CN113917006B - Method for detecting content of isopropyl isothiocyanate in cefathiamidine - Google Patents
Method for detecting content of isopropyl isothiocyanate in cefathiamidine Download PDFInfo
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Abstract
The invention discloses a detection method of isopropyl isothiocyanate in cefathiamidine, and belongs to the technical field of analysis. The method for detecting the content of isopropyl isothiocyanate in cefathiamidine comprises the following steps: step 1: preparing a reference stock solution; step 2: preparing a derivatization reaction solution; step 3: preparing a diluent; step 4: preparing a reference substance solution; step 5: preparing a sample solution; step 6: preparing a blank solution; step 7: chromatographic conditions; step 8: and (5) measuring. The invention establishes a high performance liquid chromatography determination method for the content of isopropyl isothiocyanate in cefathiamidine, which can effectively detect the possible isopropyl isothiocyanate impurity in the cefathiamidine bulk drug, and control the isopropyl isothiocyanate in each gram of the cefathiamidine bulk drug to be less than or equal to 12 mug, thereby effectively controlling the quality of the cefathiamidine bulk drug.
Description
Technical Field
The invention relates to a method for detecting the content of isopropyl isothiocyanate in cefathiamidine, and belongs to the technical field of analysis.
Background
Cefathiamidine, also known as cephalosporin 18, chemical name (6R, 7R) -3- [ (acetoxy) methyl]-7- [ alpha- (N, N-diisopropylamidinothio) acetamido]-8-oxo-5-thia-1-azabicyclo [4.2.0]Octyl-2-ene-2-carboxylic acid betaine with molecular formula of C 19 H 28 N 4 O 6 S 2 The molecular weight is 472.59, the CAS number is 33075-00-2, the white or white-like crystalline powder is almost odorless and has hygroscopicity, and the chemical structural formula is as follows:
the starting material for synthesizing cefathiamidine is N, N-diisopropyl thiourea. The synthetic route of the N, N-diisopropyl thiourea is as follows:
thus, isopropyl isothiocyanate, a potential process impurity, is generated in cefathiamidine, and the molecular formula of the isopropyl isothiocyanate is C 4 H 7 NS, molecular weight 101.17, CAS number 2253-73-8.
In the 2020 edition of Chinese pharmacopoeia, the quality standard of cefathiamidine does not control isopropyl isothiocyanate, and the analysis method of isopropyl isothiocyanate in the cefathiamidine is not carried in the related literature. At present, the main detection methods of isothiocyanate compounds according to related documents are gas chromatography, liquid chromatography and capacity analysis, but are not applicable to detection in cefathiamidine, and specifically comprise the following steps:
in the gas chromatography, the cefathiamidine is easy to dissolve in water and has poor solubility in organic solvents, and isopropyl isothiocyanate can be degraded by hydrolysis reaction with water, so that the solvents for dissolving the cefathiamidine and the isopropyl isothiocyanate cannot be combined. Therefore, gas chromatography is not suitable for detecting the content of isopropyl isothiocyanate in cefathiamidine.
The positive liquid chromatography method cannot detect the cefathiamidine because of poor solubility in the organic solvent. Reverse liquid chromatography is not suitable for detecting the content of isopropyl isothiocyanate in cefathiamidine because the mobile phase is mostly aqueous solution and isopropyl isothiocyanate cannot be directly detected.
The capacity analysis method has the characteristics of simple equipment, convenient operation and the like, but has lower sensitivity and is commonly used for constant analysis. The limit of isopropyl isothiocyanate in cefathiamidine is 0.0012%, and the detection sensitivity requirement is high, so that the volume titration method is not suitable for detecting the content of isopropyl isothiocyanate in cefathiamidine.
In view of the above, it is necessary to develop a time-saving detection method suitable for monitoring the isopropyl isothiocyanate content in cefathiamidine, so as to overcome the shortcomings of the prior art.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting the content of isopropyl isothiocyanate in cefathiamidine.
The technical scheme for solving the technical problems is as follows: a method for detecting the content of isopropyl isothiocyanate in cefathiamidine comprises the following steps:
step 1: preparing reference stock solution
Weighing isopropyl isothiocyanate reference substance, adding acetone to prepare a solution containing 2.4 mug isopropyl isothiocyanate per 1ml serving as reference substance stock solution;
step 2: preparation of the derivatization reaction solution
The mixed solution of diethylamine and acetone in a volume ratio of 1:9 is used as a derivatization reaction solution;
step 3: preparation of the diluent
The mixed solution of phosphate buffer solution and acetonitrile in the volume ratio of 1:1 is used as a diluent;
step 4: preparing reference substance solution
Precisely transferring 1ml of the reference substance stock solution in the step 1, placing the reference substance stock solution in a 10ml volumetric flask, adding 1ml of the derivatization reaction solution in the step 2, diluting the solution to a fixed volume to a scale by using the diluent in the step 3 after water bath, and taking the solution as a reference substance solution;
step 5: preparation of test solutions
Precisely weighing 200 mg+/-0.5 mg of cefathiamidine to be measured, placing in a 10ml volumetric flask, adding 1ml of the derivatization reaction solution in the step 2, after water bath, fixing the volume to a scale by using the diluent in the step 3, and shaking uniformly to obtain a sample solution;
step 6: preparation of blank solution
Precisely transferring 1ml of the derivatization reaction solution in the step 2, placing the derivatization reaction solution in a 10ml volumetric flask, and after water bath, fixing the volume to a scale by using the diluent in the step 3 to serve as a blank solution;
step 7: chromatographic conditions
The chromatographic column is C 18 The column is subjected to gradient elution by taking phosphate buffer as a mobile phase A and acetonitrile as a mobile phase B, the temperature of the column is 25 ℃, the flow rate is 1.0ml/min, and the detection wavelength is 240nm by an ultraviolet detector;
step 8: measurement
Respectively sucking 20 mu l of the reference substance solution in the step 4 and 20 mu l of the sample solution in the step 5, injecting into a high performance liquid chromatograph, and reading data; the peak shape of the reference substance solution is symmetrical, the theoretical plate number is more than 2000, the sample solution chromatogram has isopropyl isothiocyanate peak, and the retention time of the isopropyl isothiocyanate peak in the reference substance solution is consistent; blank solution chromatogram, no peak same as isopropyl isothiocyanate control appears, namely blank solution is free from interference;
and calculating the content of isopropyl isothiocyanate in the cefathiamidine sample by adopting an external standard method according to the concentration of the reference substance solution and the corresponding chromatographic peak area of the sample solution.
