Method for simultaneously determining contents of three effective components in Shuanghuanglian oral liquid

Abstract

The invention discloses a method for simultaneously determining the contents of three active ingredients of chlorogenic acid, baicalin and forsythin in Shuanghuanglian oral liquid, which adopts high performance liquid chromatography, takes octadecylsilane chemically bonded silica as a filler, takes a glacial acetic acid solution with the concentration of 0.5% (g / ml) as a mobile phase A and takes acetonitrile as a mobile phase B for gradient elution, and the detection wavelength is 298 nm. The method is high in specificity, high in precision, good in accuracy, simple and convenient to operate, low in cost and short in consumed time, the quality of the Shuanghuanglian oral liquid can be conveniently, rapidly and effectively controlled, and the stability of the product quality and the safety and effectiveness of medication are guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection and relates to a method for simultaneously determining the contents of three active ingredients in Shuanghuanglian oral liquid. Background Art

[0002] Shuanghuanglian oral liquid is a pure Chinese medicine preparation, composed of three Chinese medicines: Scutellaria baicalensis, honeysuckle, and forsythia. Honeysuckle is sweet and cold in nature, aromatic and dispersing, good at clearing heat evil from the lung meridian, and is the main medicine; Scutellaria baicalensis is bitter and cold in nature, good at clearing lung fire and upper-jiao heat; Forsythia suspensa is bitter and slightly cold in nature, dispersing wind-heat in the upper jiao, and has the effect of clearing heat and detoxifying, and the two are the ministerial medicines. The three medicines are used together to achieve the effects of relieving exterior symptoms with pungent and cool properties, clearing heat and detoxifying. Shuanghuanglian oral liquid has been widely used in veterinary clinics, such as treating various bacterial and viral diseases and colds and fevers in dogs, pigs, chickens, and sheep, and has achieved significant therapeutic effects.

[0003] Chlorogenic acid, forsythin and baicalin are the main active ingredients of Shuanghuanglian oral liquid. However, there are few reports on methods for simultaneously determining the contents of these three ingredients. In the Chinese Pharmacopoeia of Veterinary Medicine, the contents of these three active ingredients under Shuanghuanglian oral liquid need to be determined separately by two methods, which requires a long determination time or requires the use of two high-performance liquid chromatographs to work simultaneously.

[0004] In order to better control product quality and ensure product efficacy, it is necessary to establish a method for simultaneously determining the contents of three active ingredients, chlorogenic acid, baicalin and forsythin, in Shuanghuanglian oral liquid. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for simultaneously determining the contents of three active ingredients, namely chlorogenic acid, baicalin and forsythin, in Shuanghuanglian oral liquid. The method is simple to operate, low in cost and time, and can conveniently, quickly and effectively control the quality of Shuanghuanglian oral liquid.

[0006] After research, the present invention provides the following technical solutions:

[0007] 1. A method for simultaneously determining the contents of three active ingredients in Shuanghuanglian oral liquid, characterized in that: the contents of chlorogenic acid, baicalin and forsythin are determined by high performance liquid chromatography;

[0008] 1) Chromatographic conditions

[0009] Octadecylsilane bonded silica gel is used as filler;

[0010] Use 0.5% (g / ml) glacial acetic acid solution as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution according to the following procedure:

[0011] 0-3.5min: the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%;

[0012] 3.5-6min: The volume fraction of mobile phase A changes linearly from 90% to 85%, and the volume fraction of mobile phase B changes linearly from 10% to 15%;

[0013] 6-15min: Mobile phase A volume fraction is 85%, mobile phase B volume fraction is 15%;

[0014] 15-17min: The volume fraction of mobile phase A changes linearly from 85% to 78%, and the volume fraction of mobile phase B changes linearly from 15% to 22%;

[0015] 17-25min: the volume fraction of mobile phase A is 78%, and the volume fraction of mobile phase B is 22%;

[0016] 25-27min: the volume fraction of mobile phase A changes linearly from 78% to 90%, and the volume fraction of mobile phase B changes linearly from 22% to 10%;

[0017] 27-30min: Mobile phase A volume fraction is 90%, mobile phase B volume fraction is 10%;

[0018] The detection wavelength is 298nm;

[0019] 2) Preparation of mixed reference solution

[0020] Accurately weigh chlorogenic acid reference substance, baicalin reference substance, and forsythin reference substance, and dissolve them in 50% (ml / ml) methanol to prepare single reference substance solutions. Then accurately pipette each single reference substance solution and mix them, and dilute them with 50% (ml / ml) methanol to prepare a mixed reference substance solution.

