CN108872436B - Method for analyzing content of D (-) p-hydroxyphenylglycine in D (-) p-hydroxyphenylglycine methyl ester - Google Patents
Method for analyzing content of D (-) p-hydroxyphenylglycine in D (-) p-hydroxyphenylglycine methyl ester Download PDFInfo
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Abstract
The invention discloses an HPLC method for determining the content of D (-) p-hydroxyphenylglycine in D (-) p-hydroxyphenylglycine methyl ester. Preparing D (-) p-hydroxyphenylglycine methyl ester into a solution by using a dilute acid solution, adopting a chemically bonded stationary phase chromatographic column, adding organic base into buffer salt, adjusting the pH to 0.8-12 by using acid to obtain buffer salt solution, carrying out gradient elution on the buffer salt solution and an organic phase modifier in a proper proportion, detecting at a proper detection wavelength, a proper flow rate and a proper column temperature, and calculating the content of a sample according to a spectrogram. The method can effectively separate the D (-) p-hydroxyphenylglycine methyl ester and the D (-) p-hydroxyphenylglycine and accurately measure the content of the D (-) p-hydroxyphenylglycine, has the advantages of simplicity, reliability, rapidness, high sensitivity, good reproducibility and specificity, and establishes a foundation for establishing the quality standard of the D (-) p-hydroxyphenylglycine methyl ester.
Description
Technical Field
The invention relates to a method for determining and analyzing the content of D (-) p-hydroxyphenylglycine in D (-) methyl p-hydroxyphenylglycine, belonging to the field of pharmaceutical detection in pharmaceutical and chemical industries.
Background
The D (-) p-hydroxyphenylglycine methyl ester is an organic intermediate synthetic body, is prepared from D (-) p-hydroxyphenylglycine and methanol, can be used for synthesizing amoxicillin, has similar antibacterial activity to sensitive bacteria to chloramphenicol and thiamphenicol, but is still sensitive to bacteria resistant to chloramphenicol and thiamphenicol, such as escherichia coli, klebsiella, pasteurella haemolytica, staphylococcus aureus, actinobacillus pleuropneumoniae, salmonella typhi and the like. However, the yield and quality of D (-) p-hydroxyphenylglycine in the product are directly influenced.
The CN201310725200.9 patent mentions the synthesis of β -lactone amide antibiotic enzyme using D (-) p-hydroxyphenylglycine esters as raw materials, which has the defect of difficult product filtration, etc., and the residue of D (-) p-hydroxyphenylglycine is not studied, although it is discussed for the isomer case due to the small amount of L-p-hydroxyphenylglycine ester and unreacted D (-) p-hydroxyphenylglycine in the raw materials.
In the patent CN201210196387.3, it is mentioned that the D (-) p-hydroxyphenylglycine residue is controlled by TLC in the middle of the product and the content of the finished product of D (-) p-hydroxyphenylglycine methyl ester is analyzed by HPLC, but the D (-) p-hydroxyphenylglycine residue is not controlled in the finished product, and the TLC is used for controlling the residue with poor precision and cannot be accurately and quantitatively analyzed.
At present, the method for measuring and analyzing the content of the D (-) p-hydroxyphenylglycine in the D (-) methyl p-hydroxyphenylglycine ester has not been elaborated in detail. In summary, in order to match the development of the use of D (-) p-hydroxyphenylglycine methyl ester, the present invention has conducted a study on a method for measuring and analyzing the content of D (-) p-hydroxyphenylglycine in D (-) p-hydroxyphenylglycine methyl ester.
Disclosure of Invention
The invention provides an analysis method of the content of D (-) p-hydroxyphenylglycine in D (-) methyl p-hydroxyphenylglycine, which is simple to operate, accurate in result, good in reproducibility and high in sensitivity.
