A method for detecting HPLC fingerprint of coriander seed

Abstract

This invention discloses an HPLC fingerprinting method for the detection of coriander seeds. Using uniform chromatographic conditions, it achieves simultaneous fingerprinting and evaluation, enabling rapid and accurate quality assessment. The similarity of 15 batches of coriander seed samples ranged from 0.842 to 1.000, indicating some fluctuation between batches, but overall good characteristic similarity was maintained. This invention employs gradient elution, using 0.1% phosphoric acid water as mobile phase A and acetonitrile as mobile phase B; under the same elution conditions, two components are detected simultaneously, achieving good peak separation and a stable baseline. The method involves no extraction, concentration, or evaporation, is simple, rapid, accurate, reproducible, easy to master, and highly versatile, effectively improving detection efficiency, reducing detection costs, and minimizing environmental pollution. This method has high precision and good repeatability, making it suitable for quality control of traditional Chinese medicine, market supervision, and standardized production of medicinal materials, possessing high practical value and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality testing technology, specifically involving the construction of a fingerprint spectrum of coriander seeds and the determination of their content. That is, by using a high-performance liquid chromatography (HPLC) instrument with gradient elution, chlorogenic acid and rutin in coriander seeds can be simultaneously determined and a fingerprint spectrum can be constructed using the same elution conditions and the same wavelength. Background Technology

[0002] Coriander seeds are the dried fruit of *Coriandrum Sativum* L., a plant belonging to the genus *Coriandrum* in the family Apiaceae. They are neutral in nature and have a pungent and sour taste. Coriander seeds are used to release exterior pathogens, promote rash eruption, strengthen the stomach and aid digestion, prevent spoilage and relieve pain, and suppress cough and expectorate phlegm. They are used for nasal congestion due to colds, palpitations and shortness of breath, chest and abdominal tightness, cough and wheezing, injuries from falls and blows, poor eruption of smallpox rashes, loss of appetite, and toothache. Coriander seeds are mainly used to dispel wind and regulate qi, harmonize the stomach and aid digestion, relieve abdominal pain, stomachache, expel parasites, and kill bacteria.

[0003] Current research on coriander seeds mainly focuses on volatile oil components, with limited studies on quality and fingerprint analysis. The 2016 edition of the "Jiangsu Province Standard for Traditional Chinese Medicine" only uses volatile oil content as the quality control standard, which is insufficient to reflect the overall quality of the medicinal material.

[0004] To ensure the high quality of coriander seed medicinal materials and improve the quality standards of medicinal materials, this invention aims to conduct quantitative research on chlorogenic acid and rutin in coriander seed medicinal materials and construct fingerprint spectra. Summary of the Invention

[0005] This invention, based on the aforementioned background, provides a simple and rapid pretreatment method to obtain the test solution. High-performance liquid chromatography (HPLC) with a diode array detector is used, employing a gradient elution with A (0.1% phosphoric acid)-B (acetonitrile) as the mobile phase. The elution program is as follows: 0.01-12 min, 5%-16% B; 12-41 min, 16% B; 41-56 min, 16%-40% B; 56-61 min, 40% B; 61-62 min, 40%-5% B; 62-65 min, 5% B. Simultaneously, the construction of coriander seed fingerprint chromatograms, identification of common peaks, and determination of multiple component contents are achieved.

[0006] This invention is achieved through the following technical solution: An HPLC fingerprint detection method for coriander seeds includes the following steps: 1) Screening of extraction solvent and detection wavelength This experiment used 25% methanol, 50% methanol, 70% methanol, methanol, 25% ethanol, 50% ethanol, 70% ethanol, and ethanol reagents for ultrasonic extraction of samples. It was found that the baseline was stable, the chromatographic peaks were well-shaped, and the number of peaks was high under the 70% methanol condition. Therefore, 70% methanol was chosen as the extraction solvent (see...). Figure 1The experimental sample mainly contained flavonoids and phenolic acids. A photodiode array detector was used for ultraviolet full-wavelength scanning. The results showed that at a wavelength of 254 nm, there were a variety of chromatographic peaks, and the separation of each characteristic peak was good and the peak shape was symmetrical. Therefore, 254 nm was determined as the detection wavelength.

