Method for determining content of active ingredients in trifoliate orange

Abstract

The invention provides a method for determining the content of active ingredients in trifoliate orange. The method adopts high performance liquid chromatography (HPLC) to simultaneously determine naringin, trifoliate orange glycoside and imperatorin in the trifoliate orange. The method comprises the following steps: 1) preparing a reference solution and a test solution; 2) HPLC detection: adopting a DimonsilC18 chromatographic column, taking methanol-1% glacial acetic acid as a mobile phase for gradient elution, and controlling the flow rate to be 1.0 mL / min, the column temperature to be 30 DEG C and the detection wavelength to be 283 nm. The method has the beneficial effects that the three active ingredients of naringin, trifoliate glycoside and imperatorin in the trifoliate orange are simultaneously determined, and compared with single-ingredient determination, the quality of the trifoliate orange can be more comprehensively reflected; by optimizing chromatographic conditions and an extraction method, good separation and accurate determination of the three components are ensured, and the method has good specificity and linear relation, high accuracy and good reproducibility, and can provide reliable technical support for quality control, medicinal material screening and resource development of trifoliate orange.

Description

Technical Field

[0001] This invention mainly relates to the field of traditional Chinese medicine component detection technology, specifically to a method for simultaneously determining the content of three active components in tangerine peel, hesperidin and imperatorin in tangerine peel using high performance liquid chromatography (HPLC). Background Technology

[0002] With the advancement of the modernization of traditional Chinese medicine, the quality control of medicinal materials has shifted from traditional "morphological identification" to "component quantification." Content determination, as a core means of quality control, directly impacts the clinical efficacy and safety of medicinal materials and preparations. Studies have shown that while the content of naringin in different batches of tangerine peel may be similar, the content of other key components such as hesperidin and imperatorin varies significantly. Evaluating based on a single component may result in "qualified but substandard" medicinal materials entering the market, posing risks to clinical use.

[0003] The Jiangsu Provincial Standards for Traditional Chinese Medicine (2016 Edition) uses naringin content as the quality control indicator for *Citrus medica* (wort), while hesperidin and imperatorin are not required. However, *Citrus medica*, with its multiple components and multi-target effects, requires a more comprehensive overall quality standard system. This experiment establishes for the first time a high-performance liquid chromatography (HPLC) method for the simultaneous determination of naringin, hesperidin, and imperatorin in *Citrus medica* slices. This method is accurate, simple, and rapid, suitable for the content determination of *Citrus medica*, and can be used for quality control of the medicinal material and its prepared medicines. It is of great significance for the development and utilization of *Citrus medica* medicinal resources.

[0004] It should be noted that the above content falls within the scope of the inventor's technical knowledge. Due to the vast and complex nature of the technical content in this field, the above content of this application does not necessarily constitute prior art. Summary of the Invention

[0005] 1. The technical problem that the invention aims to solve: This invention provides a method for determining the content of active ingredients in tangerine peel, thereby solving the technical problems existing in the background art.

[0006] 2. Technical Solution: To achieve the above objectives, the technical solution provided by this invention is: a method for determining the content of active ingredients in tangerine peel, comprising the following steps: Step 1: Preparation of reference solutions: Prepare stock solutions of single reference standards for naringin, hesperidin, and imperatorin, respectively, mix them, and dilute with methanol to obtain a mixed reference solution; Step 2, Preparation of test solution: Take the fine powder of Citrus reticulata, add methanol and extract by ultrasonication, then filter to obtain the test solution; Step 3, HPLC detection: Inject the reference solution and the test solution into the high performance liquid chromatograph, record the chromatogram, and calculate the content according to the external standard method; The HPLC detection conditions included: a Diamonsil C18 column with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm; a mobile phase of methanol-1% glacial acetic acid with gradient elution; a flow rate of 1.0 ml / min; a column temperature of 30 °C; and a detection wavelength of 283 nm.

