A method for simultaneously determining the contents of 12 components in Changqing capsules by HPLC

Abstract

The application discloses a method for simultaneously determining the contents of 12 components in Longqing capsules by HPLC. The method comprises simultaneously determining the contents of paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, coptisine, jatrorrhizine hydrochloride, gallic acid, rutin, kaempferol, luteolin and phellodendrine hydrochloride in the Longqing capsules, gradient elution is carried out by adopting a chromatographic column with octadecylsilane-bonded silica gel as a filler, 0.1% phosphoric acid as mobile phase A and a methanol solution as mobile phase B, the column temperature is 35 DEG C, and the detection wavelength is 270 nm, and the HPLC content determination method is established, so that a multi-index quality control method of the Longqing capsules is established. The method is simple, stable and reliable, has high detection efficiency, and has reference value for establishing the quality standard and control system of the Longqing capsules based on the theory of traditional Chinese medicine.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation content determination technology, and in particular to a method for simultaneously determining the content of 12 components in Longqing capsules using HPLC. Background Technology

[0002] Longqing Capsules are a nationally improved new drug, derived from the "Interpretation of Clinical Pathway Therapeutic Drugs". [1] Composed of 10 Chinese medicinal herbs including Patrinia scabiosifolia, Hedyotis diffusa, Lonicera japonica, Coptis chinensis, Phellodendron chinense, Paeonia suffruticosa, Paeonia lactiflora, and Agrimonia pilosa, this medicine has the effects of clearing heat and detoxifying, cooling blood and promoting urination. It is mainly used for urinary tract infections caused by damp-heat in the lower abdomen, with symptoms such as frequent urination and lower back pain.

[0003] Currently, alkaloids such as berberine hydrochloride and berberine hydrochloride are mainly used as quality control indicators. [2][3] Traditional Chinese medicine treatment is based on syndrome differentiation and treatment, and the properties and meridians of Chinese herbs. The main component of the chief herb in the formula, Patrinia scabiosaefolia, is caffeic acid, which has the effect of clearing heat and detoxifying. [4][8] Rutin and kaempferol in the herb *Hedyotis diffusa* have diuretic and dampness-removing effects. [9]

[10] Berberine hydrochloride and berberine hydrochloride in Coptis chinensis and Phellodendron amurense have the effects of clearing heat and detoxifying, and reducing internal heat.

[16]

[17] Chlorogenic acid in honeysuckle has detoxifying and anti-inflammatory effects.

[11] -

[14] The adjuvant ingredient, red peony root, contains paeoniflorin, which has the effects of promoting blood circulation, removing blood stasis, relieving fever, and relieving spasms.

[15]

[21] The main component of peony bark, paeonol, has hemostatic and blood-stasis-removing effects.

[18]

[19] Agrimonia pilosa contains luteolin, a component of which has anti-inflammatory and detoxifying effects.

[20]

[23] All the above indicators are consistent with the main therapeutic effects of the principal, assistant, adjuvant, and guiding herbs in the formula. In order to further reflect the principal herb's main function, the assistant herb assists the principal herb in clearing heat and promoting urination, and the adjuvant herb not only assists the principal and assistant herbs but also treats concurrent symptoms, so that all the herbs work together to achieve the effects of clearing heat and detoxifying, cooling blood and promoting urination.

[0004] For example, Chinese patent number CN202111667325.1, entitled "An Improved Method for Quality Detection of Longqing Capsules," uses high-performance liquid chromatography (HPLC) to determine the content of berberine hydrochloride, 23-acetylalizool B, and 23-acetylalizool C. However, this method can only detect a limited number of three components and requires two different detection methods. It cannot simultaneously detect multiple indicators, resulting in low detection efficiency, wasted time and costs. Furthermore, it fails to reflect the correspondence between each indicator component and the principal, assistant, and adjuvant herbs in the formula, and cannot better control and guarantee product quality.

[0005] Therefore, this invention utilizes HPLC to simultaneously determine 12 types and multiple indicators of components in Longqing Capsules, including paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, coptisine, cypermethrin hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride, as well as flavonoids, alkaloids, and organic acids, providing a basis for further improving the quality control methods of Longqing Capsules. Summary of the Invention

[0006] The purpose of this invention is to provide a method for simultaneously determining the content of 12 components in Longqing capsules using HPLC.