A method for analyzing the content of D (-) p-hydroxyphenylglycine in D (-) methyl p-hydroxyphenylglycine comprises the following steps:
(1) diluting a to-be-detected sample of D (-) p-hydroxyphenylglycine methyl ester to obtain a test solution;
(2) analyzing the test solution in the step (1) by adopting high performance liquid chromatography to obtain liquid chromatography data;
the mobile phase used by the high performance liquid chromatography is a mixed solution of a buffer salt solution and an organic phase modifier;
the preparation method of the buffer salt solution comprises the following steps:
mixing buffer salt and organic base, and then adding acid to adjust the pH value to 2.0-6.0 to obtain the salt;
(3) and (3) calculating the content of the D (-) p-hydroxyphenylglycine in the sample to be detected according to the liquid chromatography data obtained in the step (2).
In step (1), a control solution (if necessary) and a sample solution may be prepared from 0.1% phosphoric acid aqueous solution, methanol, an initial elution ratio solution, and various buffer salt solutions having a pH range of 0.8 to 7, preferably 0.1% phosphoric acid or methanol.
Preferably, in step (2), the chromatographic column used for the analysis is a chemically bonded stationary phase chromatographic column: ultimate XB-phenyl (ii), preferably a C18, C8, C3, AQ or phenyl column, more preferably a phenyl column; the column length is preferably 30-250 mm (more preferably 250 mm); the inner diameter of the column is preferably 1.0-10 mm (more preferably 4.6 mm); the granularity range of the chromatographic column packing is 1.8-10 mu m (more preferably 5 mu m);
the detector is a variable wavelength detector, the detection wavelength is 190-360 nm, preferably 220-230 nm, and more preferably 226 nm;
the flow rate of the mobile phase is 1.0 to 2.0mL/min, and more preferably 1.5 mL/min.
Preferably, in the step (2), the buffer salt is selected from acetate, phosphate or sulfate of sodium, potassium or ammonium, preferably monopotassium phosphate, and the concentration of the buffer salt in water is 0-1000 mM (more preferably 10 mM);
the organic base is selected from one of ethylenediamine, diethylamine, triethylamine and salts thereof, and more preferably triethylamine and salts thereof; the content of the organic alkali in the water is 0-10% (more preferably 0.1%) by volume or the concentration of the salt thereof is 0-1000 mM (more preferably 10 mM);
the organic phase modifier is selected from one of methanol, acetonitrile, tetrahydrofuran, ethanol, isopropanol, glycol and acetone.
Preferably, the buffer salt is selected from potassium dihydrogen phosphate, the organic base is selected from triethylamine and salts thereof, and the organic phase modifier is selected from acetonitrile or methanol.
Preferably, the buffer salt is 10-100 mM potassium dihydrogen phosphate aqueous solution;
the organic base is 0.1-1% of triethylamine water solution, and the pH value of the system is adjusted to 2.0-6.0 by phosphoric acid.
The acid used to adjust the pH of the buffered salt solution is selected from phosphoric acid, sulfuric acid, acetic acid, formic acid, trifluoroacetic acid, perchloric acid or hydrochloric acid, preferably phosphoric acid; the pH range is 0.8 to 12, preferably 3.0 to 5.0.
Preferably, the analysis is performed by a gradient elution method,
the initial elution ratio of the buffer salt solution to the organic phase modifier in the mobile phase is 100: 0 to 50: 50, end elution ratio 0: 100-100: 0, as a further preference, the initial elution ratio is preferably 98: 2, the end elution ratio is preferably 80: 20.
in the actual operation process, in order to reduce the influence of the solution mixing on the analysis process, the buffer salt solution and the organic phase modifier are generally not directly mixed, but a mobile phase A and a mobile phase B with different buffer salt concentrations are prepared first, and then the two phases are mixed for analysis. Preferably, in mobile phase a, the buffer salt solution: acetonitrile 98: 2; mobile phase B buffered saline solution: acetonitrile 80: 20.
Preferably, the content of D (-) p-hydroxyphenylglycine is calculated by an area normalization method.
Preferably, the content of D (-) p-hydroxyphenylglycine is calculated by the following formula: y 10.64660x + 0.72535;
wherein y represents a peak area and x represents a content.
Compared with the prior art, the invention has the beneficial effects that:
(1) the D (-) p-hydroxyphenylglycine methyl ester sample is not required to be specially treated, and only needs to be accurately weighed and then dissolved and diluted by a diluent.