[0007] Furthermore, in step one, the preparation method of the naringin reference standard stock solution is as follows: accurately weigh 8 mg of naringin reference standard, place it in a 50 ml volumetric flask, dissolve it in methanol and dilute it to the mark.

[0008] Furthermore, in step one, the preparation method of the hesperidin reference standard stock solution is as follows: accurately weigh 4 mg of hesperidin reference standard, place it in a 5 ml volumetric flask, dissolve it in methanol and dilute it to the mark.

[0009] Furthermore, in step one, the preparation method of the imperatorin reference stock solution is as follows: accurately weigh 7 mg of imperatorin reference standard, place it in a 100 ml volumetric flask, dissolve it in methanol and dilute it to the mark.

[0010] Furthermore, in step two, the preparation of the test solution is as follows: take 0.5g of fine powder of Citrus reticulata, accurately weigh it, place it in a 50ml volumetric flask, add 40ml of methanol, sonicate for 30 minutes, cool it, dilute it to the mark with methanol, filter it through a No. 4 sieve, and take the filtrate.

[0011] Furthermore, in step three, the gradient elution program is as follows: 0-60 min, methanol ratio is 5% to 95%, 1% glacial acetic acid ratio is 95% to 5%; 60-70 min, methanol ratio is 95% to 5%, 1% glacial acetic acid ratio is 5% to 95%; at 70 min, methanol ratio is 5%, 1% glacial acetic acid ratio is 95%.

[0012] Furthermore, in step three, the HPLC injection volume is 10 µl.

[0013] Furthermore, the linear range of naringin is 5.98~29.90 μg / ml, the linear range of hesperidin is 34.18~170.88 μg / ml, and the linear range of imperatorin is 2.69~13.45 μg / ml.

[0014] Furthermore, the recovery rates of the method were: naringin 98.21%, hesperidin 99.97%, imperatorin 99.93%, with RSDs ≤1.38% for all.

[0015] 3. Beneficial effects: Compared with the prior art, the technical solution provided by this invention has the following advantages: This method enables the simultaneous determination of three active components in tangerine peel: naringin, hesperidin, and imperatorin. Compared with the determination of a single component, this method can more comprehensively reflect the quality of tangerine peel. By optimizing chromatographic conditions and extraction methods, the good separation and accurate determination of the three components are ensured, which can provide reliable technical support for the quality control, medicinal material screening, and resource development of tangerine peel.

[0016] It should be noted that the structures not described in this invention are not related to the design points and improvement directions of this invention, and are the same as or can be implemented using existing technologies, so they will not be elaborated here. Attached Figure Description

[0017] Figure 1 The HPLC chromatograms of reference standard A and tangerine sample B of the present invention are shown below. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more complete description of the invention will be given below with reference to the accompanying drawings, which illustrate several embodiments of the invention. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the invention will be more thorough and complete.

[0019] The core technical solution of this invention includes the following steps: Preparation of instruments and materials Instruments: High performance liquid chromatograph (including quaternary pump and diode array detector), ultrasonic cleaner (such as KQ-250B model), analytical balance (accuracy 0.0001g), volumetric flasks (5ml, 50ml, 100ml); Reagents: Methanol (chromatographic grade), 1% glacial acetic acid solution (analytical grade, 1 ml of glacial acetic acid diluted with water to 100 ml), redistilled water; reference standards included naringin (purity 93.2%), hesperidin (purity 96.0%), and imperatorin (purity 99.6%). Samples: Take 15 batches of Citrus reticulata slices, which are nearly mature fruits of Citrus reticulata, a plant of the Rutaceae family, and pass them through a No. 4 sieve for later use.

