A gradient wavelength rapid quantitative detection method for different processing methods of rehmannia glutinosa

Abstract

The present application relates to the technical field of detection methods, and provides a gradient wavelength rapid quantitative detection method for different processing methods of Rehmannia glutinosa, which simultaneously detects the contents of 10 chemical components in different processing methods of R. glutinosa by using ultra-high performance liquid chromatography; the different processing methods of R. glutinosa include fresh R. glutinosa, freeze-dried R. glutinosa, sun-dried R. glutinosa, oven-dried R. glutinosa and cooked R. glutinosa; the chemical components include catalpol, 5-hydroxymethyl furfural, rehderioside D, rehderioside A, leucopelargonidin, strigosin C, pyroebigerosid A1, verbascoside, pyroebigerosid B1 and isoverbascoside; the gradient wavelength detection method provided by the present application can quickly, efficiently and simultaneously determine the contents of 10 components in different processing methods of R. glutinosa, and is particularly suitable for quality control of R. glutinosa, and solves the problems of complicated operation, long time consumption and single detection component in the prior art.

Description

Technical Field

[0001] This invention relates to the field of detection methods, specifically to a rapid quantitative detection method for Rehmannia glutinosa processed in different ways using gradient wavelengths. Background Technology

[0002] Rehmannia ( Rehmannia glutinosa Libosch. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is listed as a superior-grade herb. It is the fresh or dried tuberous root of a herbaceous plant in the Scrophulariaceae family. It is a traditional and widely used Chinese medicinal material with a long history of medicinal application and diverse processing methods. It mainly contains iridoid glycosides, polysaccharides, and amino acids. Depending on the processing method, Rehmannia glutinosa can be divided into fresh Rehmannia glutinosa, raw Rehmannia glutinosa, and processed Rehmannia glutinosa, with significant differences in their chemical composition and pharmacological effects. Fresh Rehmannia glutinosa is cold in nature and sweet and bitter in taste. Its functions include clearing heat and cooling blood, promoting body fluid production and moistening dryness. It is often used for febrile diseases with yin deficiency, red tongue, and thirst. Raw Rehmannia glutinosa is cold in nature and sweet in taste. Its functions include clearing heat and cooling blood, nourishing yin and promoting body fluid production. It is the dried form of fresh Rehmannia glutinosa, with similar effects but slightly milder coldness. Processed Rehmannia glutinosa is slightly warm in nature and sweet in taste. Its functions include nourishing yin and replenishing blood, benefiting essence and filling marrow. It is made by stewing or steaming raw Rehmannia glutinosa with wine, changing its medicinal properties from cold to warm and its effects from clearing to replenishing. Current research on the chemical components of Rehmannia glutinosa mainly focuses on HPLC comparisons between raw and processed Rehmannia glutinosa, which has limitations such as incomplete coverage, simplistic methods, and insufficient depth. Furthermore, the quality control of Rehmannia glutinosa remains unclear. The determination of the content of Chinese medicinal materials is of great practical significance for increasing the quality control of Chinese medicinal materials, improving the overall level of the Chinese medicine industry, and enabling Chinese medicine to go global. Establishing methods for determining the content of effective components is beneficial for comprehensively understanding the quality of Rehmannia glutinosa.

[0003] Existing methods often suffer from problems such as cumbersome operation, long time consumption, and limited detection of single components, so a new detection method needs to be developed. Summary of the Invention

[0004] To address the above technical problems, this invention provides a rapid quantitative detection method for Rehmannia glutinosa processed in different ways using gradient wavelengths. The gradient wavelength detection method provided by this invention can quickly and efficiently determine the content of 10 components in Rehmannia glutinosa processed in different ways, and is particularly suitable for the quality control of Rehmannia glutinosa. It solves the problems of cumbersome operation, long time consumption and single component detection in existing methods.

[0005] The specific technical solution of the present invention is as follows: According to one aspect of the present invention, the present invention provides a rapid quantitative detection method for Rehmannia glutinosa processed by different methods using gradient wavelengths, which utilizes ultra-high performance liquid chromatography to simultaneously detect the content of 10 chemical components in Rehmannia glutinosa processed by different methods; the Rehmannia glutinosa processed by different methods includes fresh Rehmannia glutinosa, freeze-dried Rehmannia glutinosa, sun-dried Rehmannia glutinosa, oven-dried Rehmannia glutinosa, and processed Rehmannia glutinosa.