[0007] This invention is achieved by adopting the following technical solution:

[0008] The present invention provides a method for simultaneously determining the content of 12 components in Longqing capsules using HPLC, including the method for simultaneously determining the content of paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, berberine, berberine hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride in Longqing capsules, specifically comprising the following steps:

[0009] (1) Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel; gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol solution as mobile phase B; column temperature: 35℃; detection wavelength: 270nm; flow rate: 1.0mL / min; injection volume: 10μL;

[0010] (2) Preparation of reference solution: Accurately weigh an appropriate amount of reference standard and add methanol to prepare a mixed reference solution containing paeoniflorin 0.174 mg / mL, paeonol 0.007 mg / mL, chlorogenic acid 0.160 mg / mL, caffeic acid 0.037 mg / mL, berberine hydrochloride 0.166 mg / mL, berberine 0.028 mg / mL, berberine hydrochloride 0.027 mg / mL, gallic acid 0.069 mg / mL, rutin 0.029 mg / mL, kaempferol 0.004 mg / mL, luteolin 0.006 mg / mL, and berberine hydrochloride 0.110 mg / mL.

[0011] (3) Preparation of test solution: Take an appropriate amount of Longqing capsules, grind them into a fine powder, take 0.5-1.5g, weigh accurately, place in a stoppered conical flask, add 50-150mL of methanol accurately, weigh, sonicate for 30-60min, cool, add methanol to make up the weight, shake well, filter, and the test solution is obtained.

[0012] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0013] The chromatographic column mentioned in step (1) of this invention is: ShimNex CS C 18 Chromatographic column.

[0014] The ShimNex CS C of this invention 18 The chromatographic column has the following specifications: 4.6 mm × 250 mm, 5 μm.

[0015] The gradient elution procedure described in this invention is as follows:

[0016]

[0017] The detection wavelength described in this invention can also be 230nm.

[0018] The detection wavelength described in this invention can also be 250nm.

[0019] Preferably, the preparation of the test solution of the present invention is as follows: take an appropriate amount of Longqing capsules, grind them into a fine powder, take 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 100mL of methanol, weigh it, sonicate for 30min, cool it, make up the weight with methanol, shake it well, filter it, and the solution is obtained.

[0020] The ultrasonic conditions described in this invention are: power of 200-300W and frequency of 30-50kHz.

[0021] Preferably, the ultrasonic conditions described in this invention are: power of 250W and frequency of 40kHz.

[0022] The filtration described in this invention uses a 0.22μm microporous membrane.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. The HPLC content determination method established in this invention can simultaneously determine the content of 12 index components in Longqing Capsules, including paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, coptisine, cypermethrin hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride. This establishes a multi-index quality control method for Longqing Capsules and can serve as a reference for constructing a quality standard and control system for Longqing Capsules based on traditional Chinese medicine theory.

[0025] 2. This invention employs high-performance liquid chromatography (HPLC) with C0.05... 18The chromatographic column was 35℃; the wavelength was 270nm; and the mobile phase was methanol-0.1% phosphoric acid solution, with gradient elution. Results: The 12 components of Longqing capsules showed good linearity within the investigated range (r≥0.9995), with precision RSD of 0.47–1.48%, repeatability RSD of 0.60–1.60%, stability RSD of 0.34–1.43%, and average recoveries of 97.83%–100.52% with RSD of 0.9%–2.1%. This indicates that the method is highly specific, sensitive, repeatable, simple, stable, and reliable.

[0026] 3. This invention screened and investigated chromatographic conditions, finding that the methanol-0.1% phosphoric acid solution provided a relatively stable baseline, exhibited the best resolution of compound peaks after elution with methanol in the organic phase, and reduced tailing of the analyte peaks after acidification with added phosphoric acid in the inorganic phase. Therefore, this invention selected methanol-0.1% phosphoric acid solution as the mobile phase. Due to the significant differences in retention times among components in reversed-phase chromatography, a gradient elution method was employed after reviewing the literature. This ensured good separation of the analyte peaks from adjacent impurity peaks while maintaining a stable baseline. Attached Figure Description

[0027] Figure 1 HPLC chromatogram of the mixed reference standard (where 1 represents gallic acid peak, 2 represents berberine hydrochloride peak, 3 represents chlorogenic acid peak, 4 represents caffeic acid peak, 5 represents paeoniflorin peak, 6 represents berberine peak, 7 represents berberine hydrochloride peak, 8 represents berberine hydrochloride peak, 9 represents luteolin peak, 10 represents paeonol peak, 11 represents rutin peak, and 12 represents kaempferol peak)