(2) The method provided by the invention has the advantages that the content of the D (-) p-hydroxyphenylglycine in the D (-) p-hydroxyphenylglycine methyl ester is obtained, the operation is simple, the result is accurate, the reproducibility is good, and the sensitivity is high.
Drawings
FIG. 1 is a systematic adaptive chromatogram of D (-) p-hydroxyphenylglycine methyl ester in example 2, with a main peak-off time of 4.781min and a D (-) p-hydroxyphenylglycine peak-off time of 2.154 min.
FIG. 2 is a chromatogram of the mobile phase buffer solution of example 2 at pH 3.3 with a peak time of 4.733min for the main peak and 2.156min for D (-) p-hydroxyphenylglycine.
FIG. 3 is a chromatogram of the sample solution of example 3 at a mobile phase flow rate of 1.4mL/min showing a main peak time of 5.080min and a D (-) p-hydroxyphenylglycine peak time of 2.307 min.
FIG. 4 is a chromatogram of the solution of the standard sample of example 4 at a column temperature of 20 ℃ showing that the peak-off time of the main peak is 4.984min and the peak-off time of D (-) p-hydroxyphenylglycine is 2.166 min.
FIG. 5 is a chromatogram of oxidative degradation of D (-) p-hydroxyphenylglycine methyl ester sample, with a main peak-off time of 4.804min and a D (-) p-hydroxyphenylglycine peak-off time of 2.164 min.
FIG. 6 is a chromatogram of D (-) p-hydroxyphenylglycine methyl ester sample subjected to alkali degradation, wherein the peak-off time of the main peak is 4.793min, and the peak-off time of D (-) p-hydroxyphenylglycine is 2.152 min.
FIG. 7 is a chromatogram of D (-) p-hydroxyphenylglycine methyl ester sample obtained after acid degradation, wherein the peak-off time of the main peak is 4.796min, and the peak-off time of D (-) p-hydroxyphenylglycine is 2.156 min.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
The instrument used in each example is an Angilent high performance liquid chromatograph; the D (-) p-hydroxyphenylglycine methyl ester reference substance is prepared by self, and the content is more than 99.5 percent.
In the case of gradient elution, the ratio between the mobile phases is a volume ratio unless otherwise specified.
Example 1
Under selected chromatographic conditions: variable wavelength ultraviolet detector, detection wavelength: 226 nm; flow rate: 1.5 ml/min; column temperature: 25 ℃; sample introduction volume: 5 mu l of the solution; a chromatographic column: ultimate XB-phenyl (ii);
buffer salt solution: 10mM potassium dihydrogen phosphate +10mM triethylamine hydrochloride (solvent is water), pH adjusted to 3.5 with phosphoric acid.
Mobile phase A: buffer salt solution: acetonitrile 98: 2, mobile phase B: buffer salt solution: acetonitrile 80: 20. Elution was performed in a gradient ratio. The gradient elution procedure is shown in the following table:
| time (min) | 0 | 3 | 15 | 17 |
| Mobile phase A,% | 100 | 100 | 0 | 0 |
| Mobile phase B,% | 0 | 0 | 100 | 100 |
D (-) p-hydroxyphenylglycine stock solution: weighing 50mg of D (-) p-hydroxyphenylglycine reference substance into a 100ml volumetric flask, adding 0.1% phosphoric acid for dissolving to a constant volume, and shaking up.
System adaptation solution: weighing 50mg of D (-) p-hydroxyphenylglycine methyl ester reference substance into a 50ml volumetric flask, adding 1ml of D (-) p-hydroxyphenylglycine stock solution, then adding 0.1% phosphoric acid to the scale, and shaking up. (D (-) p-hydroxyphenylglycine concentration 0.01 mg/ml).
Test solution preparation: 50mg of D (-) p-hydroxyphenylglycine methyl ester sample is weighed into a 50ml volumetric flask, 0.1% phosphoric acid is added for dissolving to a constant volume, and the mixture is shaken up (the concentration is 1 mg/ml).