[0021] 3) Preparation of test solution

[0022] Accurately measure Shuanghuanglian oral liquid, add 50% (ml / ml) methanol, ultrasonically treat for 20 minutes, cool to 10-30°C, dilute with 50% (ml / ml) methanol, mix well, filter, and take the filtrate as the test solution;

[0023] 4) Determination method

[0024] Accurately pipette the mixed reference solution and test solution separately, inject into liquid chromatograph, record the chromatogram, and calculate the contents of chlorogenic acid, baicalin and forsythin in the test sample by peak area according to the external standard method.

[0025] The beneficial effects of the present invention are as follows: the present invention provides a method for simultaneously determining the contents of three active ingredients, namely chlorogenic acid, baicalin and forsythin, in Shuanghuanglian oral liquid, which has strong specificity, high precision, good accuracy, simple operation, inexpensive and easily available reagents and consumables, low cost, short time consumption, and can conveniently, quickly and effectively control the quality of Shuanghuanglian oral liquid, and ensure the stability of product quality and the safety and effectiveness of medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the chromatogram of chlorogenic acid reference solution (detection wavelength 274nm).

[0027] Figure 2 This is the chromatogram of baicalin reference solution (detection wavelength 324nm).

[0028] Figure 3 This is the chromatogram of the Forsythiaside reference solution (detection wavelength 278nm).

[0029] Figure 4 It is the chromatogram of the mixed reference solution (detection wavelength 298nm).

[0030] Figure 5 It is the chromatogram of the test solution (detection wavelength 298nm).

[0031] Figure 6 This is the chromatogram of the negative sample solution lacking Forsythia suspensa.

[0032] Figure 7 This is the chromatogram of the negative sample solution lacking Scutellaria baicalensis.

[0033] Figure 8 The chromatogram of the negative sample solution lacking chlorogenic acid. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and beneficial effects of the present invention more clear, the preferred embodiments of the present invention are described in detail below.

[0035] The chlorogenic acid reference substance used in the preferred embodiment was purchased from the China Food and Drug Inspection Institute, the baicalin reference substance was purchased from the China Veterinary Drug Inspection Institute, and the forsythiaside reference substance was purchased from the China Food and Biological Products Inspection Institute.

[0036] Example 1 Method for simultaneously determining the contents of three active ingredients in Shuanghuanglian oral liquid

[0037] HPLC was used to simultaneously determine the contents of chlorogenic acid, baicalin and forsythin in Shuanghuanglian oral liquid.

[0038] 1) Chromatographic conditions

[0039] Column: SunFireTM C18 (4.6mm×150mm, 5μm)

[0040] Use 0.5% (g / ml) glacial acetic acid solution as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution according to the following procedure:

[0041] 0-3.5min: the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%;

[0042] 3.5-6min: The volume fraction of mobile phase A changes linearly from 90% to 85%, and the volume fraction of mobile phase B changes linearly from 10% to 15%;

[0043] 6-15min: Mobile phase A volume fraction is 85%, mobile phase B volume fraction is 15%;

[0044] 15-17min: The volume fraction of mobile phase A changes linearly from 85% to 78%, and the volume fraction of mobile phase B changes linearly from 15% to 22%;

[0045] 17-25min: the volume fraction of mobile phase A is 78%, and the volume fraction of mobile phase B is 22%;

[0046] 25-27min: the volume fraction of mobile phase A changes linearly from 78% to 90%, and the volume fraction of mobile phase B changes linearly from 22% to 10%;

[0047] 27-30min: Mobile phase A volume fraction is 90%, mobile phase B volume fraction is 10%;

[0048] The detector is a diode array detector; the detection wavelengths of the chlorogenic acid reference solution, baicalin reference solution and forsythiaside reference solution are 274nm, 324nm and 278nm respectively; the detection wavelength of the mixed reference solution and the test solution is 298nm;

[0049] The flow rate is 1.0 ml / min.