[0006] In the above technical solution, the chemical components include catalpol, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalis leaf glycoside C, pyrodiclofenac phenylethanol glycoside A1, verbascoside, pyrodiclofenac phenylethanol glycoside B1, and isoruascoside.

[0007] In the above technical solution, during the ultra-high performance liquid chromatography (UHPLC) detection, gradient elution is performed using acetonitrile as mobile phase A and a 0.05%~0.15% (v / v) aqueous phosphoric acid solution as mobile phase B; the specific procedure for the gradient elution is as follows: From 0 to 5 minutes, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%. Within 5-7 minutes, the volume percentage of mobile phase A increases from 0% to 3%, while the volume percentage of mobile phase B decreases from 100% to 97%. Within 7-11 minutes, the volume percentage of mobile phase A increased from 3% to 5%, while the volume percentage of mobile phase B decreased from 97% to 95%. Within 11-16 minutes, the volume percentage of mobile phase A increased from 5% to 12%, while the volume percentage of mobile phase B decreased from 95% to 88%. Over 16-26 minutes, the volume percentage of mobile phase A increased from 12% to 20%, while the volume percentage of mobile phase B decreased from 88% to 80%. During 26-35 minutes, the volume percentage of mobile phase A increased from 20% to 30%, while the volume percentage of mobile phase B decreased from 80% to 70%.

[0008] In the above technical solution, the ultra-high performance liquid chromatography (UHPLC) method employs a gradient detection wavelength, specifically: 0~5min, detection wavelength is 210nm; 5~9 minutes, detection wavelength is 284nm; The detection time was 9~19.6 min, with a detection wavelength of 210 nm. The detection time was 19.6~35 min, and the detection wavelength was 330 nm.

[0009] In the above technical solution, when using ultra-high performance liquid chromatography for detection, octadecylsilane-bonded silica gel is used as the packing material, the flow rate is 0.25~0.35mL / min, and the column temperature is 25~35℃.

[0010] The above technical solution includes the following steps for the rapid quantitative detection of Rehmannia glutinosa processed using gradient wavelengths: Preparation of the test solution: Mix Rehmannia glutinosa powder and solvent, filter, and take the filtrate to obtain the test solution; Preparation of reference solution: Take the reference standard and solvent to prepare a mixed solution containing catalpol, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalis leaf glycoside C, pyrodiola phenylethanol glycoside A1, verbascoside, pyrodiola phenylethanol glycoside B1 and isoruascoside as the reference solution. The test solution and the reference solution were analyzed by ultra-high performance liquid chromatography to detect the contents of ziziphus jujuba var. spinosa, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalisin C, pyrodiclofenac phenylethanol glycoside A1, verbascoside, pyrodiclofenac phenylethanol glycoside B1, and isoruascoside.

[0011] In the above technical solution, when the test solution and the reference solution are prepared, the solvents each independently include methanol with a volume fraction of 25% to 100%.

[0012] Preferably, the solvent is methanol with a volume fraction of 25%.

[0013] In the above technical solutions, the mixing method includes ultrasound or reflux; Preferably, the mixing is performed by ultrasonic mixing at a power of 400W and a frequency of 50kHz for 30-60 minutes.

[0014] In the above technical solution, when preparing the test solution, the mass-volume ratio of Rehmannia glutinosa powder to solvent is 1g:20~30mL.

