] 1.3.3.2 Relaxation Delay Time D The determination of 1 Chromium acetylacetone (CA) is a commonly used relaxant that can accelerate the return of excited atomic nuclei to the ground state, thereby effectively shortening the relaxation delay time. Previous experimental results have shown that the key quantitative signal of magnolol... T 1 is the longest, in settings D At step 1, sufficient relaxation of the key quantitative signal of magnolol should be ensured. Subsequently, the effects of different concentrations of CA on the quantitative signals of four types of monophthalides and magnolol were investigated. T The effects of the 1 value and the full width at half maximum (FWHM) are shown in Table 3. Figure 5-6 .

[0044] Table 3. Key quantitative signals of chromium acetylacetone at different concentrations for internal standards and four types of monophthalides. T Influence of 1 value and full width at half maximum (FWHM) As can be seen from the data in Table 3, with the increase of CA concentration, the key quantitative signals of the four types of monophthalides... T As the I value gradually decreases, the corresponding half-peak width also gradually widens. Taking into account the quantitative signal of the reference standard... T Influenced by the I value and the full width at half maximum (FWHM), a CA concentration of 3 mM was selected, at which point magnolol... T 1 is 0.360 s. In quantitative NMR experiments, the relaxation delay time ( D 1) is a key parameter affecting the accuracy of two-dimensional NMR quantification; only D 1≥ 5 × T Only by setting 1 can the magnetization vector be completely restored to the equilibrium state. Therefore, based on the experimental results, the setting is... D 1 = 1.8 s.

[0045] 1.3.4 Determination of the number of sampling points TDF1 The quantitative accuracy and testing time of the HSQC experiment with different TDF1 values ​​were evaluated using representative components of four types of monophthalamides. Using the BTHB1 mixed standard sample as the research object, the hsqcetgpsisp2.2 pulse sequence was employed, with TDF1 values ​​of 64, 96, 128, 256, and 512 for determination. Table 4 shows that when TDF1 is 64 or 96, the relative error of the quantitative results is relatively large; when TDF1 is greater than 128, the error of the quantitative results is small, and the sampling time is shorter. Therefore, TDF1 of 128 was determined to be the optimal value.

[0046] Table 4. Effects of different numbers of sampling points on the accuracy and sampling time of BTHB1 samples. 1.3.5 Determination of the number of scans NS To optimize sampling efficiency, the BTHB1 mixed standard sample was used as the research object. The hsqcetgpsisp2.2 pulse sequence was employed, with scan counts of 8, 16, 32, and 64 to investigate the effect of the NS (neutral density) on accuracy and testing time. As shown in Table 5, the quantitative error was within 2% when the NS was 8-32, indicating that the NS had little impact on quantitative accuracy. When the number of scans was 64, the quantitative error was slightly larger, which may be due to the longer sampling time. Therefore, considering the combined effect of different NS values ​​on the accuracy of quantitative results and sampling efficiency, the NS value was determined to be 16 scans. (See Table 5).

[0047] Table 5. Effects of different scan numbers on the accuracy of BTHB1 sample quantification and sampling time. 1.3.6 Determination of Sampling Method The key parameters for the HSQC qNMR method for the four types of monophthalide components were determined as follows: the quantitative method was the internal standard method, the internal standard was magnolol (added at 1.50 mM), the relaxation reagent was chromium acetylacetone (CA, added at 3 mM), and the relaxation delay time was ( D =1.8s), coupling constant (CNST2 = 156), number of sampling points (TDF1 = 128), number of scans (NS = 16).

[0048] 1.4 Methodological Validation 1.4.1 Linear Prepare six mixed standard solutions of different concentrations according to "1.2.3" "1) 4 types of monophthalide standard solutions", taking 400 ml of each. µ L was transferred to a 5 mm NMR tube, and the HSQC spectra of each standard solution were determined using the HSQC sampling method defined in "1.3.6". (The last part, "2n", appears to be an incomplete sentence or fragment and is left untranslated.) 苯酞 / n 厚朴酚 (where n is the amount of substance of monophthalides and magnolol) The ratio is plotted on the x-axis, and the ratio of the quantitative signal volume of monophthalides to the quantitative signal volume of magnolol is plotted on the y-axis to show the linear relationship. The results show a good linear relationship, as shown in Table 6. Figure 7 .

[0049] Table 6 Results of the Linearity Study 1.4.2 Limit of Detection and Limit of Quantification The spectra obtained from the detection of four types of monophthalide mixed standard solutions (BT-Ⅰ (0.119 mM), BT-Ⅱ (0.026 mM), BT-Ⅲ (0.065 mM), and BT-Ⅳ (0.032 mM)) under section “1.2.3” were used. The signal-to-noise ratio (S / N) of the quantitative signal for BT-Ⅰ was 2.63, for BT-Ⅱ it was 2.38, for BT-Ⅲ it was 3.34, and for BT-Ⅳ it was 2.26. A S / N of 10 was used as the limit of quantitation, and a S / N of 3 was used as the limit of detection. The limits of detection and quantitation for the four types of phthalide components were obtained, and the results are shown in Table 7.