[0007] 2) Preparation of test solution Take 1.0g of coriander seed powder (passed through a No. 4 sieve), accurately weigh it, accurately add 50mL of 70% methanol, weigh it, extract it by sonication for 30min, cool it, weigh it again, make up the weight loss with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product. 3) Preparation of reference solution Take appropriate amounts of chlorogenic acid and rutin reference standards, accurately weigh them, and add methanol to prepare a mixed reference solution containing 0.05 mg of each per 1 mL (content determination). 4) HPLC detection Chromatographic column: C18 column, Acclaim TM 120 C18 5µm 120A 4.6×250mm; Mobile phase: A (0.1% phosphoric acid) - B (acetonitrile); The elution program is as follows: 0.01-12 min, 5%-16% B; 12-41 min, 16% B; 41-56 min, 16%-40% B; 56-61 min, 40% B; 61-62 min, 40%-5% B; 62-65 min, 5% B.

[0008] Detection wavelength: 254 nm; Column temperature: 30 ℃; Flow rate: 0.8 mL / min; Injection volume: 10 μL; 5) Fingerprint pattern establishment and common peak calibration Fifteen batches of coriander seeds were analyzed. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software was used for common peak matching, similarity calculation, and generation of a control fingerprint chromatogram. Common peaks were identified (see...). Figure 2-3 ).

[0009] 6) Common Peak Identification The retention time comparison method of reference standards was used to qualitatively identify common characteristic peaks; 7) Content determination After identifying the common characteristic peaks, the two components were determined to be chlorogenic acid and rutin. An external standard curve was established, and the contents of chlorogenic acid and rutin were determined (see [link to standard curve]). Figure 4-7 (Table 1-3).

[0010] 8) Methodological Examination Under the chromatographic conditions described above, precision, repeatability, stability, and recovery of the reference standard were investigated. The feasibility of the method was evaluated by the relative retention time (RSD) of the common peaks. The results showed that the RSDs were all <3.0% (see Tables 4-8), indicating that the method has good precision and repeatability, and the samples are stable within 24 hours, meeting the requirements for fingerprint chromatogram and content determination studies.

[0011] The innovative points and beneficial effects of this invention are as follows: 1) Gradient elution is employed, using 0.1% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B. Under the same elution conditions, both components are detected simultaneously, achieving good peak separation and a stable baseline. The method involves no extraction, concentration, or evaporation; it is simple, rapid, accurate, reproducible, easy to master, and highly versatile. It effectively improves detection efficiency, reduces detection costs, and minimizes environmental pollution.

[0012] 2) Precision, repeatability and stability were tested. The RSD of the relative retention time of the common peak was less than 3.0%, which indicates that the method has good precision and repeatability. The sample is stable within 24 hours and the results are reliable, which meets the requirements of fingerprint spectroscopy research.

[0013] 3) Literature review revealed that current research on coriander seeds primarily focuses on volatile oil components, with limited studies on quality and fingerprint spectroscopy. The 2016 edition of the *Jiangsu Province Standard for Traditional Chinese Medicine* only uses volatile oil content as the quality control standard for content determination, which is insufficient to reflect the overall quality of the medicinal material. This is the first report of simultaneously determining two components (chlorogenic acid and rutin) and constructing fingerprint spectra, with a total of 22 peaks in the fingerprint spectrum. This significantly improves detection efficiency and reduces instrument costs. More importantly, it upgrades the process from one-time testing and evaluation to one-time testing and dual evaluation, achieving twice the result with half the effort.