[0028] Figure 2 HPLC chromatogram of the test sample (where 1 represents gallic acid peak, 2 represents berberine hydrochloride peak, 3 represents chlorogenic acid peak, 4 represents caffeic acid peak, 5 represents paeoniflorin peak, 6 represents berberine peak, 7 represents berberine hydrochloride peak, 8 represents berberine hydrochloride peak, 9 represents luteolin peak, 10 represents paeonol peak, 11 represents rutin peak, and 12 represents kaempferol peak)

[0029] Figure 3 HPLC chromatogram of honeysuckle-deficient negative sample (where 1 represents gallic acid peak, 2 represents berberine hydrochloride peak, 3 represents chlorogenic acid peak, 4 represents caffeic acid peak, 5 represents paeoniflorin peak, 6 represents berberine peak, 7 represents berberine hydrochloride peak, 8 represents berberine hydrochloride peak, 9 represents luteolin peak, 10 represents paeonol peak, 11 represents rutin peak, and 12 represents kaempferol peak)

[0030] Figure 4HPLC chromatogram of a negative sample of Coptis chinensis and Phellodendron amurense (where 1 represents gallic acid peak, 2 represents berberine hydrochloride peak, 3 represents chlorogenic acid peak, 4 represents caffeic acid peak, 5 represents paeoniflorin peak, 6 represents berberine peak, 7 represents berberine hydrochloride peak, 8 represents berberine hydrochloride peak, 9 represents luteolin peak, 10 represents paeonol peak, 11 represents rutin peak, and 12 represents kaempferol peak).

[0031] Figure 5 HPLC chromatogram of a negative sample lacking peony bark (where 1 represents gallic acid peak, 2 represents berberine hydrochloride peak, 3 represents chlorogenic acid peak, 4 represents caffeic acid peak, 5 represents paeoniflorin peak, 6 represents berberine peak, 7 represents berberine hydrochloride peak, 8 represents berberine hydrochloride peak, 9 represents luteolin peak, 10 represents paeonol peak, 11 represents rutin peak, and 12 represents kaempferol peak).

[0032] Figure 6 HPLC chromatogram of negative samples lacking honeysuckle, coptis, phellodendron, and peony bark (where 1 represents gallic acid peak, 2 represents berberine hydrochloride peak, 3 represents chlorogenic acid peak, 4 represents caffeic acid peak, 5 represents paeoniflorin peak, 6 represents berberine peak, 7 represents berberine hydrochloride peak, 8 represents berberine hydrochloride peak, 9 represents luteolin peak, 10 represents paeonol peak, 11 represents rutin peak, and 12 represents kaempferol peak).

[0033] Figure 7 Precision test to examine chromatograms

[0034] Figure 8 Repeatability test to examine chromatograms

[0035] Figure 9 Stability test to examine chromatograms

[0036] Figure 10 Chromatogram of recovery test

[0037] Figure 11 Chromatogram obtained using acetonitrile (0.3% formic acid + 0.3% triethylamine) - water (0.3% formic acid + 0.3% triethylamine) as the mobile phase.

[0038] Figure 12 Chromatogram obtained using methanol-0.1% formic acid as the mobile phase.

[0039] Figure 13 Chromatogram obtained from gradient elution 1

[0040] Figure 14 Chromatogram obtained from gradient elution 2

[0041] Figure 15 Chromatogram obtained by gradient elution 3

[0042] Figure 16 Chromatogram obtained from gradient elution 4

[0043] Figure 17 Chromatogram obtained from gradient elution 5 Detailed Implementation

[0044] Example 1: Method for simultaneous determination of 12 components in Longqing capsules using HPLC

[0045] (1) Chromatographic conditions and system suitability test: ShimNex CS C 18 (4.6 mm × 250 mm, 5 μm); gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol solution as mobile phase B; column temperature: 35 ℃; detection wavelength: 270 nm; flow rate: 1.0 mL / min; injection volume: 10 μL;

[0046] The gradient elution procedure is as follows:

[0047]

[0048] (2) Preparation of reference solution: Accurately weigh an appropriate amount of reference standard and add methanol to prepare a mixed reference solution containing paeoniflorin 0.174 mg / mL, paeonol 0.007 mg / mL, chlorogenic acid 0.160 mg / mL, caffeic acid 0.037 mg / mL, berberine hydrochloride 0.166 mg / mL, berberine 0.028 mg / mL, berberine hydrochloride 0.027 mg / mL, gallic acid 0.069 mg / mL, rutin 0.029 mg / mL, kaempferol 0.004 mg / mL, luteolin 0.006 mg / mL, and berberine hydrochloride 0.110 mg / mL.