[0015] Compared with existing technologies, this invention provides a rapid quantitative detection method for Rehmannia glutinosa processed using different methods with gradient wavelengths. First, the content of 10 components in 45 batches of Rehmannia glutinosa processed using different methods (fresh, freeze-dried, sun-dried, oven-dried, and steamed) was measured. Different processing methods significantly affected the components in Rehmannia glutinosa. 5-hydroxymethylfurfural (5-HMF) was not detected in fresh, freeze-dried, and oven-dried Rehmannia glutinosa, while a small amount was detected in sun-dried Rehmannia glutinosa. The content of 5-HMF increased sharply in processed Rehmannia glutinosa. With prolonged heating time and increased heating temperature, the content of components such as catalpol gradually decreased, and verbascoside and isoverascoside were not even detected in processed Rehmannia glutinosa. Therefore, catalpol and 5-HMF can be used as key indicator components to effectively distinguish Rehmannia glutinosa products obtained through different processing methods. Fresh Rehmannia glutinosa has the highest content of catalpol and does not contain 5-hydroxymethylfurfural. As the processing progresses, the content of catalpol in raw and processed Rehmannia glutinosa decreases while the content of 5-hydroxymethylfurfural increases. This invention can provide strong technical support for the standardized production of processed Rehmannia glutinosa products, rational clinical use, and intellectual property protection.

[0016] Secondly, this invention establishes a rapid method for determining the chemical composition of Rehmannia glutinosa using gradient wavelength-UPLC. The method is simple and fast, providing a scientific basis for comprehensively improving the quality control standards of Rehmannia glutinosa. Attached Figure Description

[0017] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0018] Figure 1 This is the chromatogram of Rehmannia glutinosa used as a test sample in Example 1 of the present invention. In the figure, I is the chromatogram of the mixed reference standard, and II is the chromatogram of the Rehmannia glutinosa sample (batch number PFM250721-1). Peak 1 is catalpol, peak 2 is 5-hydroxymethylfurfural, peak 3 is rehmannia glycoside D, peak 4 is rehmannia glycoside A, peak 5 is leonurin, peak 6 is digitalis leaf glycoside C, peak 7 is pyrodiclofenac phenylethanol glycoside A1, peak 8 is verbascoside, peak 9 is pyrodiclofenac phenylethanol glycoside B1, and peak 10 is isoruascoside. Figure 2 The following are chromatograms of Rehmannia glutinosa processed in different ways as test samples in Example 1 of the present invention. In the diagram, A represents sun-dried Rehmannia glutinosa, B represents fresh Rehmannia glutinosa, C represents processed Rehmannia glutinosa, D represents oven-dried Rehmannia glutinosa, and E represents freeze-dried Rehmannia glutinosa. Peak 1 is catalpol, peak 2 is 5-hydroxymethylfurfural, peak 3 is rehmannia glycoside D, peak 4 is rehmannia glycoside A, peak 5 is leonurin, peak 6 is digitalis leaf glycoside C, peak 7 is pyrodiclofenac phenylethanol glycoside A1, peak 8 is verbascoside, peak 9 is pyrodiclofenac phenylethanol glycoside B1, and peak 10 is isoruascoside. Figure 3 This is the maximum absorption spectrum of catalpol in the Rehmannia glutinosa component of Example 1 of the present invention; Figure 4 This is the maximum absorption spectrum of 5-hydroxymethylfurfural in the Rehmannia glutinosa component of Example 1 of the present invention; Figure 5 This is the maximum absorption spectrum of rehmannia glycoside D in the rehmannia component of Example 1 of the present invention; Figure 6 This is the maximum absorption spectrum of rehmannia glycoside A in the rehmannia component of Example 1 of the present invention; Figure 7 This is the maximum absorption spectrum of leonurin in the Rehmannia glutinosa component of Example 1 of the present invention; Figure 8 This is the maximum absorption spectrum of digitoxin C in the Rehmannia component of Example 1 of the present invention; Figure 9 This is the maximum absorption spectrum of pyrophoric acid glycoside A1 in the Rehmannia component of Example 1 of the present invention; Figure 10 This is the maximum absorption spectrum of verbascoside in the Rehmannia glutinosa component of Example 1 of the present invention; Figure 11 This is the maximum absorption spectrum of pyrophoric acid glycoside B1 in the Rehmannia component of Example 1 of the present invention; Figure 12 This is the maximum absorption spectrum of isoverroside in the Rehmannia glutinosa component of Example 1 of the present invention; Figure 13 The images show chromatograms of test samples prepared with different solvents during the selection of extraction solvents in Example 1 of this invention. In the figure, peak 1 is catalpol, peak 2 is 5-hydroxymethylfurfural, peak 3 is rehmannia glycoside D, peak 4 is rehmannia glycoside A, peak 5 is leonurin, peak 6 is digitalis leaf glycoside C, peak 7 is pyrethroid phenylethanol glycoside A1, peak 8 is verbascoside, peak 9 is pyrethroid phenylethanol glycoside B1, and peak 10 is verbascoside. Figure 14 This is a comparative chromatogram of the detection methods of the present invention and the prior art. In the figure, A is the chromatogram of the detection method of Comparative Example 1, and B is the chromatogram of the detection method of Example 1 of the present invention. Peak 1 is catalpol, peak 2 is 5-hydroxymethylfurfural, peak 3 is rehmannia glycoside D, peak 4 is rehmannia glycoside A, peak 5 is leonurin, peak 6 is digitalis leaf glycoside C, peak 7 is pyrodiclofenac phenylethanol glycoside A1, peak 8 is verbascoside, peak 9 is pyrodiclofenac phenylethanol glycoside B1, and peak 10 is isoruascoside. Figure 15 This is the chromatogram of Comparative Example 2 of the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention more apparent, the invention is described in detail below. It should be understood that the invention is not limited to the description herein.