[0020] Preparation of mixed reference solution Step 1. Preparation of a single reference standard stock solution Take naringin reference standard (purity 93.2%), place it in a desiccator for equilibration for 24 hours, accurately weigh 8.0 mg (electronic balance accuracy 0.0001 g), transfer it to a 50 ml volumetric flask; add methanol and sonicate (power 250 W, time 5 minutes), cool, dilute with methanol to the mark, shake well, and obtain naringin stock solution (concentration: 0.16 mg / ml, actual concentration after conversion according to purity is 0.1491 mg / ml). Take 4.0 mg of hesperidin reference standard (purity 96.0%), transfer it to a 5 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well to obtain hesperidin stock solution (concentration: 0.8 mg / ml, actual concentration after conversion is 0.768 mg / ml). Take imperatorin reference standard (purity 99.6%), accurately weigh 7.0 mg, transfer to a 100 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well to obtain imperatorin stock solution (concentration: 0.07 mg / ml, actual concentration after conversion is 0.0697 mg / ml).

[0021] Step 2. Preparation of mixed reference solution Accurately measure 1 ml of the above-mentioned naringin stock solution, 1 ml of hesperidin stock solution, and 1 ml of imperatorin stock solution, and place them together in a 5 ml volumetric flask. Dilute to the mark with methanol and shake well to obtain a mixed reference solution. The concentrations of the three components in this solution were calculated to be: naringin 29.82 μg / ml, hesperidin 153.6 μg / ml, and imperatorin 13.94 μg / ml, respectively.

[0022] Preparation of test solution Step 1. Sample Pretreatment Take the sliced ​​tangerine peel (batch number: 15041403, place of origin: Jiangsu), pulverize it and pass it through a No. 4 sieve (sieve hole inner diameter 250±9.9μm). Place the fine powder in a weighing bottle and dry it in an oven at 60℃ for 2 hours. Then transfer it to a desiccator to cool to room temperature for later use.

[0023] Step 2. Extraction and Filtering Accurately weigh 0.5002 g of the above fine powder and place it in a 50 ml volumetric flask. Add 40 ml of methanol, seal the flask, and place it in an ultrasonic cleaner (KQ-250B type, power 250W, frequency 40kHz) for ultrasonic treatment for 30 minutes (keeping the water temperature below 40℃ during the process). After removing the flask, let it cool to room temperature, replenish the lost weight with methanol, and shake well. Filter the above solution through a 0.45 μm organic phase filter membrane, discard about 2 ml of the initial filtrate, and use the subsequent filtrate as the test solution.

[0024] HPLC detection and result analysis Step 1. Instrument and Chromatographic Conditions Setup Instrument: Waterse2695 high performance liquid chromatograph (equipped with 2998 diode array detector); Chromatographic column: Diamonsil C18 (250mm×4.6mm, 5μm, batch number: 20210315); Mobile phase: Phase A is methanol (chromatographic grade, batch number: 20210508), and Phase B is 1% glacial acetic acid solution (take 1 ml of glacial acetic acid, dilute with water to 100 ml, and filter through a 0.45 μm filter membrane). As shown in Table 1, the gradient elution program (set according to the instrument control software) is as follows: 0 min: Phase A 5%, Phase B 95% (flow rate 1.0 ml / min); to 60 min: Phase A 95%, Phase B 5% (flow rate 1.0 ml / min); to 70 min: Phase A switches to 5%, Phase B switches to 95% (to equilibrate the column for the next injection). Detection wavelength: 283nm (determined by full-wavelength scanning with a diode array detector; at this wavelength, the response values ​​of the three components are the highest and the baseline is stable). Column temperature: 30℃ (precisely controlled via a column temperature chamber); Injection volume: 10µl; Column equilibration time: Equilibrate for 30 minutes before each injection to ensure baseline stability (fluctuation range ≤ ±0.001AU).

[0025]

[0026] Step 2. Sample Injection and Data Recording like Figure 1 As shown in A, take the mixed reference solution, inject it three times consecutively, record the chromatogram, and calculate the average peak area of ​​the three times; like Figure 1 As shown in B, take the test solution, inject it once, record the chromatogram, and read the peak area at the corresponding retention time.