[0049] (3) Preparation of test solution: Take an appropriate amount of Longqing capsules, grind them into a fine powder, take 0.5-1.5g, weigh accurately, place in a stoppered conical flask, accurately add 50-150ml of methanol, weigh, sonicate for 30-60min (250W, 40kHz), cool, add methanol to make up the weight, shake well, filter, and the test solution is obtained.

[0050] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0051] Example 2: HPLC method for simultaneous determination of the content of 12 components in Longqing capsules

[0052] (1) Chromatographic conditions and system suitability test: ShimNex CS C 18(4.6 mm × 250 mm, 5 μm); gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol solution as mobile phase B; column temperature: 35 ℃; detection wavelength: 250 nm; flow rate: 1.0 mL / min; injection volume: 10 μL;

[0053] The gradient elution procedure is the same as in Example 1.

[0054] (2) Preparation of reference solution: Accurately weigh an appropriate amount of reference standard and add methanol to prepare a mixed reference solution containing paeoniflorin 0.174 mg / mL, paeonol 0.007 mg / mL, chlorogenic acid 0.160 mg / mL, caffeic acid 0.037 mg / mL, berberine hydrochloride 0.166 mg / mL, berberine 0.028 mg / mL, berberine hydrochloride 0.027 mg / mL, gallic acid 0.069 mg / mL, rutin 0.029 mg / mL, kaempferol 0.004 mg / mL, luteolin 0.006 mg / mL, and berberine hydrochloride 0.110 mg / mL.

[0055] (3) Preparation of test solution: Take an appropriate amount of Longqing capsules, grind them into a fine powder, take 1.5g, weigh it accurately, put it in a stoppered conical flask, add 150ml of methanol accurately, weigh it, sonicate for 60min (300W, 50kHz), cool it, make up the weight with methanol, shake it well, filter it, and the test solution is obtained.

[0056] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0057] Example 3: HPLC method for simultaneous determination of the content of 12 components in Longqing capsules

[0058] (1) Chromatographic conditions and system suitability test: ShimNex CS C 18 (4.6 mm × 250 mm, 5 μm); gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol solution as mobile phase B; column temperature: 35 ℃; detection wavelength: 230 nm; flow rate: 1.0 mL / min; injection volume: 10 μL;

[0059] The gradient elution procedure is the same as in Example 1.

[0060] (2) Preparation of reference solution: Accurately weigh an appropriate amount of reference standard and add methanol to prepare a mixed reference solution containing paeoniflorin 0.174 mg / mL, paeonol 0.007 mg / mL, chlorogenic acid 0.160 mg / mL, caffeic acid 0.037 mg / mL, berberine hydrochloride 0.166 mg / mL, berberine 0.028 mg / mL, berberine hydrochloride 0.027 mg / mL, gallic acid 0.069 mg / mL, rutin 0.029 mg / mL, kaempferol 0.004 mg / mL, luteolin 0.006 mg / mL, and berberine hydrochloride 0.110 mg / mL.

[0061] (3) Preparation of test solution: Take an appropriate amount of Longqing capsules, grind them into a fine powder, take 0.5g, weigh it accurately, place it in a stoppered conical flask, add 50ml of methanol accurately, weigh it, sonicate for 30min (200W, 30kHz), cool it, make up the weight with methanol, shake it well, filter it, and the test solution is obtained.

