CN112595793B - Earthworm injection detection method based on phenol determination - Google Patents

Earthworm injection detection method based on phenol determination Download PDF

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CN112595793B
CN112595793B CN202011631330.2A CN202011631330A CN112595793B CN 112595793 B CN112595793 B CN 112595793B CN 202011631330 A CN202011631330 A CN 202011631330A CN 112595793 B CN112595793 B CN 112595793B
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mobile phase
phenol
volume fraction
injection
earthworm
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CN112595793A (en
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苗瑞娟
张雷
陈华
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South China University of Technology SCUT
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

Abstract

The invention provides a earthworm injection detection method based on phenol determination, which adopts liquid chromatography to separate phenol in earthworm injection and then determines the phenol at 270 nm; the stationary phase of the chromatographic column is octadecylsilane chemically bonded silica, and the elution mode is gradient elution; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 20mM diammonium phosphate, and the mobile phase B is 40% acetonitrile. The phenol is separated from the earthworm injection and then measured, and the detection result has good linear relation, high detection precision, good stability, good repeatability, good durability, simple and convenient operation, stability and reliability. According to the phenol measurement result, whether the addition amount of phenol in the earthworm injection meets the product standard or not is judged, so that the quality of the finished earthworm injection can be effectively controlled, and the medication safety is ensured.

Description

Earthworm injection detection method based on phenol determination
Technical Field
The invention relates to the technical field of medicines, in particular to a earthworm injection detection method based on phenol determination.
Background
The Lumbricus injection contains various components such as nucleoside, amino acid, protein and polypeptide, lipid, enzymes and microelements, has effects of relieving asthma and cough, lowering blood pressure, etc., and can be used for treating cough and asthma caused by bronchial asthma. Phenol was added as a bacteriostatic.
Hypoxanthine and nitrogen-containing compounds are main active ingredients of earthworm injection for relieving asthma and cough, and the existing quality standard content determination part only comprises the steps of determining the total nitrogen content by a nitrogen determination method, determining amino nitrogen by a titration method and determining the content of valine by a thin-layer chromatography.
Phenol as a bacteriostatic agent has certain toxicity and corrosivity, the phenol is not checked and the content of the phenol is not measured by the existing quality standard, the phenol measuring method in the four parts of the 2020 edition of Chinese pharmacopoeia is an oxidation-reduction titration method and a back-titration method, and the defects of complicated experimental operation, easy oxidation of phenol, multiple interference factors and the like exist. "determination of phenol content in earthworm injection by reversed-phase high performance liquid chromatography" of Panyunjing, volume 32, 4 of 2013, discloses a method for determining phenol content in earthworm injection, which uses methanol-water (60:40) as a mobile phase, although the linear relationship of phenol in a concentration range is good, the average recovery rate is 99.46%, the RSD is 0.25% (n is 9), nucleoside components can not be completely separated under the condition, and the result reliability is low.
In order to effectively control the quality of the finished earthworm injection and reduce the potential safety hazard of medication, the novel method can detect the content of phenol while measuring the effective components, and provides reliable experimental basis for the comprehensive control of the quality of the earthworm injection.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: separating phenol from Lumbricus injection, and measuring to control quality of Lumbricus injection.
In order to solve the technical problems, the invention adopts the technical scheme that:
a detection method of Lumbricus injection based on phenol determination comprises separating phenol in Lumbricus injection by liquid chromatography, and determining at 270 nm; the stationary phase of the chromatographic column is octadecylsilane chemically bonded silica, and the elution mode is gradient elution; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 20mM diammonium hydrogen phosphate, and the mobile phase B is 40% acetonitrile; and monitoring the quality of the earthworm injection according to the measurement result of the phenol content.
Further, the liquid chromatography also simultaneously separates hypoxanthine, uridine, inosine and guanosine in the earthworm injection, wherein the hypoxanthine, uridine, inosine and guanosine are all measured at 248 nm; and monitoring the quality of the earthworm injection according to the content measurement results of hypoxanthine, uridine, inosine and guanosine respectively.
Further, the elution procedure of the gradient elution is as follows:
0min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
15min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
30min, wherein the volume fraction of the mobile phase A is 90%, and the volume fraction of the mobile phase B is 10%;
31min, wherein the volume fraction of the mobile phase A is 20%, and the volume fraction of the mobile phase B is 80%;
45min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%;
46min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
for 60min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
the flow rate was 1.0 mL/min-1。
In the liquid chromatography, the solvent used for sample treatment was 20mM diammonium phosphate.