[0050] 2) Preparation of mixed reference solution

[0051] Accurately weigh the chlorogenic acid reference substance, baicalin reference substance, and forsythin reference substance, and dissolve them in 50% (ml / ml) methanol to prepare a single reference substance solution. Then accurately pipette each single reference substance solution and mix them, and dilute them with 50% (ml / ml) methanol to prepare a mixed reference substance solution containing 10 μg chlorogenic acid, 200 μg baicalin, and 60 μg forsythin per 1 ml;

[0052] 3) Preparation of test solution

[0053] Accurately measure 1 ml of Shuanghuanglian oral liquid in a 50 ml volumetric bottle, add 50% (ml / ml) methanol, ultrasonically treat for 20 min, cool to room temperature, dilute to scale with 50% (ml / ml) methanol, shake well, filter, and take the filtrate as the test solution;

[0054] 4) Determination method

[0055] Accurately pipette 10 μl of chlorogenic acid reference solution, baicalin reference solution, forsythiaside reference solution, mixed reference solution and test solution respectively, inject into liquid chromatograph, record the chromatogram, and calculate the contents of chlorogenic acid, baicalin and forsythiaside in the test sample by peak area according to the external standard method.

[0056] Figure 1 It is the chromatogram of chlorogenic acid reference solution (detection wavelength 274nm); Figure 2 is the chromatogram of baicalin reference solution (detection wavelength 324nm); Figure 3 This is the chromatogram of the forsythiaside reference solution (detection wavelength 278nm); Figure 4 and Figure 5 The chromatograms are respectively of the mixed reference solution and the test solution (detection wavelength 298nm).

[0057] Example 2 Methodological Investigation

[0058] 1. Exclusivity

[0059] The following three negative samples were prepared: a negative sample lacking Forsythia suspensa (mixed with honeysuckle and scutellaria baicalensis), a negative sample lacking scutellaria baicalensis (mixed with honeysuckle and Forsythia suspensa), and a negative sample lacking chlorogenic acid (mixed with scutellaria baicalensis and Forsythia suspensa). The above three negative samples were taken respectively, and negative sample solutions were prepared according to the preparation method of the test solution in Example 1, and the samples were measured according to the chromatographic conditions in Example 1, and the chromatograms were recorded.

[0060] The chromatograms of the three negative sample solutions are as follows: Figures 6 to 8 As shown. Figure 1 to Figure 3 and Figure 6 to Figure 8 Comparative analysis shows that no interference peaks appear in the chromatograms of the negative sample solutions at the retention time of the negative control, and the chromatographic peaks appearing in the figures are consistent with the retention time of the main peaks in the chromatograms of the corresponding control, thereby proving that there is no interference between the active ingredients at the retention time positions, proving that the method of the present invention has strong specificity.

[0061] 2. Linear Relationship

[0062] Accurately measure 0.5, 1, 2, 4, 6, and 8 ml of the mixed reference substance solution in Example 1 respectively and place them in a 10 ml volumetric flask, add 50% (ml / ml) methanol to dilute, and prepare a series of mixed reference substance dilutions. Perform the measurement according to the chromatographic conditions in Example 1, and record the chromatogram. Draw a standard curve with the injection volume X of the mixed reference substance dilution as the abscissa and the peak area Y as the ordinate to obtain the regression equation.

[0063] The results are shown in Table 1. Chlorogenic acid, baicalin, and forsythin showed good linear relationships with the peak area in the concentration ranges of 0.1-20, 5-250, and 3-80 μg / ml, respectively. The correlation coefficients R 2 All are greater than 0.999.