[0027] Step 3. Content Calculation Calculation formula using the external standard method: The content (%) of the component in the test sample = (C × V × D × 10) -6 ) / m×100% in: C represents the concentration of the reference solution (μg / ml); V is the final volume of the test solution (50 ml); D is the dilution factor (D=1 in this example); m is the sample size (g) of the test sample.

[0028] Methodological validation experiments Examining linear relationships As shown in Table 2, the mixed reference standard stock solution was diluted to five concentration gradients according to the specified ratio, and each gradient was injected and measured. A standard curve was plotted with concentration (X) on the x-axis and peak area (Y) on the y-axis. The results showed that the three components had a good linear relationship (R²>0.995), and... Figure 1The trend of peak area change with concentration is consistent in A.

[0029]

[0030] Precision test The same mixed standard solution was injected 6 times consecutively, and the peak area RSD was calculated: naringin 1.25%, hesperidin 1.88%, and imperatorin 1.07%, indicating that the instrument precision met the requirements.

[0031] Repeatability test Six test solutions were prepared in parallel using the same batch of tangerine samples (batch number 15041403) according to the above method. The contents were determined and the RSD was calculated: naringin 2.15%, hesperidin 1.76%, imperatorin 1.52%, which proved that the method has good repeatability.

[0032] Stability test The test solution was injected at 0, 2, 4, 8 and 12 hours, respectively. The peak area RSDs were 1.01%, 1.07% and 0.93%, respectively, all <2%, indicating that the solution was stable within 12 hours.

[0033] Recovery test As shown in Table 3, 0.25g of a sample with known content was taken, and a certain amount of reference standard was precisely added. The sample was processed and determined according to the test sample preparation method. The recovery rate was calculated, and the average recovery rate was 98.21%~99.97%, with RSD≤1.38%, which meets the requirements for traditional Chinese medicine testing.

[0034]

[0035] Sample content determination As shown in Tables 4 and 5, 15 batches of samples were taken and tested according to the above method. The experimental results show that this method is suitable for the determination of hesperidin, naringin, and imperatorin in tangerine peel. Significant differences in components were observed between batches, indicating that there are considerable differences in the quality of tangerine peel slices from different manufacturers.

[0036] The following are the sources and test results of 15 batches of samples:

[0037] Table 5. Test results of 15 batches of samples (n=2)

[0038] This method enables the simultaneous determination of three key active ingredients: naringin, hesperidin, and imperatorin, covering core pharmacodynamic groups such as flavonoids and coumarins. Through multi-indicator joint control, it can more accurately reflect the intrinsic quality differences in tangerine peel slices. Experimental data shows that the contents of the three components fluctuated significantly in 15 batches of samples (e.g., naringin 0.31%~1.17%, imperatorin 0.01%~0.10%). Evaluation of a single component may lead to misjudgment of quality, while this method can comprehensively capture these differences, providing a scientific basis for the grading and screening of medicinal materials.

[0039] To address the complex composition of Citrus aurantium, a combination of Diamonsil C18 and methanol-1% glacial acetic acid mobile phase was selected through screening. This, combined with a dedicated detection wavelength of 283 nm, resulted in peak resolutions >1.5 for all three components, with symmetrical peak shapes (tailing factor 0.9–1.1), effectively avoiding interference from impurity peaks. Method validation results showed average recoveries of 98.21%–99.97% for the three components, with RSDs ≤1.38%, far exceeding industry standards and ensuring accurate quantification of trace components.

[0040] For medicinal herb cultivation, this method can guide the optimization of cultivation techniques (such as determining the optimal harvest time) by analyzing the differences in composition of samples from different producing areas and harvesting periods. For processing, it can evaluate the impact of processing techniques on components (such as the effect of drying temperature on the stability of imperatorin). For pharmaceutical production, it can be used for incoming inspection of raw materials and quality monitoring of finished products to avoid instability in efficacy caused by fluctuations in raw materials.