[0020] In the gradient wavelength rapid quantitative detection method for Rehmannia glutinosa processed by different methods of the present invention, ultra-high performance liquid chromatography (UHPLC) is used to simultaneously detect the contents of 10 chemical components (cattailol, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalisin C, pyrophoric acid phenylethanoid glycoside A1, verbascoside, pyrophoric acid phenylethanoid glycoside B1, and isoruascoside) in Rehmannia glutinosa processed by different methods. The different processing methods of Rehmannia glutinosa include fresh Rehmannia glutinosa, freeze-dried Rehmannia glutinosa, sun-dried Rehmannia glutinosa, oven-dried Rehmannia glutinosa, and processed Rehmannia glutinosa. In the UHPLC detection, acetonitrile is used as mobile phase A and 0.05%~0.15% (v / v) phosphoric acid aqueous solution is used as mobile phase B for gradient elution. In the ultra-high performance liquid chromatography (UHPLC) detection, the volume fraction of the phosphoric acid aqueous solution is 0.05%~0.15%, preferably 0.07%~0.11%, more preferably 0.08%~0.10%, and most preferably 0.10%.

[0021] In ultra-high performance liquid chromatography (UHPLC) detection, octadecylsilane-bonded silica gel is used as the packing material, and the flow rate is 0.25~0.35 mL / min. Within the range of 0.25~0.35 mL / min, 0.25 mL / min, 0.30 mL / min, 0.35 mL / min, etc., or the range of 0.25~0.30 mL / min, 0.30~0.35 mL / min, etc., can be selected. The column temperature is 25~35℃. Within the range of 25~35℃, 25℃, 27℃, 29℃, 31℃, 33℃, 35℃, etc., or the range of 25~27℃, 27~29℃, 29~31℃, 31~33℃, 33~35℃, etc., can be selected.

[0022] During the extraction of the test sample solution, the mixing method can be ultrasonic or reflux. Ultrasonic mixing is based on the cavitation, mechanical vibration, and thermal effects of ultrasound in the liquid, which breaks down interfacial resistance and accelerates extraction; reflux mixing achieves extraction based on the liquid's boiling point and heat transfer. Ultrasonic mixing is preferred, with ultrasonication at a power of 400W and a frequency of 50kHz for 30-60 minutes. Within the 30-60 minute range, different time values ​​can be selected, such as 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, and 60 min, with 60 minutes being the preferred value.

[0023] Example 1 1. Instruments Ultimate 3000 high performance liquid chromatograph (quaternary pump, diode array detector); Waters ACQUITY UPLC H-class Plus (quaternary pump, diode array detector); Mettler XPE26 analytical balance (0.001 mg); Mettler XS105DU analytical balance (0.01 mg); Millipore ultrapure water system (USA).