[0014] 4) The key technology of this invention is that acetonitrile in the mobile phase has the characteristic of preserving the peak retention time of various chemical components. When combined with phosphoric acid solution, it can make the acidic components exist as molecules and eliminate the tailing of ions. Through infinitely repeated gradient ratio studies between acetonitrile and phosphoric acid of multiple concentrations, an elution condition was explored that can make multiple chemical components with large polarity differences present in the same mobile phase, and the peak retention time is appropriate, the baseline is stable, and the reproducibility is good. Attached Figure Description

[0015] Figure 1 Chromatogram of coriander seed solvent screening; Figure 2 HPLC fingerprints of 15 batches of coriander seeds; Figure 3 HPLC fingerprint of coriander seeds (reference); Figure 4Linear relationship diagram of chlorogenic acid; Figure 5 Linear relationship diagram of rutin; Figure 6 Mixed control HPLC chromatogram; Figure 7 HPLC chromatogram of coriander seeds (with peak 10: chlorogenic acid, peak 17: rutin); Figure 2-3 In the graph, the horizontal axis represents the retention time (minutes), and the vertical axis represents the response value (mV). Figure 4-5 In the figure, the vertical axis represents the peak area; the horizontal axis represents the injection volume (ng). Figure 6-7 The horizontal axis represents the retention time (minutes), and the vertical axis represents the response value (mAU). Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: Screening of Extraction Solution and Detection Wavelength This experiment used 25% methanol, 50% methanol, 70% methanol, methanol, 25% ethanol, 50% ethanol, 70% ethanol, and ethanol reagents for ultrasonic extraction of the samples. It was found that the baseline was stable and the chromatographic peaks were well-shaped under 70% methanol conditions; therefore, 70% methanol was selected as the extraction solvent. The experimental samples mainly contained flavonoids and phenolic acids. A photodiode array detector was used for ultraviolet full-wavelength scanning. The results showed a rich variety of chromatographic peaks at 254 nm, with good separation and symmetrical peak shapes for each characteristic peak; therefore, 254 nm was determined as the detection wavelength.

[0019] Example 2 Preparation of test and reference solutions 1) Preparation of test solution Take 1.0g of coriander seed powder (passed through a No. 4 sieve), accurately weigh it, accurately add 50mL of 70% methanol, weigh it, extract it by sonication for 30min, cool it, weigh it again, make up the weight loss with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product. 2) Preparation of reference solution Take appropriate amounts of chlorogenic acid and rutin reference standards, weigh them accurately, and add methanol to prepare a mixed reference solution containing 0.05 mg of each per 1 mL (for content determination).

[0020] Example 3 Chromatographic conditions Chromatographic column: C18 column, Acclaim TM 120 C18 5µm 120A 4.6×250mm; Mobile phase: A (0.1% phosphoric acid) - B (acetonitrile); The elution program is as follows: 0.01-12 min, 5%-16% B; 12-41 min, 16% B; 41-56 min, 16%-40% B; 56-61 min, 40% B; 61-62 min, 40%-5% B; 62-65 min, 5% B.

[0021] Detection wavelength: 254 nm; Column temperature: 30 ℃; Flow rate: 0.8 mL / min; Injection volume: 10 μL.

[0022] Example 4: Fingerprint patterns and similarity Fifteen batches of coriander seeds were tested. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software was used to match common peaks, calculate similarity, and generate a control fingerprint spectrum. Common peaks were identified separately.

[0023] Twenty-two common peaks were identified in coriander seeds. The similarity evaluation results showed that the similarity of 15 batches of coriander seed samples ranged from 0.842 to 1.000, indicating that there were some fluctuations between batches, but the overall similarity of characteristics was still good.

[0024] Example 5: Common Peak Identification and Content Determination A total of 22 peaks were observed, and characteristic peaks of chlorogenic acid and rutin were identified. A standard curve (for determining the content of chlorogenic acid and rutin) was established using the external standard method (Table 1-3). Table 1. Linear Relationship and Regression Equation of Chlorogenic Acid

[0025] Table 2. Linear Relationship and Regression Equation of Rutin

[0026] Table 3. Content of coriander seeds in 15 batches

[0027] Example 6 Methodological Investigation 1) Precision test Take an appropriate amount of the chlorogenic acid and rutin mixed standard solution and inject it into 6 consecutive injections. The precision peak area was calculated, and the results showed that the RSD was less than 3.0% (see Table 4), indicating good precision.