Further, the concentration of hypoxanthine in the sample to be tested is controlled to 3.8-37 μ g/mL during sample treatment-1The concentration of uridine was controlled to 3.8-37. mu.g/mL-1The concentration of inosine was controlled to 19 to 187. mu.g.mL-1The concentration of guanosine is controlled to be 2.5-24 mu g/mL-1The concentration of phenol was controlled to 122--1。
Further, in the liquid chromatography, the sample volume of the sample to be detected is 10 μ L; the injector temperature was 15 ℃; the column temperature was 30 ℃.
Further, the chromatographic column is a Titank C18 chromatographic column with the specification of 250X 4.6mm and 5 μm.
The invention has the beneficial effects that: the phenol is separated from the earthworm injection and then measured, and the detection result has good linear relation, high detection precision, good stability, good repeatability, good durability, simple and convenient operation, stability and reliability. According to the phenol measurement result, whether the addition amount of phenol in the earthworm injection meets the product standard or not is judged, so that the quality of the finished earthworm injection can be effectively controlled, and the medication safety is ensured.
Drawings
The detailed structure of the invention is described in detail below with reference to the accompanying drawings
FIG. 1 is HPLC chromatograms of a reference solution and a test solution of the earthworm injection detection method based on phenol determination, wherein A is the chromatogram of the reference solution at 248nm, B is the chromatogram of the test solution at 248nm, C is the chromatogram of the reference solution at 270nm, and D is the chromatogram of the test solution at 270 nm; 1-hypoxanthine (hypoxanthine), 2-uridine (uridine), 3-inosine (inosine), 4-guanosine (guanosine), 5-phenol (phenol).
Fig. 2 is an HPLC chromatogram (λ 200nm to 400nm) of a control solution of the method for detecting earthworm injection based on phenol assay according to the present invention.
Detailed Description
The technical solution of the present invention will be further explained in detail based on the detailed embodiments of the technical contents, the structural features, the achieved objects and the effects of the present invention, and with reference to the accompanying drawings.
Examples
A detection method of Lumbricus injection based on phenol determination comprises separating phenol in Lumbricus injection by liquid chromatography, and determining at 270 nm; the liquid chromatography also separates hypoxanthine, uridine, inosine and guanosine in the earthworm injection respectively, and the hypoxanthine, the uridine, the inosine and the guanosine are all measured at 248 nm; and monitoring the quality of the earthworm injection according to the content measurement results of hypoxanthine, uridine, inosine, guanosine and phenol respectively.
The chromatographic column used is a Titank C18 chromatographic column with the specification of 250X 4.6mm and 5 μm. The stationary phase of the chromatographic column is octadecylsilane chemically bonded silica, and the elution mode is gradient elution; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 20mM diammonium hydrogen phosphate, and the mobile phase B is 40% acetonitrile; the elution procedure for the gradient elution was:
0min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
15min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
30min, wherein the volume fraction of the mobile phase A is 90%, and the volume fraction of the mobile phase B is 10%;
31min, wherein the volume fraction of the mobile phase A is 20%, and the volume fraction of the mobile phase B is 80%;
45min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%;
46min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
for 60min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
the flow rate was 1.0 mL/min-1The sample volume of the sample to be detected is 10 mu L; the injector temperature was 15 ℃; the column temperature was 30 ℃.
When the sample is processed, the concentration of hypoxanthine in the sample to be detected is controlled to be 3.8-37 mu g/mL-1The concentration of uridine was controlled to 3.8-37. mu.g/mL-1The concentration of inosine was controlled to 19 to 187. mu.g.mL-1The concentration of guanosine is controlled to be 2.5-24 mu g/mL-1The concentration of phenol was controlled to 122--1. The solvent used for controlling the concentration of each component is 20mM diammonium hydrogen phosphate.
To further illustrate the feasibility of the solution of the present application, the following experimental examples are given:
test examples
1 Instrument and reagent
1.1 instruments
High performance liquid chromatograph A, a Waters e 2695-2998 model high performance liquid chromatograph (Waters corporation, USA); high performance liquid chromatograph B, a Waters e 2695-2489 high performance liquid chromatograph (Waters corporation, USA); electronic analytical balances of CPA225D type and ME5 type (Satorius corporation); ultra pure water machine (Milli-Q).