[0024] 2. Reagents and reagents Reagents: Methanol (Sinopharm Chemical Reagent Co., Ltd., batch number: 10014118, analytical grade); Acetonitrile (Thermo Fisher Scientific (China) Co., Ltd., batch number: F22M2A201, chromatographic grade); Water was ultrapure water; The detailed specifications of the reference standard are shown in Table 1 below; Table 1. Specifications of the Reference Standard

[0025] The detailed specifications of the test sample are shown in Table 2 below.

[0026] Table 2 Sample Specification Information

[0027] 3. Chromatographic conditions Octadecylsilane-bonded silica gel (Waters ACQUITY UPLC BEH C18; 1.7 μm, 2.1 × 100 mm) was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% (v / v) aqueous phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 3, with a flow rate of 0.3 mL / min; the gradient detection wavelengths are shown in Table 4; the column temperature was 30 °C. The theoretical plate number, calculated based on the catalpol peak, should not be less than 5000. Table 3 Gradient elution - mobile phase ratio

[0028] Table 4 Gradient Wavelength Switching Time

[0029] 4. Reference solution The preparation process of the reference solution includes: weighing the reference standard accurately, adding 25% (v / v) methanol aqueous solution to prepare a mixed solution containing 50 μg of catalpol, 1 μg of 5-hydroxymethylfurfural, 70 μg of rehmannia glycoside D, 24 μg of rehmannia glycoside A, 27 μg of leonurin, 12 μg of digitalis leaf glycoside C, 11 μg of pyrodiclofenac phenylethanoid glycoside A1, 12 μg of verbascoside, 11 μg of pyrodiclofenac phenylethanoid glycoside B1, and 5 μg of isoratascoside, which serves as the reference solution.

[0030] 5. Test solution The preparation process of the test solution includes: taking 1g of the test sample, accurately weighing it, placing it in a stoppered conical flask, accurately adding 25mL of a 25% methanol aqueous solution, weighing it, sonicating it at a power of 400W and a frequency of 50kHz for 60min, removing it, cooling it, replenishing the lost weight with a 25% methanol aqueous solution, shaking it well, filtering it, and taking the filtrate to obtain the test solution.

[0031] Accurately pipette 1 µL each of the reference solution and the test solution, inject them into the ultra-high performance liquid chromatograph, and measure to obtain the chromatogram.

[0032] Among them, Rehmannia glutinosa (batch number PFM250721-1) was used as the test sample and subjected to ultra-high performance liquid chromatography (UHPLC) detection according to the above procedure. The chromatogram is shown in [reference needed]. Figure 1 ; In the method for content determination established in this invention, gradient wavelengths of 210 nm, 284 nm, and 330 nm are selected as the determination wavelengths; acetonitrile-0.1% phosphoric acid is used as the mobile phase with gradient elution; and 25% methanol is preferred as the extraction solvent, with ultrasonic treatment for 60 min. The established method ensures the stability and accuracy of the effective components in Rehmannia glutinosa, and uses ultra-high performance liquid chromatography (UPLC) for rapid determination, reflecting its overall chemical characteristics. The method is simple and rapid, providing a scientific basis for comprehensively improving its quality. Furthermore, the above embodiments have undergone methodological validation according to pharmacopoeia requirements, and the results show that the method is accurate, reliable, sensitive, and specific, with all indicators meeting the requirements of quality control. This gradient wavelength determination method is applicable to the quality control of the traditional Chinese medicine Rehmannia glutinosa.

[0033] The contents of 10 components in 45 batches of Rehmannia glutinosa samples processed using different methods, including fresh, freeze-dried, sun-dried, oven-dried, and steamed, were determined according to Table 2. Chromatograms of different processing methods are shown in Table 2. Figure 2 The content results are shown in Tables 5-6.

[0034] Table 5. Results of Rehmannia glutinosa content determination (mg / g)

[0035] Table 6. Results of Rehmannia glutinosa content determination (mg / g)

[0036] The results showed that different processing methods significantly affected the components of Rehmannia glutinosa. 5-hydroxymethylfurfural (5-HMF) was not detected in fresh, freeze-dried, or oven-dried Rehmannia glutinosa, while a small amount was detected in sun-dried Rehmannia glutinosa. The amount of 5-HMF in processed Rehmannia glutinosa increased dramatically. With prolonged heating time and increased heating temperature, the content of components such as catalpol gradually decreased, and verbascoside and isoverascoside were not even detected in processed Rehmannia glutinosa. This invention establishes a rapid method for determining the chemical components of Rehmannia glutinosa using gradient wavelength-UPLC. The method is simple and fast, providing a scientific basis for comprehensively improving the quality control standards of Rehmannia glutinosa.