[0064] Table 1 Linear relationship investigation results

[0065] Tested ingredients Regression equation Linear range <![CDATA[R 2 ]]> Chlorogenic acid Y=298431X-39824 0.1–20 μg / mL 0.9995 Baicalin Y=21032X-12266 5–250 μg / mL 0.9996 Forsythiaside Y=5627.5X-38.015 3–80 μg / mL 0.9995

[0066] 3. Precision

[0067] Take the three single reference substance solutions in Example 1, and continuously inject and measure 6 times according to the chromatographic conditions in Example 1, record the chromatogram, and calculate the relative standard deviation (RSD) based on the 6 peak areas of the chromatographic peaks of each active ingredient.

[0068] The results showed that the RSDs of the peak areas of the three active ingredients, chlorogenic acid, baicalin and forsythin, were all less than 1, and were 0.84%, 0.37% and 0.63% respectively, proving that the method of the present invention has good precision.

[0069] 4. Sample recovery rate

[0070] Accurately measure 1 ml of Shuanghuanglian oral liquid sample with known content into a 50 ml volumetric flask, a total of 6 portions, accurately add each single reference solution respectively, prepare the test solution according to the method described in Example 1, and measure according to the chromatographic conditions in Example 1, record the chromatogram, calculate the content of each measured component, and calculate the average sample recovery rate of the measured component based on the amount of reference added.

[0071] The results are shown in Table 2. The average recoveries of the three active ingredients were all greater than 98%, and RSD was ≤ 1.3%, which proved that the method of the present invention has high accuracy.

[0072] Table 2 Results of sample recovery investigation

[0073] Tested ingredients Chlorogenic acid Baicalin Forsythiaside Average recovery rate (%) 98.96 99.07 99.85 RSD(%) 1.26 1.04 0.97

[0074] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for simultaneously determining the contents of three active ingredients in Shuanghuanglian oral liquid, characterized in that: The contents of chlorogenic acid, baicalin and forsythin were determined by HPLC; 1) Chromatographic conditions Octadecylsilane bonded silica gel is used as filler; With 0.5% (g / ml) glacial acetic acid solution as mobile phase A and acetonitrile as mobile phase B, gradient elution was performed according to the following program: 0-3.5 min: mobile phase A volume fraction was 90%, mobile phase B volume fraction was 10%; 3.5-6min: The volume fraction of mobile phase A changes linearly from 90% to 85%, and the volume fraction of mobile phase B changes linearly from 10% to 15%; 6-15min: Mobile phase A volume fraction is 85%, mobile phase B volume fraction is 15%; 15-17min: The volume fraction of mobile phase A changes linearly from 85% to 78%, and the volume fraction of mobile phase B changes linearly from 15% to 22%; 17-25min: the volume fraction of mobile phase A is 78%, and the volume fraction of mobile phase B is 22%; 25-27min: the volume fraction of mobile phase A changes linearly from 78% to 90%, and the volume fraction of mobile phase B changes linearly from 22% to 10%; 27-30min: Mobile phase A volume fraction is 90%, mobile phase B volume fraction is 10%; The detection wavelength is 298nm; 2) Preparation of mixed reference solution Accurately weigh chlorogenic acid reference substance, baicalin reference substance, and forsythin reference substance, and dissolve them in 50% (ml / ml) methanol to prepare single reference substance solutions. Then accurately pipette each single reference substance solution and mix them, and dilute them with 50% (ml / ml) methanol to prepare a mixed reference substance solution. 3) Preparation of test solution Accurately measure Shuanghuanglian oral liquid, add 50% (ml / ml) methanol, ultrasonically treat for 20 minutes, cool to 10-30°C, dilute with 50% (ml / ml) methanol, mix well, filter, and take the filtrate as the test solution; 4) Determination method Accurately pipette the mixed reference solution and test solution separately, inject into liquid chromatograph, record the chromatogram, and calculate the contents of chlorogenic acid, baicalin and forsythin in the test sample by peak area according to the external standard method.

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