[0062] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0063] Example 4: HPLC method for simultaneous determination of the content of 12 components in Longqing capsules

[0064] (1) Chromatographic conditions and system suitability test: ShimNex CS C 18 (4.6 mm × 250 mm, 5 μm); gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol solution as mobile phase B; column temperature: 35 ℃; detection wavelength: 270 nm; flow rate: 1.0 mL / min; injection volume: 10 μL;

[0065] The gradient elution procedure is as follows:

[0066]

[0067] (2) Preparation of reference solution: Accurately weigh an appropriate amount of reference standard and add methanol to prepare a mixed reference solution containing paeoniflorin 0.174 mg / mL, paeonol 0.007 mg / mL, chlorogenic acid 0.160 mg / mL, caffeic acid 0.037 mg / mL, berberine hydrochloride 0.166 mg / mL, berberine 0.028 mg / mL, berberine hydrochloride 0.027 mg / mL, gallic acid 0.069 mg / mL, rutin 0.029 mg / mL, kaempferol 0.004 mg / mL, luteolin 0.006 mg / mL, and berberine hydrochloride 0.110 mg / mL.

[0068] (3) Preparation of test solution: Take an appropriate amount of Longqing capsules, grind them into a fine powder, take 1g, weigh it accurately, place it in a stoppered conical flask, add 100mL of methanol accurately, weigh it, sonicate for 30min (250W, 40kHz), cool it, make up the weight with methanol, shake it well, filter it, and the test solution is obtained.

[0069] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0070] Example 5: Method for simultaneous determination of the content of 12 components in Longqing capsules using HPLC

[0071] (1) Chromatographic conditions and system suitability test: ShimNex CS C 18 (4.6 mm × 250 mm, 5 μm); gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol solution as mobile phase B; column temperature: 35 ℃; detection wavelength: 270 nm; flow rate: 1.0 mL / min; injection volume: 10 μL;

[0072] The gradient elution procedure is as follows:

[0073]

[0074]

[0075] (2) Preparation of reference solution: Accurately weigh an appropriate amount of reference standard and add methanol to prepare a mixed reference solution containing paeoniflorin 0.174 mg / mL, paeonol 0.007 mg / mL, chlorogenic acid 0.160 mg / mL, caffeic acid 0.037 mg / mL, berberine hydrochloride 0.166 mg / mL, berberine 0.028 mg / mL, berberine hydrochloride 0.027 mg / mL, gallic acid 0.069 mg / mL, rutin 0.029 mg / mL, kaempferol 0.004 mg / mL, luteolin 0.006 mg / mL, and berberine hydrochloride 0.110 mg / mL.

[0076] (3) Preparation of test solution: Take an appropriate amount of Longqing capsules, grind them into a fine powder, take 1g, weigh it accurately, place it in a stoppered conical flask, add 100ml of methanol accurately, weigh it, sonicate for 30min (250W, 40kHz), cool it, add methanol to make up the weight, shake it well, filter it, and the test solution is obtained.

[0077] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0078] To further verify the effectiveness of the present invention, a series of verification tests were conducted, as follows:

[0079] 1. Materials and Instruments

[0080] 1.1 Instruments

[0081] Agilent 1260 high-performance liquid chromatograph (Agilent Technologies); Diamonsil C18 column; ShimNex CS C column 18 AL-104 electronic balance (Mettler-Toledo); Discovery DV215CD analytical balance (Shanghai Ohaus Instruments Co., Ltd.); KQ5200 ultrasonic cleaner (Beijing Innovation Ultrasonic Electronics Research Institute).

[0082] 1.2 Drugs and Reagents

[0083] Paeoniflorin (batch number: wkq18032104), paeonol (batch number: 11708-200506), chlorogenic acid (batch number: wkq23022707), caffeic acid (batch number: 110885-200102), berberine hydrochloride (CO2515160405), berberine (batch number: wkq23030705), berberine hydrochloride (batch number: wkq22102601), gallic acid (batch number: wkq19010307), rutin (wkq17051605), kaempferol (batch number: wkq19011609), luteolin (batch number: wkq23021710), and berberine hydrochloride (batch number: wkq23032109) reference standards. Longqing Capsules (0.5g / capsule, batch numbers 20220203, 20220506, 20220815) were provided by Guizhou Yuancheng Pharmaceutical Co., Ltd. Acetonitrile, methanol (chromatographic grade, Tianjin Kemio Chemical Reagent Co., Ltd.), and water were purified water; all other reagents were analytical grade.

[0084] 2. Methods and Results

[0085] 2.1 Chromatographic conditions

[0086] ShimNex CS C 18 The chromatographic column was 4.6 mm × 250 mm, 5 μm; the column temperature was 35 ℃; the detection wavelength was 270 nm; the mobile phase was 0.1% phosphoric acid (A)-methanol (B) solution, and the gradient elution was shown in Table 1; the flow rate was 1.0 ml / min, and the injection volume was 10 μL.