[0037] 6. Method Determination 6.1 Wavelength Selection Ten reference solutions were taken and their spectra were scanned in the range of 200 nm to 400 nm. The maximum absorbance of the ten reference solutions is shown in the figure. Figures 3-12 .

[0038] The results showed that citronellol, rehmannia glycoside D, rehmannia glycoside A, and leonurin had good peak shapes and good separation at 210 nm; 5-hydroxymethylfurfural had good peak shapes and good separation at 284 nm; while the remaining five reference standards (digitalis glycoside C, pyrodiclofenac phenylethanol glycoside A1, verbascoside, pyrodiclofenac phenylethanol glycoside B1, and isoruascoside) had good peak shapes and good separation at 330 nm. Therefore, the gradient wavelengths of 210 nm, 284 nm, and 330 nm were selected as the determination wavelengths for this method.

[0039] 6.2 Selection of Extraction Solvent Accurately weigh 1g of Rehmannia glutinosa powder (batch number PFM250721-1) and place it in a stoppered conical flask. Accurately add 25mL each of methanol, 75% methanol (75% methanol aqueous solution), 50% methanol (50% methanol aqueous solution), and 25% methanol (25% methanol aqueous solution). Sonicate the solution at 400W and 50kHz for 60 minutes to prepare the test solution. See the comparison chart of different solvents. Figure 13 As shown, the measurement results are shown in Table 7.

[0040] Table 7. Effects of different extraction solvents on the content of Rehmannia glutinosa components (mg / g)

[0041] The results showed that the components in Rehmannia glutinosa were well represented in 25% methanol extract. Other solvents, such as citronellol, showed poor peak shapes and lower contents of digitalis glycoside C, pyrodiclofenac phenylethanoid A1, and verbascoside. Therefore, 25% methanol was chosen as the extraction solvent.

[0042] 6.3 Selection of Extraction Method 1 g of Rehmannia glutinosa powder (batch number PFM250721-1) was accurately weighed and placed in a stoppered conical flask. 25 mL of 25% methanol aqueous solution was accurately added. The effects of ultrasound (power 400 W, frequency 50 kHz, time 60 min) and reflux (time 60 min) on the components of Rehmannia glutinosa were investigated. The extraction conditions and determination results are shown in Table 8.

[0043] Table 8. Effects of different extraction methods on the content of Rehmannia glutinosa components (mg / g)

[0044] The results showed that there was no significant difference in the content of Rehmannia glutinosa components obtained by different extraction methods. Considering simplicity and ease of operation, ultrasound was selected as the extraction method in this study.

[0045] 6.4 Selection of extraction time Accurately weigh 1g of Rehmannia glutinosa powder (batch number PFM250721-1), place it in a stoppered conical flask, and accurately add 25mL of 25% methanol aqueous solution. Ultrasonic treatment (400W power, 50kHz frequency) was then applied for 30min, 45min, and 60min to prepare the test solution. The results are shown in Table 9. Table 9. Effects of different extraction times on the content of Rehmannia glutinosa components (mg / g)

[0046] The results showed that ultrasonic treatment for 60 min resulted in better extraction; therefore, the ultrasonic treatment time was set at 60 min.

[0047] 7. Methodological Examination 7.1 Examination of Linear Relationships Weigh out 12.658 mg of ziziphus acetyl alcohol, 5.041 mg of 5-hydroxymethylfurfural, 15.065 mg of rehmannia glycoside D, 3.035 mg of rehmannia glycoside A, 17.544 mg of leonurin, 3.231 mg of digitalisin C, 3.038 mg of pyrodiclofenac phenylethanoid A1, 5.061 mg of verbascoside, 2.947 mg of pyrodiclofenac phenylethanoid B1, and 6.252 mg of isoverascoside, respectively, and prepare a series of concentration solutions with 25% (v / v) methanol aqueous solution. Inject the solutions and record the peak areas. Plot a standard curve with the peak area integral value on the ordinate and the injection amount of the reference standard on the abscissa. The results are shown in Tables 10-14.