1.2 chromatography columns
A chromatographic column a: titank C18 (250X 4.6mm, 5 μm) (S/N: 126BA90150), column b: titank C18(250 mm. times.4.6 mm, 5 μm) (S/N: 126BA90130)
1.3 reagent acetonitrile (chromatographically pure, Honeywell, USA); 20mM diammonium phosphate is domestic analytically pure.
1.4 hypoxanthine control (batch No. 140661-201704, content 100%), uridine control (batch No. 110887-201803, content 99.5%), inosine control (batch No. 140669-201606, content 100%), guanosine control (batch No. 111977-201501, content 93.6%), and phenol control (batch No. 100509-201804, content 100%) all purchased from China food and drug testing institute.
1.5 sample earthworm injection provided by Guangdong Xinfeng pharmaceutical industry Co., Ltd, the batches are 180301, 191201, 191202, 200101, 200102, 200103, 200104, 200105, 200106, 200107 and 200108 respectively, and the specifications are as follows: 2 mL/piece.
1.6 preparation of control stock solutions: respectively and precisely weighing appropriate amounts of hypoxanthine, uridine, inosine and guanosine reference substances, placing the reference substances into the same 25mL volumetric flask, dissolving the reference substances by using 20mM diammonium hydrogen phosphate, and fixing the volume to a scale to prepare a mixed reference substance stock solution containing 0.18mg of hypoxanthine, 0.17mg of uridine, 0.93mg of inosine and 0.13mg of guanosine per 1 mL. Accurately weighing a proper amount of phenol reference substance, placing the phenol reference substance in a 10mL volumetric flask, dissolving the phenol reference substance by using the mobile phase A, and fixing the volume to a scale to prepare a phenol reference substance stock solution containing 6.0mg of phenol per 1 mL.
1.7 preparation of control solutions: precisely measuring 1mL of the mixed reference stock solution and 1.6 mL of the phenol reference stock solution respectively, placing the mixed reference stock solution and the phenol reference stock solution into the same 10mL volumetric flask, diluting the mixed reference stock solution to a scale by using 20mM diammonium phosphate, and shaking the mixed reference stock solution and the phenol reference stock solution uniformly to obtain a reference solution.
1.8 preparation of test solution: precisely measuring 2mL of earthworm injection in 1.5, adding into a 10mL volumetric flask, diluting to a constant volume to a scale with 20mM diammonium hydrogen phosphate, shaking up, and filtering to obtain a test solution.
1.9 determination: respectively injecting the reference solution and the sample solution into a high performance liquid chromatograph for determination to obtain peak areas A of 4 nucleosides and phenol in the sample solutionTest articleAnd the corresponding chromatographic peak area A in the reference solutionReference substance；
The content of nucleoside or phenol in the earthworm injection is calculated according to the following formula:
Figure GDA0003297901640000051
in equation (1): x-content of nucleoside or phenol in injection, MR-the amount of the reference, w-the purity of the reference, As-the area of the peak of the nucleoside or phenol in the test solution, Ns-the dilution factor of the test solution, ARPeak area of nucleoside or phenol in control solution, NR-dilution factor of the control solution.
2 methods and results
2.1 HPLC chromatographic conditions
2.1.1 determination of elution conditions
The chromatographic column is Titank C18 (250X 4.6mm, 5 μm); mobile phase: 20mM hydrogen phosphateDiammonium (a) -40% acetonitrile (B); flow rate: 1.0 mL/min-1(ii) a Column temperature: 30 ℃; λ is 248nm (hypoxanthine, uridine, inosine, guanosine) and 270nm (phenol); sample injector temperature: 15 ℃; sample introduction amount: 10 mu L of the solution; the gradient elution procedure is shown in table 1.
The chromatograms of the control solution and the test solution at 248nm and 270nm under the chromatographic conditions of 2.1.1 are shown in FIG. 1. As can be seen from FIG. 1, hypoxanthine, uridine, inosine, guanosine, and phenol can be completely separated from the peaks without interfering with each other by using the condition of "2.1.1".