[0087] Table 1 Gradient elution program

[0088]

[0089] 2.2 Solution Preparation

[0090] 2.2.1 Mixed reference solution

[0091] Accurately weigh an appropriate amount of reference standard and add methanol to prepare a mixed reference solution containing paeoniflorin 0.174 mg / ml, paeonol 0.007 mg / ml, chlorogenic acid 0.160 mg / ml, caffeic acid 0.037 mg / ml, berberine hydrochloride 0.166 mg / ml, berberine 0.028 mg / ml, berberine hydrochloride 0.027 mg / ml, gallic acid 0.069 mg / ml, rutin 0.029 mg / ml, kaempferol 0.004 mg / ml, luteolin 0.006 mg / ml, and berberine hydrochloride 0.110 mg / ml.

[0092] 2.2.2 Test solution

[0093] Take an appropriate amount of Longqing capsules, grind them into a fine powder, take about 1.0g, weigh it accurately, place it in a stoppered conical flask, accurately add 100ml of methanol, weigh it, sonicate for 30min, cool it, make up the weight with methanol, shake it well, filter it, and you will get the product.

[0094] 2.2.3 Negative control solution

[0095] According to the pharmacopoeia [2] Prepare negative test solutions for honeysuckle, peony bark, coptis and phellodendron as described in section “2.2.2”.

[0096] 2.3 System Suitability and Specificity Test

[0097] The sample was injected and determined according to the chromatographic conditions in section "2.1". The results are shown below. Figures 1 to 6 The figure shows that the chromatographic peaks of the 12 components were completely separated and there was no interference, indicating that the method has good specificity.

[0098] 2.4 Examination of Linear Relationships

[0099] Accurately pipette an appropriate amount of the mixed reference solution and dilute it to six different concentrations. Determine the concentrations according to the chromatographic conditions in section "2.1". Perform regression analysis with the injection volume (X, μg) of each reference standard as the abscissa and the peak area (Y) as the ordinate. The results are shown in Table 2. Table 2 shows that all components exhibit good linearity within the detection limit (r ≥ 0.9995).

[0100] Table 2. Results of linear relationships for each component

[0101]

[0102]

[0103] 2.5 Precision Test

[0104] Six portions of the mixed reference solution were taken and injected for determination under the chromatographic conditions described in section "2.1". The RSDs of the peak areas for paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, berberine, berberine hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride were 1.48%, 1.43%, 0.59%, 1.07%, 0.47%, 1.34%, 1.15%, 0.91%, 1.13%, 0.53%, 1.25%, and 1.28%, respectively, indicating good instrument precision. See Figure 7 .

[0105] 2.6 Repeatability Test

[0106] Six test solutions were prepared in parallel using the same batch of Longqing capsules according to the method in section "2.2.2". The solutions were then injected and analyzed under the chromatographic conditions in section "2.1". The peak areas RSDs of paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, berberine, berberine hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride were 1.04%, 0.60%, 1.44%, 0.65%, 1.22%, 0.96%, 1.60%, 0.95%, 1.04%, 0.56%, 0.81%, and 1.54%, respectively, indicating good reproducibility of the method. (See...) Figure 8 .

[0107] 2.7 Stability Test

[0108] The same batch of Longqing capsules were used to prepare test solutions according to the method in section "2.2.2". The solutions were then injected and analyzed at 0, 4, 8, 12, and 24 hours under the chromatographic conditions in section "2.1". The RSDs of the peak areas for paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, berberine, berberine hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride were 1.21%, 0.75%, 1.24%, 0.84%, 1.01%, 0.99%, 1.75%, 0.53%, 0.34%, 1.43%, 1.31%, and 0.89%, respectively, indicating that the test solutions were stable within 24 hours. (See [link to relevant documentation]). Figure 9 .