[0048] Table 10 Results of Linearity Study

[0049] Table 11 Results of Linearity Study

[0050] Table 12 Results of Linearity Study

[0051] Table 13 Results of Linearity Study

[0052] Table 14 Results of Linearity Study

[0053] The results showed that the detection of 10 components in Rehmannia glutinosa—including catalpol, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalisin C, pyrodiclofenac phenylethanol glycoside A1, verbascoside, pyrodiclofenac phenylethanol glycoside B1, and isoruascoside—exhibited good linear relationships.

[0054] 7.2 Repeatability Test Take 1g of Rehmannia glutinosa powder (batch number PFM250721-1), accurately weigh it, and prepare the test solution according to the method of "5. Test Solution". Determine the content (mg / g) of 10 components and calculate the RSD value. The results are shown in Table 15.

[0055] Table 15 Results of repeatability tests (unit: mg / g)

[0056] The results showed that the RSD values ​​of each component were all less than 2.5%, indicating that the method had good repeatability.

[0057] 7.3 Stability Test The same test solution was injected at 0h, 1h, 2h, 4h, 8h, 12h and 24h for ultra-high performance liquid chromatography determination. The results are shown in Table 16.

[0058] Table 16 Results of the stability test of the test sample solution

[0059] The results showed that the RSD of the peak areas of catalpol, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalis leaf glycoside C, pyrodiclofenac phenylethanol glycoside A1, verbascoside, pyrodiclofenac phenylethanol glycoside B1, and isoruascoside were all less than 3%, indicating that the sample solution was stable within 24 hours.

[0060] 7.4 Accuracy Test Take 1g of Rehmannia glutinosa powder with known content (batch number PFM250721-1), take 9 portions of the powder, accurately weigh them, add high, medium and low concentration mixed reference solutions, and prepare the test solution according to the method of "5. Test Solution". Calculate the recovery rate. The results are shown in Tables 17-26. The results show that the method has good repeatability.

[0061] Table 17 Results of the catalpol recovery test

[0062] Table 18 Results of 5-Hydroxymethylfurfural Recovery Test

[0063] Table 19 Results of the recovery rate test of rehmannia glycoside D

[0064] Table 20 Results of the recovery rate test of rehmannia glycoside A

[0065] Table 21 Results of Leonurus ginsenoside recovery test

[0066] Table 22 Results of the recovery rate test of digitalis glycoside C

[0067] Table 23 Results of the recovery test of pyrethroid phenylethanoid A1

[0068] Table 24 Results of Verbascoside Recovery Test

[0069] Table 25 Results of the recovery test of pyrethroid phenylethanoid B1

[0070] Table 26 Results of the isobarbiturin recovery test

[0071] Comparative Example 1 The prior art discloses the detection of Rehmannia glutinosa characteristic spectral data, such as the patent application with patent number 202310785282.X, which describes a method for constructing and identifying characteristic spectral data of raw or processed Rehmannia glutinosa. When this prior art method is used for detection, the results are as follows: Figure 14 As shown; The comparison revealed that digitoxin C, pyrophoric acid phenylethanoid A1, verbascoside, pyrophoric acid phenylethanoid B1, and isoruascoside eluted after 14 minutes, with the maximum absorption of these five components at 330 nm. In contrast, the wavelength of the comparative scheme from 9.5 to 40 minutes was 200 nm to 220 nm, which is not the maximum absorption wavelength of these five components. Furthermore, the separation of rehmannia glycoside D, rehmannia glycoside A, and leonurin was not good, affecting the accuracy of component quantification.

[0072] Comparative Example 2 The difference between this comparative example and Example 1 is that a gradient wavelength was not used; instead, a detection wavelength of 203 nm was employed. The results are as follows: Figure 15 As shown, the results indicate that the chromatogram baseline obtained without using gradient wavelengths is not stable, and the chromatogram separation is poor.