TABLE 1 gradient elution procedure
Figure GDA0003297901640000061
2.1.2 determination of the detection wavelength
Taking the reference solution, scanning with PDA detector at full wavelength, and detecting according to the chromatographic conditions under the item "2.1.1". The full-wavelength scan (λ 200nm to 400nm) is shown in detail in fig. 2, and as can be seen from the results in fig. 2, λmax (hypoxanthine)＝249.6nm，λmax (uridine)＝261.4nm，λmax (inosine)＝248.4nm，λmax (guanosine)＝253.1nm，λmax (phenol)For 270.9nm, the wavelengths at 248nm were chosen for measurement of hypoxanthine, uridine, inosine and guanosine, and the wavelength at 270nm was chosen for measurement of phenol.
2.2 methodological investigation
TABLE 2 investigation of the Linear relationship of the components
Figure GDA0003297901640000062
2.2.1 Linear relationship examination, precisely sucking the mixed reference stock solution and the phenol reference stock solution in sequence, respectively 0.2mL, 0.5mL, 0.8mL, 1.0mL, 1.2mL, 1.5mL and 2.0mL, placing the mixed reference stock solution and the phenol reference stock solution in a 10mL volumetric flask, diluting with the mobile phase A, fixing the volume to a scale, and shaking up to obtain a series of mixed reference solutions. Injecting sample according to HPLC chromatographic condition under item "2.1.1", and recording chromatogram. In comparison with the mass concentration X (μ g. mL)-1) The abscissa represents the area of the peak, and the ordinate represents the area of the peak. The detailed results are shown in Table 2.
2.2.2 precision absorption of the reference solution, continuous sample injection of 6 needles according to HPLC chromatographic conditions, recording chromatographic peak areas of each component, and calculating RSD. As a result, the peak areas RSD (n ═ 6) of hypoxanthine, uridine, inosine, guanosine, and phenol were 0.22%, 0.69%, 0.10%, 0.47%, and 0.06%, respectively, and were all less than 2%, indicating that the method was accurate. The detailed results are shown in Table 3.
TABLE 3 precision test results of each component
Figure GDA0003297901640000071
2.2.3 Stable earthworm injection (batch 180301), preparing a test solution according to the preparation method of the test solution, placing the test solution in a sample injector at 15 ℃, injecting 10 mu L of the sample in 0h, 4h, 8h, 12h, 16h and 24h respectively, recording the chromatographic peak area of each component, and calculating RSD. As a result, the peak areas RSD (n ═ 6) of hypoxanthine, uridine, inosine, guanosine, and phenol were 0.30%, 0.87%, 0.25%, 0.41%, and 0.35%, respectively, indicating that the test article solution was stable within 24 hours. The details are shown in Table 4.
TABLE 4 stability test results of various components in earthworm injection
Figure GDA0003297901640000072
2.2.4 repeatedly taking the same Diolong injection (batch 180301), preparing 6 parts of test solution in parallel according to the preparation method of the test solution under the item '1.8', injecting samples according to the HPLC chromatographic condition under the item '2.1.1', recording a chromatogram, and calculating the average content and RSD of each component. As a result, the average contents of hypoxanthine, uridine, inosine, guanosine, and phenol (n ═ 6) in the earthworm injection were 0.0868 mg/mL, respectively-1、0.0788mg·mL-1、0.3786mg·mL-1、0.0546mg·mL-1、2.7811mg·mL-1RSD is respectively 0.50%, 0.60%, 0.61%0.57% and 0.49% of the total amount of the components, which are less than 2%, and the detailed results are shown in Table 5. As can be seen from Table 5, the method is very reproducible.
TABLE 5 results of the repeatability experiment of each component in the test solution of earthworm injection
Figure GDA0003297901640000081
2.2.5 sample application recovery ratio 1mL of Lumbricus injection (batch 180301) with known content is precisely absorbed, placed in 10mL volumetric flask, and mixed reference stock solution and phenol reference stock solution (5 components are approximately 80%, 100% and 120% of the sample content) are added according to low, medium and high 3 concentrations, and three test solutions are prepared in parallel at each concentration, and the detailed preparation method is shown in Table 6.
TABLE 6 method for preparing experimental sample solution for sample recovery rate
Figure GDA0003297901640000082
According to HPLC chromatographic conditions, 10. mu.L of sample is injected, the chromatographic peak area is recorded and the average recovery rate and RSD value are calculated. The average recovery rates (n is 9) of hypoxanthine, uridine, inosine, guanosine and phenol were 99.90%, 97.67%, 98.69%, 96.44% and 97.79%, respectively, and RSDs were 1.13%, 1.16%, 0.64%, 1.90% and 0.87%, respectively, which were less than 2%, indicating that the method has good accuracy, and the detailed data are shown in table 7.