Adding a standard sample solution: weighing 50mg of D (-) p-hydroxyphenylglycine methyl ester sample into a 50mL volumetric flask, transferring 1.0mL of D (-) p-hydroxyphenylglycine stock solution into the same volumetric flask, dissolving and diluting the D (-) p-hydroxyphenylglycine stock solution to a scale by using 0.1% phosphoric acid, and shaking up. (the concentration of D (-) methylparaben was 1mg/ml, and the concentration of D (-) p-hydroxyphenylglycine was 0.01 mg/ml).
Example 2
Under selected chromatographic conditions: a variable wavelength ultraviolet detector with the detection wavelength of 226 nm; flow rate: 1.5 ml/min; column temperature: 25 ℃; sample introduction volume: 5 mu l of the solution; a chromatographic column: ultimate XB-phenyl (ii);
buffer salt solution: 10mM potassium dihydrogen phosphate +10mM triethylamine hydrochloride, adjusted to pH 3.5. + -. 0.2 with phosphoric acid.
The mobile phase is mobile phase A: buffer salt solution: acetonitrile 98: 2, mobile phase B: buffer salt solution: acetonitrile 80: 20. Eluting in gradient proportion, and introducing into system adaptive solution 1 needle respectively, wherein the tailing factor of p-hydroxyphenylglycine/methyl p-hydroxyphenylglycine is less than or equal to 2.0, and the separation degree of p-hydroxyphenylglycine from the nearest peak is not less than 1.5; adding 2 needles of standard sample solution, and requiring that the absolute difference value of the detection result of the standard sample solution is less than or equal to 0.1 percent and the maximum single unknown impurity is less than or equal to 0.05 percent. (solution preparation is described in example 1)
System adaptive solution
Sample labeling solution
When the pH value of the buffer salt solution is 3.5, a chromatogram obtained by adding the system adaptive solution is shown in figure 1, and when the pH value is 3.3, one chromatogram obtained by adding the standard sample solution is shown in figure 2.
Example 3
Under selected chromatographic conditions: a variable wavelength ultraviolet detector with the detection wavelength of 226 nm; flow rate: the flow rates were changed to 1.4ml/min and 1.6 ml/min. Column temperature: 25 ℃; sample introduction volume: 5 mu l of the solution; a chromatographic column: ultimate XB-phenyl (ii);
buffer salt solution: 10mM potassium dihydrogen phosphate +10mM triethylamine hydrochloride, adjusted to pH 3.5 with phosphoric acid.
The mobile phase is mobile phase A: buffer salt solution: acetonitrile 98: 2, mobile phase B: buffer salt solution: acetonitrile 80: 20. Respectively feeding into 1 needle of system adaptive solution, wherein D (-) p-hydroxyphenylglycine/D (-) methyl p-hydroxyphenylglycine tail factor is not more than 2.0, the separation degree of D (-) p-hydroxyphenylglycine and the nearest peak is not less than 1.5, and adding 2 needles of standard solution into a sample; the absolute difference of the detection results of the test solutions is marked, the D (-) p-hydroxyphenylglycine is required to be less than or equal to 0.1 percent, the maximum single unknown impurity is less than or equal to 0.05 percent, the gradient elution ratio is shown in example 1, and the results are shown in the following table (the solution preparation is shown in example 1):
system adaptive solution
Sample labeling solution
Wherein, when the mobile phase is 1.4mL/min, one of the chromatograms obtained by adding the standard sample solution is shown in figure 3.
Example 4
Under selected chromatographic conditions: variable wavelength ultraviolet detector, detection wavelength: 226 nm; flow rate: 1.5 ml/min; column temperature: 20 ℃ and 30 ℃; sample introduction volume: 5 mu l of the solution; a chromatographic column: ultimate XB-phenyl (ii);
the mobile phase is mobile phase A: phosphate buffer salt (1.36 g of potassium dihydrogen phosphate was weighed into 1L of water, 1.37g of triethylamine hydrochloride was added, and pH was adjusted to 3.5 with phosphoric acid): acetonitrile 98: 2. mobile phase B: phosphate buffer salt (1.36 g of potassium dihydrogen phosphate was weighed into 1L of water, 1.37g of triethylamine hydrochloride was added, and pH was adjusted to 3.5 with phosphoric acid): acetonitrile 80: 20.