[0109] 2.8 Recovery Experiment

[0110] Accurately weigh six parallel portions of the same batch of this product, each approximately 0.5 g, containing known amounts of each component. Place each portion in a stoppered Erlenmeyer flask. Add three portions of each reference standard (each with a different amount) to the amounts corresponding to 80%, 100%, and 120% of the content of paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, coptisine, purslane hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride, respectively. Prepare the test solutions according to the method in section "2.2.2". Inject and determine the chromatographic peak areas of each component under the chromatographic conditions in section "2.1". Calculate the chromatographic peak areas of each component. The average recoveries were 99.36%, 98.32%, 97.83%, 98.09%, 99.93%, 100.52%, 98.93%, 99.51%, 99.24%, 99.85%, 100.05%, and 99.24%, respectively, with RSDs of 1.3%, 1.9%, 2.1%, 1.9%, 1.7%, 2.1%, 1.5%, 1.8%, 1.9%, 1.4%, 0.9%, and 1.9%, respectively. The results show that the recoveries of the 12 components were good and basically met the requirements of the experimental methodology. (See...) Figure 10 .

[0111] 2.9 Sample Content Determination

[0112] Take three batches of Longqing capsules, prepare test solutions according to the method in section "2.2.2", analyze them according to the chromatographic conditions in section "2.1", calculate the content, and the results are shown in Table 3.

[0113] Table 3. Content of the analyte in the samples (mg·g) -1 (n=3)

[0114]

[0115] 3. Discussion

[0116] 3.1 Selection of chromatographic conditions

[0117] This experiment simultaneously determined 12 components of Longqing capsules. During the experiment, the separation effects of different ratio gradient elutions of eight mobile phases were compared based on reagent polarity and elution degree: methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution, methanol-0.1% formic acid solution, acetonitrile-0.1% formic acid solution, acetonitrile (0.3% formic acid and 0.3% triethylamine)-water (0.3% formic acid and 0.3% triethylamine), and methanol (0.3% formic acid and 0.3% triethylamine)-water (0.3% formic acid and 0.3% triethylamine). The results showed that the separation degree of acetonitrile (0.3% formic acid and 0.3% triethylamine)-water (0.3% formic acid and 0.3% triethylamine) was very poor (see...). Figure 11 The baseline for methanol-0.1% formic acid was unstable and the resolution was poor (see [reference needed]). Figure 12The baseline of the methanol-0.1% phosphoric acid solution was relatively stable. Elution with methanol in the organic phase resulted in the best separation of the compound peaks. Furthermore, the addition of phosphoric acid to the inorganic phase reduced peak tailing after acidification. Therefore, the methanol-0.1% phosphoric acid solution was chosen as the mobile phase (see...). Figures 1 to 2 Since the retention times of each component in reversed-phase chromatography vary greatly, a gradient elution method was adopted after reviewing the literature. This method can achieve better separation between the peak of the analyte and other adjacent impurity peaks, and ensure a stable baseline.

[0118] This experiment investigated the program for the mobile phase "methanol-0.1% phosphoric acid solution" selected in the previous study, and set up 5 gradient elution programs as follows:

[0119] Gradient 1: Methanol, 0-20 min: 5%-10%; 20-35 min: 10%-20%; 35-60 min: 20%-25%; 60-85 min: 25%-28%; 85-100 min: 28%-42%; 100-120 min: 42%-70%;

[0120] Gradient 2: Methanol, 0-15 min: B, 7%-10%; 15-20 min: 10%-17%; 20-30 min: 17%-20%; 30-60 min: 20%-25%; 60-85 min: 25%-28%; 85-105 min: 28%-42%; 105-120 min: 42%-70%;

[0121] Gradient 3: Methanol, 0-15 min: B, 7%-10%; 15-20 min: 10%-17%; 20-25 min: 17%-20%; 25-40 min: 20%-25%; 40-60 min: 25%-28%; 60-80 min: 28%-42%; 80-100 min: 42%-70%;

[0122] Gradient 4: Methanol, 0-20 min: B, 5%-10%; 20-35 min: 10%-20%; 35-60 min: 20%-25%; 60-85 min: 25%-28%; 85-110 min: 28%-42%; 110-125 min: 42%-70%;

[0123] Gradient 5: Methanol, 0-15 min: B, 7%-10%; 15-20 min: 10%-17%; 20-25 min: 17%-20%; 25-50 min: 20%-25%; 50-70 min: 25%-28%; 70-90 min: 28%-42%; 90-110 min: 42%-70%;

[0124] Results: The gradient 5 elution program yielded the best chromatographic separation. Therefore, the preferred elution program of this invention is the optimal one. See Figures 13 to 17 .