[0028] Table 4 Precision Results

[0029] 2) Stability test Take an appropriate amount of the test solution of this product and inject it for determination at 0, 5, 9, 12, 14, 19, 21 and 24 hours after preparation. Calculate the peak area of ​​each phase. The relative retention time difference within 24 hours is not significant, and the RSD of each chromatographic peak is less than 3.0% (see Table 5). This indicates that the test solution has good stability within 24 hours.

[0030] Table 5 Stability Results

[0031] 3) Repeatability test Nine portions of the same batch of coriander seed powder were accurately weighed, and a certain amount of a known concentration of standard solution was added to prepare the solution before injection. The repeatability of the content calculations showed little difference, with RSDs all less than 3.0% (see Table 6), indicating good repeatability.

[0032] Table 6 Repeatability Results

[0033] 4) Recovery test Six portions of the same batch of coriander seed powder were accurately weighed and used to prepare test solutions, which were then injected. Content calculations were performed, and the RSDs were all less than 3.0% (see Tables 7-8), indicating good recovery.

[0034] Table 7 Results of chlorogenic acid recovery

[0035] Table 8 Rutin Recovery Results

[0036] It should be understood that, in order to simplify this disclosure and aid in understanding one or more of the various aspects of the invention, features of the invention are sometimes grouped together in a single embodiment or in its description in the foregoing description of exemplary embodiments of the invention. However, this method of disclosure should not be construed as reflecting an intention that the claimed invention requires more features than expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in fewer than all the features of the foregoingly disclosed embodiments. Therefore, the claims, following the detailed description, are hereby expressly incorporated into that detailed description, wherein each claim itself is a separate embodiment of the invention.

[0037] Although the invention has been described with reference to a limited number of embodiments, those skilled in the art will understand from the foregoing description that other embodiments are conceivable within the scope of the invention described herein. Furthermore, it should be noted that the language used in this specification has been chosen primarily for readability and instructional purposes, and not for the purpose of interpreting or limiting the subject matter of the invention. Therefore, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the invention is illustrative and not restrictive, and the scope of the invention is defined by the appended claims.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting coriander seeds using HPLC fingerprinting, characterized in that, Includes the following steps: 1) Preparation of test solution: Take coriander seed powder, extract it with 70% methanol by ultrasonic extraction for 30 min, filter it, and take the filtrate; 2) Preparation of reference solution: Take appropriate amounts of chlorogenic acid and rutin reference standards, accurately weigh them, and add methanol to prepare a mixed reference solution containing 0.05 mg of each per 1 mL; 3) HPLC detection: A C18 column was used with gradient elution of acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase, and the detection wavelength was 254 nm. 4) Establish a fingerprint spectrum of coriander seeds and identify 22 common peaks; 5) Common peak identification was performed using a reference standard comparison; 6) Determination of the content of indicator components using the external standard method; 7) Conduct similarity calculations and methodological investigations.

2. The method according to claim 1, characterized in that, The gradient elution program is A (0.1% phosphoric acid) - B (acetonitrile): 0.01-12 min, 5%-16% B; 12-41 min, 16% B; 41-56 min, 16%-40% B; 56-61 min, 40% B; 61-62 min, 40%-5% B; 62-65 min, 5% B.

3. The method according to claim 1, characterized in that, The column temperature was 30℃, the flow rate was 0.8 mL / min, and the injection volume was 10 μL.

4. The method according to claim 1, characterized in that, The fingerprint similarity of 15 batches of coriander seeds ranged from 0.842 to 1.

000.

5. The method according to claim 1, characterized in that, The identified common peaks include chlorogenic acid and rutin.

6. The method according to claim 1, characterized in that, The methodological evaluation showed that the RSD was less than 3.0%, and the precision, repeatability, and stability were all good.

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