Respectively feeding into 1 needle of system adaptive solution, wherein D (-) p-hydroxyphenylglycine/D (-) methyl p-hydroxyphenylglycine tail factor is not more than 2.0, the separation degree of D (-) p-hydroxyphenylglycine and the nearest peak is not less than 1.5, and adding 2 needles of standard solution into a sample; the absolute difference of the detection results of the test solutions is marked, the D (-) p-hydroxyphenylglycine is required to be less than or equal to 0.1 percent, the maximum single unknown impurity is less than or equal to 0.05 percent, the gradient elution ratio is shown in example 1, and the results are shown in the following table (the solution preparation is shown in example 1):
system adaptive solution
Sample labeling solution
Wherein, when the column temperature is 20 ℃, one of chromatograms obtained by adding the standard sample solution is shown in figure 4.
Example 5 academic investigation of the method for determining the content of D (-) p-hydroxyphenylglycine in D (-) methyl p-hydroxyphenylglycine by the method.
Specificity
Oxidative degradation:
a sample of 50mgD (-) methylparaben was weighed into a 50ml volumetric flask, and (2ml) of commercially available H2O2(5%) after standing for 16h, 1mol/l sulfurous acid (3.5ml) was addedSodium hydrogen solution, using 0.1% phosphoric acid to fix the volume to the scale, shaking up and filtering, and simultaneously making blank.
Detecting data
Results of oxidative degradation test
aRelative deviation from undegraded ═ purity of degradation front peak-purity of degradation rear peak)/purity of degradation front peak
One of the chromatograms obtained after oxidative degradation is shown in FIG. 5.
Alkali degradation:
weighing a 50mgD (-) p-hydroxyphenylglycine methyl ester sample into a 50ml volumetric flask, adding (2ml)0.1mol/L NaOH solution, adjusting the pH to be neutral (pH 6-7) by using 0.1mol/L HCl solution after (6min), fixing the volume to the scale by using 0.1% phosphoric acid, shaking up and filtering.
Detecting data
Results of alkali degradation test
The chromatogram after base degradation is shown in FIG. 6.
Acid degradation:
weighing a 50mgD (-) p-hydroxyphenylglycine methyl ester sample into a 50ml volumetric flask, adding (2ml)0.1mol/L HCl solution, adjusting the pH to be neutral (pH is approximately equal to 6-7) by using 0.1mol/LNaOH after 16h, fixing the volume to the scale by using 0.1% phosphoric acid, shaking up and filtering.
Detecting data
Results of acid degradation test
The chromatogram after acid degradation is shown in FIG. 7.
The sample is obviously degraded under the conditions of strong oxidizing property, acidity and alkalinity, in addition, high-temperature, high-humidity and strong light irradiation tests are carried out, the appearance is obviously changed at high temperature, and the obvious change is not seen under the conditions of high humidity and strong light irradiation; the relative deviation of the peak purity of the D (-) p-hydroxyphenylglycine methyl ester after degradation under the conditions of high temperature, high humidity and strong light irradiation and the relative deviation without degradation are less than 5 percent, and the separation degree of the D (-) p-hydroxyphenylglycine/D (-) p-hydroxyphenylglycine methyl ester from the adjacent peak is more than 1.5.
Investigation of linear relationships
Accurately transferring the D (-) p-hydroxyphenylglycine stock solution, preparing the D (-) p-hydroxyphenylglycine stock solution into 20%, 50%, 100% and 150% concentration solutions respectively, taking the 4 concentration solutions and the quantitative Limit (LOQ) concentration solution as 5 concentration points, feeding the concentration points into 3 needles respectively, and performing linear regression on the concentration of the solution to be measured by using the average value of the peak area of each impurity in the map; the method comprises the following steps: the correlation coefficient R is more than or equal to 0.995, the intercept is less than or equal to 25 percent, and the residual standard deviation is less than or equal to 5 percent.