[0050] Table 7 Results of Limit of Detection and Limit of Quantification 1.4.3 Repeatability Take sample DJ-1 and prepare 6 parallel sample solutions according to the test solution preparation method under section "1.2.4". HSQC spectra were measured, and the RSD value was calculated based on the integrated peak volume ratio of the quantitative signals of phthalide components and magnolol in the sample to determine the repeatability of the method. According to the Chinese Pharmacopoeia (2020 edition) 9101 Analytical Method Validation Guidelines, if the content of the analyte in the test sample is greater than 1%, the RSD value should be less than 2%, and if the content of the analyte is less than 0.01%, the RSD should be less than 4%, to ensure the reliability of the repeatability evaluation results. The contents of BT-Ⅰ and BT-Ⅲ components in Ligusticum chuanxiong are at the 1% level, and the repeatability RSDs are both less than 2%. Since the contents of BT-Ⅱ and BT-Ⅳ components in Ligusticum chuanxiong are less than 0.01%, the measured RSD values ​​are 2.96% and 2.95%, respectively, which meet the pharmacopoeia standards. In summary, the experimental results for the four types of phthalide components show good repeatability (see Table 8).

[0051] Table 8 Repeatability Experiments 1.4.4 Stability Test Take sample DJ-1 and prepare one test solution according to the method in section "1.2.4". HSQC spectra were measured at 0, 4, 8, 16, 24, 36, and 48 h. The integrated peak volume ratios of the key quantitative signals of the four monophthalide components and the quantitative signal of magnolol in the sample were recorded, and the RSD values ​​were calculated. The results showed that components BT-I, BT-II, and BT-III exhibited good stability within 48 h, with RSDs of 1.99%, 1.60%, and 1.92%, respectively. The RSD value of component BT-IV was 3.16%. Due to the low content of component BT-IV in the sample, its relative deviation may be slightly larger, but it is still within an acceptable range. Overall, the stability of this method meets the requirements for content determination.

[0052] Table 9 Stability Test 1.4.6 Quantitative Accuracy Verification Because the content of the four monophthalide components in Ligusticum chuanxiong varies depending on factors such as origin, processing, and harvesting time, and the differences in the content of the four monophthalide components can be significant, proving only one mixing ratio of the four monophthalide components is far from sufficient. To ensure that this method can truly and accurately reflect the actual content of the four monophthalide components in the sample, multiple mixed reference standards of representative components of the four monophthalide components in different ratios were used for verification. The analysis shows that although the quantification error of the low-content BT-IV component (especially in the ratios of 100:20:20:24 and 200:5:50:6) is relatively large, its impact on the total amount of the four components is small; moreover, in most mixing ratios, the RSD values ​​of the main components BT-I and BT-III are less than 2%, and the RSD of the total amount of the four phthalides is also stable within 2%, indicating that the method has good overall accuracy.

[0053] Table 10 Accuracy Verification Experiment ① Table 11 Accuracy Verification Experiment ② 1.4.7 Sample Testing Fifteen batches of Ligusticum chuanxiong samples were prepared according to the method described in section "1.2.4". HSQC spectra of four types of monophthalide components in Ligusticum chuanxiong samples from different origins were collected according to law, and the molar content of each component was calculated using the internal standard method. Since NMR quantification yields molar content, it needs to be converted to percentage content. Therefore, the percentage content of each component in Ligusticum chuanxiong from different origins was calculated using ligustilide (MW: 190.24), butenylphthalide (MW: 188.22), ligustilide-A (MW: 192.25), and butylphthalide (MW: 190.24) as reference molar masses.

[0054] The results showed that BT-I and BT-III were the main phthalide components, with significantly higher contents than BT-II and BT-IV. Among the various origins of Ligusticum chuanxiong, the highest contents of BT-I and BT-III components were found in Leshan City, Sichuan Province, exceeding 1.45% and 0.53%, respectively. The total contents of the four types of phthalide components in Ligusticum chuanxiong from different origins were all above 1.0%, with significantly higher contents in Leshan and Shifang City, Sichuan Province, compared to other origins. All these results met the requirements for ligustilide as specified in the Chinese Pharmacopoeia (2025 edition). The results are shown in Table 12.

[0055] Table 12 Content of four types of monophthalide components in Ligusticum chuanxiong 1.5 Conclusion This invention establishes a novel rapid two-dimensional NMR quantification method for simultaneously quantifying four types of phthalide components in Ligusticum chuanxiong based on HSQC NMR technology. This method enables simultaneous and accurate quantification of these four phthalide components in Ligusticum chuanxiong, and boasts advantages such as high sampling efficiency and good repeatability. This invention is applicable to the rapid and accurate quantification of phthalide components in Ligusticum chuanxiong and its processed medicinal slices using HSQC technology.