[0125] 3.2 Selection of detection wavelength

[0126] The chromatogram of Longqing capsules obtained in this experiment showed that the elution order of the 12 components was gallic acid, berberine hydrochloride, chlorogenic acid, caffeic acid, paeoniflorin, berberine, berberine hydrochloride, luteolin, paeonol, rutin, and kaempferol. Ultraviolet scanning was performed at a detection wavelength of 200–400 nm to examine the chromatograms of each component. Common peaks were found at 230 nm, 250 nm, and 270 nm. However, based on the sensitivity and content of each component, 270 nm was ultimately selected as the detection wavelength.

[0127] 3.3 Conclusion

[0128] Based on the traditional Chinese medicine formula theory of "principal, assistant, adjuvant, and guide," this study selected Longqing Capsules for multi-index content determination. This experiment used HPLC to simultaneously determine 12 components in Longqing Capsules. The method is simple, easy to perform, and the results are reliable. This provides a basis for simultaneously controlling the quality of 12 components in Longqing Capsules: paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, coptisine, cypermethrin hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride.

[0129] The embodiments described herein are merely preferred embodiments of the invention and are not intended to limit the concept and scope of the invention. Any modifications and improvements made by those skilled in the art to the technical solutions of the invention without departing from the design concept of the invention should fall within the protection scope of the invention.

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Claims

1. A method for simultaneously determining the content of 12 components in Longqing capsules using HPLC, characterized in that, The method for simultaneously determining the contents of paeoniflorin, paeonol, chlorogenic acid, caffeic acid, berberine hydrochloride, coptisine, berberine hydrochloride, gallic acid, rutin, kaempferol, luteolin, and berberine hydrochloride in Longqing capsules includes the following steps: (1) Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel; gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol solution as mobile phase B; column temperature: 35℃; detection wavelength: 270nm; flow rate: 1.0mL / min; injection volume: 10μL; The gradient elution procedure is as follows: ； (2) Preparation of reference solution: Accurately weigh an appropriate amount of reference standard and add methanol to prepare a mixed reference solution containing paeoniflorin 0.174 mg / ml, paeonol 0.007 mg / ml, chlorogenic acid 0.160 mg / ml, caffeic acid 0.037 mg / ml, berberine hydrochloride 0.166 mg / ml, berberine 0.028 mg / ml, berberine hydrochloride 0.027 mg / ml, gallic acid 0.069 mg / ml, rutin 0.029 mg / ml, kaempferol 0.004 mg / ml, luteolin 0.006 mg / ml, and berberine hydrochloride 0.110 mg / ml. (3) Preparation of test solution: Take an appropriate amount of Longqing capsules, grind them into a fine powder, take 0.5-1.5g, weigh accurately, place in a stoppered conical flask, add 50-150ml of methanol accurately, weigh, sonicate for 30-60min, cool, add methanol to make up the weight, shake well, filter, and the test solution is obtained. (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

2. The method for simultaneously determining the content of 12 components in Longqing capsules using HPLC according to claim 1, characterized in that, The chromatographic column mentioned in step (1) is: ShimNex CS C 18 Chromatographic column.

3. The method for simultaneously determining the content of 12 components in Longqing capsules using HPLC according to claim 2, characterized in that, The ShimNex CS C 18 The chromatographic column specifications are: 4.6mm × 250mm, 5µm.

4. The method for simultaneously determining the content of 12 components in Longqing capsules using HPLC according to claim 1, characterized in that, The preparation of the test solution in step (3) is as follows: take an appropriate amount of Longqing capsules, grind them into a fine powder, take 1g, weigh it accurately, place it in a stoppered conical flask, add 100ml of methanol accurately, weigh it, sonicate for 30min, cool it, add methanol to make up the weight, shake it well, filter it, and the solution is obtained.

5. The method for simultaneously determining the content of 12 components in Longqing capsules using HPLC according to claim 1, characterized in that, The ultrasonic conditions are: power of 200-300W and frequency of 30-50kHz.

6. The method for simultaneously determining the content of 12 components in Longqing capsules using HPLC according to claim 5, characterized in that, The ultrasonic conditions are: power of 250W and frequency of 40kHz.

7. The method for simultaneously determining the content of 12 components in Longqing capsules using HPLC according to claim 1, characterized in that, The filtration is performed using a 0.22μm microporous membrane.

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