[0041] In summary, the above methods can completely reproduce the detection process of this invention, and the experimental results are stable and reliable. This method is suitable for routine detection and quality control of active ingredients in tangerine peel, and a quality control system applicable to the entire tangerine peel industry chain has been constructed. Its scientific, practical and standardized characteristics are of great significance for improving the quality control level of traditional Chinese medicine and ensuring clinical efficacy.

[0042] The above-described embodiments are merely illustrative of certain implementations of the present invention, and are described in a relatively specific and detailed manner. However, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements are all within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for determining the content of active ingredients in tangerine peel, characterized in that, Includes the following steps: Step 1: Preparation of reference solutions: Prepare stock solutions of single reference standards for naringin, hesperidin, and imperatorin, respectively, mix them, and dilute with methanol to obtain a mixed reference solution; Step 2, Preparation of test solution: Take the fine powder of Citrus reticulata, add methanol and extract by ultrasonication, then filter to obtain the test solution; Step 3, HPLC detection: Inject the reference solution and the test solution into the high performance liquid chromatograph, record the chromatogram, and calculate the content according to the external standard method; The HPLC detection conditions included: a Diamonsil C18 column with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm; a mobile phase of methanol-1% glacial acetic acid with gradient elution; a flow rate of 1.0 ml / min; a column temperature of 30 °C; and a detection wavelength of 283 nm.

2. The method for determining the content of active ingredients in tangerine peel according to claim 1, characterized in that: In step one, the preparation method of the naringin reference standard stock solution is as follows: accurately weigh 8 mg of naringin reference standard, place it in a 50 ml volumetric flask, dissolve it in methanol and dilute it to the mark.

3. The method for determining the content of active ingredients in tangerine peel according to claim 1, characterized in that: In step one, the preparation method of the hesperidin reference standard stock solution is as follows: accurately weigh 4 mg of hesperidin reference standard, place it in a 5 ml volumetric flask, dissolve it in methanol and dilute it to the mark.

4. The method for determining the content of active ingredients in tangerine peel according to claim 1, characterized in that: In step one, the preparation method of the imperatorin reference stock solution is as follows: accurately weigh 7 mg of imperatorin reference standard, place it in a 100 ml volumetric flask, dissolve it in methanol and dilute it to the mark.

5. The method for determining the content of active ingredients in tangerine peel according to claim 1, characterized in that: In step two, the preparation of the test solution is as follows: take 0.5g of fine powder of Citrus reticulata, weigh it accurately, put it in a 50ml volumetric flask, add 40ml of methanol, sonicate for 30 minutes, cool it, dilute it with methanol to the mark, filter it through a No. 4 sieve, and take the filtrate.

6. The method for determining the content of active ingredients in tangerine peel according to claim 1, characterized in that: In step three, the gradient elution program is as follows: 0-60 min, methanol ratio is 5% to 95%, 1% glacial acetic acid ratio is 95% to 5%; 60-70 min, methanol ratio is 95% to 5%, 1% glacial acetic acid ratio is 5% to 95%; at 70 min, methanol ratio is 5%, 1% glacial acetic acid ratio is 95%.

7. The method for determining the content of active ingredients in tangerine peel according to claim 1, characterized in that: In step three, the HPLC injection volume is 10 µl.

8. The method for determining the content of active ingredients in tangerine peel according to claim 1, characterized in that: The linear range of naringin is 5.98~29.90 μg / ml, the linear range of hesperidin is 34.18~170.88 μg / ml, and the linear range of imperatorin is 2.69~13.45 μg / ml.

9. The method for determining the content of active ingredients in tangerine peel according to claim 1, characterized in that: The recovery rates of the method were: naringin 98.21%, hesperidin 99.97%, imperatorin 99.93%, with RSDs ≤1.38%.

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