Claims

1. An NMR method for simultaneously quantifying four monophthalide components in Ligusticum chuanxiong based on HSQC spectroscopy, characterized in that: It includes the following steps: 1) Preparation of internal standard solution: Take magnolol and dissolve it in DMSO- d Add a relaxation reagent to step 6 and mix well to prepare an internal standard solution. 2) Preparation of the test solution: Take Ligusticum chuanxiong, extract with methanol, filter and dry the extract, dissolve the residue in the internal standard solution obtained in step 1), and the test solution is obtained. 3) Detection: The test solution was detected using a nuclear magnetic resonance (NMR) spectrometer. The NMR parameters included: relaxation delay time: 1.8 s; 4) Quantitative analysis: Acquire HSQC spectra, integrate the quantitative signal region, and calculate the content of various monophthalide components based on the integrated value; The four types of monophthalide components are BT-I to BT-IV type monophthalides: BT-I type monophthalides are a class of compounds having the structure shown in general formula I: Formula I, where R is each independently selected from hydrogen, hydroxyl or C1–C6 hydrocarbon group, m is 0~4, and R1 is C1–C6 alkyl; BT-II type monophthalides are a class of compounds having the structure shown in general formula II: Formula II, wherein R5 are each independently selected from hydrogen or hydroxyl, m is 0~4; R2 is C1–C6 alkyl; BT-III type monophthalides are a class of compounds having the structure shown in general formula III: Formula III, wherein R is each independently selected from hydrogen, hydroxyl or C1–C6 hydrocarbon group, m is 0 to 4, and R3 is C1–C6 alkyl; BT-IV type monophthalides are a class of compounds having the structure shown in general formula IV: Formula IV, wherein R5 are each independently selected from hydrogen or hydroxyl, m is 0 to 4; R4 is C1–C6 alkyl.

2. The NMR method according to claim 1, characterized in that: In step 1), the concentration of magnolol in the internal standard solution is 1.50 mM, and the concentration of the relaxation reagent is 3.00 mM.

3. The NMR method according to claim 2, characterized in that: The relaxation reagent is chromium acetylacetone.

4. The NMR method according to claim 1, characterized in that: Step 2) The extraction is ultrasonic extraction, with a temperature not exceeding 25 ℃ and a power of 300 W.

5. The NMR method according to claim 1, characterized in that: Step 2) The mass-to-volume ratio of Ligusticum chuanxiong, methanol, filtrate, and internal standard solution is 1-5 g: 25 ml: 2 mL: 400 g. µ L.

6. The NMR method according to claim 1, characterized in that: Step 3) The NMR parameters also include: sampling mode 25% NUS, pulse sequence: hsqcetgpsisp2.2, temperature: 298 K, number of scans: 16; number of blank scans: 32, coupling constant: 156 Hz, gain: 101, spectral width: F2: 16.0 ppm, F1: 200.0 ppm, number of sampling points: F2: 2048, F1: 128; center frequency: O1P: 5.3 ppm, O2P: 115.0 ppm.

7. The NMR method according to claim 1, characterized in that: Step 4) The HSQC spectrum is subjected to phase correction and baseline correction using Topspin 4.1.4 software.

8. The NMR method according to claim 1, characterized in that: Step 4) The quantitative signal region of BT-I type monophthalide in the quantitative signal region is: δ H 5.3 ~ 5.6 ppm, δ C The quantitative signal region for BT-II type monophthalamide is 109.2 ~ 114.0 ppm. δ H 5.9 ~ 6.0 ppm, δ C The quantitative signal region for BT-III type monophthalamide is 107.8 ~ 110.8 ppm. δ H 5.0 ~ 5.2 ppm, δ C The quantitative signal region for BT-IV class monophthalamide is 79.3 ~ 83.7 ppm. δ H 5.6 ~ 5.7 ppm, δ C The quantitative signal region of magnolol is 79.4 ~ 83.6 ppm. δ H 5.8 ~ 5.9 ppm, δ C 136.9 ~ 140.2ppm.

9. The NMR method according to claim 1, characterized in that: Step 4) The content is calculated using the following formula: C S = N R / N S × C R × A S / A R In the formula: C S The molar concentration (mM) of the Ligusticum chuanxiong sample to be tested; N R The number of protons in the internal standard quantitative signal is 2; N S The number of protons in the quantitative signal of monophthalamides is 1; C R A is the molar concentration (mM) of the internal standard. S A represents the integrated peak volume of any one of the monophthalic acid quantitative signals from BT-I to BT-IV in the Ligusticum chuanxiong sample to be tested; R This is the integrated peak volume of the quantitative signal of the internal standard.

Images