TABLE 7 recovery rate of each component in Lumbricus injection
Figure GDA0003297901640000091
Figure GDA0003297901640000101
2.2.6 durable earthworm injection (batch 180301), a test solution was prepared according to the method under item "1.8", sample injection was performed according to the HPLC condition under item "2.1.1" under condition (1) (HPLC A and column a), condition (2) (HPLC A and column B), and condition (3) (HPLC B and column B), respectively, the peak areas of the components were recorded, and the content and RSD were calculated. As a result, the same Diolong injection (batch No. 180301) contained less than 2% of hypoxanthine, uridine, inosine, guanosine, and phenol in all RSD values, indicating that the method was excellent in durability. The detailed results are shown in Table 8.
Table 8 method durability results
Figure GDA0003297901640000102
3 determination of the content
Taking 10 batches of earthworm injection with different batches, respectively preparing test solution according to the preparation method of the test solution, carrying out sample injection analysis according to HPLC (high performance liquid chromatography) chromatographic conditions, recording chromatographic peak areas, and calculating the content of each component according to an external standard method. The measurement results of the contents of the components in different batches of earthworm injection are shown in Table 9.
As can be seen from the results in Table 9, the method is simple, accurate and can effectively detect the contents of 4 nucleosides and phenol in the earthworm injection simultaneously, so that the method can be used for the comprehensive control of the quality of the finished earthworm injection.
TABLE 9 measurement results of contents of components in different batches of Lumbricus injection (unit: mg. multidot.mL)-1)
Figure GDA0003297901640000111
In conclusion, the earthworm injection detection method based on phenol determination provided by the invention not only can quantitatively determine the content of 4 nucleosides and phenol in the earthworm injection at the same time, but also can completely separate 4 nucleosides, phenol and other various impurities, has strong specificity, high separation degree and sensitivity and good accuracy, and can be used for comprehensively controlling the quality of finished products of the earthworm injection. And judging whether the addition amount of the phenol in the earthworm injection meets the product standard or not according to the measurement results of hypoxanthine, uridine, inosine, guanosine and phenol, thereby effectively controlling the quality of the finished earthworm injection and ensuring the medication safety.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (4)

1. A detection method of Lumbricus injection based on phenol determination is characterized in that liquid chromatography is adopted to separate phenol in Lumbricus injection and determine at 270 nm; the stationary phase of the chromatographic column is octadecylsilane chemically bonded silica, and the elution mode is gradient elution; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 20mM diammonium hydrogen phosphate, and the mobile phase B is 40% acetonitrile; monitoring the quality of the earthworm injection according to the determination result of the phenol content;
the liquid chromatography also separates hypoxanthine, uridine, inosine and guanosine which are used for monitoring the quality of the earthworm injection in the earthworm injection respectively, and the hypoxanthine, the uridine, the inosine and the guanosine are all measured at 248 nm;
the solvent adopted during sample treatment is 20mM diammonium hydrogen phosphate; the elution procedure for the gradient elution was:
0min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
15min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
30min, wherein the volume fraction of the mobile phase A is 90%, and the volume fraction of the mobile phase B is 10%;
31min, wherein the volume fraction of the mobile phase A is 20%, and the volume fraction of the mobile phase B is 80%;
45min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%;
46min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
for 60min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0;
the flow rate was 1.0 mL/min-1。
2. The method for detecting Pheretima injection based on phenol assay of claim 1, wherein the concentration of hypoxanthine in the sample is controlled to 3.8-37 μ g/mL-1The concentration of uridine was controlled to 3.8-37. mu.g/mL-1The concentration of inosine was controlled to 19 to 187. mu.g.mL-1The concentration of guanosine is controlled to be 2.5-24 mu g/mL-1The concentration of phenol was controlled to 122--1。
3. The method for detecting earthworm injection based on phenol determination according to claim 2, wherein in the liquid chromatography, the sample volume of the sample to be detected is 10 μ L; the injector temperature was 15 ℃; the column temperature was 30 ℃.
4. The method for detecting earthworm injection based on phenol determination according to any one of claims 1 to 3, wherein the chromatographic column is a Titank C18 chromatographic column with a specification of 250 x 4.6mm and 5 μm.
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