D (-) p-hydroxyphenylglycine
Stability test
Weighing 1 part of D (-) p-hydroxyphenylglycine methyl ester sample to serve as a stable solution, and putting the stable solution at room temperature for 1 needle at intervals of 0min, 30min, 2.5h, 5h, 7h and 9.5h after the preparation is finished; 1 needle is respectively inserted for 0min, 30min, 2h, 3.5h, 5.5h, 7.5h, 10h, 12h, 16.5h, 18.5h and 20h under the refrigeration condition; the method comprises the following steps: the relative deviation D (-) p-hydroxyphenylglycine between the normalized content of the impurity peak area and the normalized content of the 0min area at each time point is less than or equal to 10 percent, the maximum single unknown impurity is less than or equal to 10 percent, and no impurity peak appears compared with the 0min solution.
Stable solution (Normal temperature)
Stabilizing solution (refrigeration)
Precision of chromatography system
Selecting the same batch of D (-) p-hydroxyphenylglycine methyl ester sample, weighing 7 parts, adding 1.0ml of D (-) p-hydroxyphenylglycine stock solution into 6 parts, adding 0.1% phosphoric acid to 50ml, and shaking up to obtain a standard sample solution. 2 needles of the test solution without the added mark are added, and 2 needles of each part of the test solution with the added mark are added; the method comprises the following steps: RSD of the detection result of D (-) p-hydroxyphenylglycine in 6 parts of standard test solution is less than or equal to 2 percent, and RSD of the detection result of the largest single unknown impurity is less than or equal to 10 percent
Non-labeled test solution
Method repeatability test
System adaptation solution: weighing 50mg of D (-) p-hydroxyphenylglycine methyl ester reference substance into a 50ml volumetric flask, adding 1ml of D (-) p-hydroxyphenylglycine stock solution, then adding 0.1% phosphoric acid to the scale, and shaking up. (D (-) p-hydroxyphenylglycine concentration is 0.01mg/ml) is continuously added into the adaptive solution 6 needles of the system; the method comprises the following steps: the RSD of the peak area of D (-) p-hydroxyphenylglycine is less than or equal to 2 percent, and the RSD of the peak area of the maximum single unknown impurity is less than or equal to 10 percent.
System precision solution
In conclusion, the method effectively separates the D (-) p-hydroxyphenylglycine methyl ester and the D (-) p-hydroxyphenylglycine under the same condition, and simultaneously measures the content of the D (-) p-hydroxyphenylglycine, and is simple, rapid and accurate. Is an ideal method for measuring the content of the D (-) p-hydroxyphenylglycine in the D (-) p-hydroxyphenylglycine methyl ester.
Claims (2)
1. A method for analyzing the content of D (-) p-hydroxyphenylglycine in D (-) methyl p-hydroxyphenylglycine ester is characterized by comprising the following steps:
(1) diluting a to-be-detected sample of D (-) p-hydroxyphenylglycine methyl ester to obtain a test solution;
(2) analyzing the test solution in the step (1) by adopting high performance liquid chromatography to obtain liquid chromatography data;
the mobile phase used by the high performance liquid chromatography is a mixed solution of a buffer salt solution and acetonitrile;
the preparation method of the buffer salt solution comprises the following steps:
10mM monopotassium phosphate +10mM triethylamine hydrochloride, wherein the solvent is water, and the pH is adjusted to 3.5 by phosphoric acid;
in the step (2), respectively preparing a mobile phase A and a mobile phase B with different concentrations, and then analyzing by adopting a gradient elution method;
in the mobile phase A, the volume ratio of the buffer salt solution to the acetonitrile is 98: 2;
in the mobile phase B, the volume ratio of the buffer salt solution to the acetonitrile is 80: 20;
the gradient elution procedure is shown in the following table:
In the step (2), the chromatographic column used for analysis is a chemically bonded stationary phase chromatographic column: ultimate XB-Phenyl;
the detector is a variable wavelength detector, and the detection wavelength is 220-230 nm;
the flow rate of the mobile phase is 1.0-2.0 mL/min;
(3) calculating the content of D (-) p-hydroxyphenylglycine in the sample to be detected according to the liquid chromatography data obtained in the step (2);
the content of the D (-) p-hydroxyphenylglycine is calculated by an area normalization method.
2. The method for analyzing the content of D (-) p-hydroxyphenylglycine in D (-) p-hydroxyphenylglycine methyl ester according to claim 1, wherein in the step (1), the sample to be tested is diluted with 0.05-0.2% phosphoric acid or methanol.
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