[0073] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid quantitative detection method for Rehmannia glutinosa processed using different methods with gradient wavelengths, characterized in that, The contents of 10 chemical components in Rehmannia glutinosa processed by different methods were simultaneously determined by ultra-high performance liquid chromatography (UHPLC). The different processing methods of Rehmannia glutinosa included fresh Rehmannia glutinosa, freeze-dried Rehmannia glutinosa, sun-dried Rehmannia glutinosa, oven-dried Rehmannia glutinosa, and processed Rehmannia glutinosa.

2. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to claim 1, characterized in that, The chemical components include catalpol, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalis leaf glycoside C, pyrodiclofenac phenylethanol glycoside A1, verbascoside, pyrodiclofenac phenylethanol glycoside B1, and isoruascoside.

3. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to claim 1, characterized in that, In the ultra-high performance liquid chromatography (UHPLC) method, gradient elution is performed using acetonitrile as mobile phase A and a 0.05%–0.15% (v / v) aqueous phosphoric acid solution as mobile phase B. The specific procedure for gradient elution is as follows: From 0 to 5 minutes, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%. Within 5-7 minutes, the volume percentage of mobile phase A increases from 0% to 3%, while the volume percentage of mobile phase B decreases from 100% to 97%. Within 7-11 minutes, the volume percentage of mobile phase A increased from 3% to 5%, while the volume percentage of mobile phase B decreased from 97% to 95%. Within 11-16 minutes, the volume percentage of mobile phase A increased from 5% to 12%, while the volume percentage of mobile phase B decreased from 95% to 88%. Over 16-26 minutes, the volume percentage of mobile phase A increased from 12% to 20%, while the volume percentage of mobile phase B decreased from 88% to 80%. During 26-35 minutes, the volume percentage of mobile phase A increased from 20% to 30%, while the volume percentage of mobile phase B decreased from 80% to 70%.

4. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to claim 2, characterized in that, The ultra-high performance liquid chromatography method employs a gradient detection wavelength, specifically: 0~5min, detection wavelength is 210nm; 5~9 minutes, detection wavelength is 284nm; The detection time was 9~19.6 min, with a detection wavelength of 210 nm. The detection time was 19.6~35 min, and the detection wavelength was 330 nm.

5. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to claim 3, characterized in that, When performing the ultra-high performance liquid chromatography (UHPLC) detection, octadecylsilane-bonded silica gel is used as the packing material, and the flow rate is 0.25~0.35 mL / min.

6. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to claim 1, characterized in that, When performing the ultra-high performance liquid chromatography (UHPLC) detection, the column temperature is 25~35℃.

7. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to any one of claims 1 to 6, characterized in that, Includes the following steps: Preparation of the test solution: Mix Rehmannia glutinosa powder and solvent, filter, and take the filtrate to obtain the test solution; Preparation of reference solution: Take the reference standard and solvent to prepare a mixed solution containing catalpol, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalis leaf glycoside C, pyrodiola phenylethanol glycoside A1, verbascoside, pyrodiola phenylethanol glycoside B1 and isoruascoside as the reference solution. The test solution and the reference solution were analyzed by ultra-high performance liquid chromatography to detect the contents of ziziphus jujuba var. spinosa, 5-hydroxymethylfurfural, rehmannia glycoside D, rehmannia glycoside A, leonurin, digitalisin C, pyrodiclofenac phenylethanol glycoside A1, verbascoside, pyrodiclofenac phenylethanol glycoside B1, and isoruascoside.

8. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to claim 7, characterized in that, When preparing the test solution and the reference solution, the solvents each independently include methanol with a volume fraction of 25% to 100%. Preferably, the solvent is methanol with a volume fraction of 25%.

9. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to claim 7, characterized in that, The mixing method includes ultrasonic or reflux; Preferably, the mixing is performed by ultrasonic mixing at a power of 400W and a frequency of 50kHz for 30-60 minutes.

10. The method for rapid quantitative detection of Rehmannia glutinosa processed by different methods using gradient wavelengths according to claim 7, characterized in that, When preparing the test solution, the mass-to-volume ratio of Rehmannia glutinosa powder to solvent is 1g:20~30mL.

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