The principle of the method for detecting the content of isopropyl isothiocyanate in cefathiamidine of the invention is as follows:
according to the invention, acetone is used as a dissolution diluent of isopropyl isothiocyanate control, a mixed solution of diethylamine and acetone in a volume ratio of 1:9 is used as a derivatization reaction solution, isopropyl isothiocyanate control solution is taken, 1ml of the derivatization reaction solution is added, and derivatization reaction is carried out in a water bath, so that thiourea is generated.
The retention time during elution is greatly affected by the column temperature, and the retention time generally moves forward with the rise of the column temperature. The chromatographic column temperature is set to be 20-30 ℃, sample injection is carried out under the identical conditions, the influence on the separation degree, peak shape, symmetry degree and the like of the sample is small, and the components can be well separated. If the temperature is too high, the degree of separation of each peak in the chromatogram decreases. The detection wavelength was set to 240nm, at which the absorption effect of the isopropyl isothiocyanate derivative was optimal.
The invention adopts phosphate buffer solution and acetonitrile as mobile phase, can realize the complete separation of cefathiamidine and impurity isopropyl isothiocyanate, the separation degree is above 1.5, the theoretical plate number is above 2000, and meanwhile, the analysis time can be reduced, and the influence of the interference between components on the accuracy of the detection result is effectively avoided.
The invention adopts an external standard method to carry out quantitative analysis, and the components with peaks can be quantitatively analyzed no matter whether all the components in the sample have peaks or not. The external standard method is the most accurate method for quantifying the reference substance because of the comparison of homogeneous components.
In conclusion, according to the principle that isopropyl isothiocyanate can react with diethylamine to generate thiourea, the thiourea content is detected by high performance liquid chromatography, and the chromatographic conditions defined by the invention are adopted to calculate the isopropyl isothiocyanate content in cefathiamidine. In addition, the invention also performs methodology verification in terms of specificity, linearity, detection and quantification limits, accuracy, precision, stability and the like. The result proves that the detection method has the advantages of high separation degree, good repeatability, strong specificity, high accuracy, high sensitivity, detection limit up to 0.003581 mug/ml, stable and reliable result and short analysis time.
The method for detecting the content of isopropyl isothiocyanate in cefathiamidine has the beneficial effects that:
1. the invention establishes a high performance liquid chromatography determination method for the content of isopropyl isothiocyanate in cefathiamidine, which can effectively detect the possible isopropyl isothiocyanate impurity in the cefathiamidine bulk drug, and control the isopropyl isothiocyanate in each gram of the cefathiamidine bulk drug to be less than or equal to 12 mug, thereby effectively controlling the quality of the cefathiamidine bulk drug.
2. The detection method has the advantages of high separation degree, good reproducibility, strong specificity, high accuracy, high sensitivity, detection limit up to 0.003581 mug/ml, stable and reliable result and short analysis time.
3. The detection method has the advantages of simple operation, mild detection conditions, wide application of detection equipment and instruments, wide sources of mobile phase raw materials, simple preparation and low cost, and is suitable for large-scale popularization and application.
On the basis of the technical scheme, the invention can be improved as follows.
Further, in step 1, the isopropyl isothiocyanate control had a purity of 97.0%.
The adoption of the method has the further beneficial effects that: the isopropyl isothiocyanate control was commercially available, for example, from Ara Ding Shiji (Shanghai) Inc., at 97.0% purity.
Further, in step 3, the preparation method of the phosphate buffer solution is as follows: 6.95g of disodium hydrogen phosphate dodecahydrate and 1.29g of citric acid monohydrate were weighed, dissolved in ultrapure water and diluted to 1000ml.
The adoption of the method has the further beneficial effects that: according to the invention, the phosphate buffer solution with the proportion is adopted as a diluent, is consistent with the initial proportion of the mobile phase, and can reduce blank interference to the greatest extent.
Further, in the step 4, the step 5 and the step 6, the temperature of the water bath is 25 ℃ and the time is 30min-40min.
The adoption of the method has the further beneficial effects that: according to the invention, the temperature and time of the water bath are adopted to carry out derivatization reaction, so that thiourea is generated after complete reaction.
In step 5, the cefathiamidine sample is in the form of injection.
The adoption of the method has the further beneficial effects that: at present, the cefathiamidine is only an injection and is suitable for being used as a cefathiamidine bulk drug.
Further, in step 7, the chromatographic column is Kromasil 100-5-C 18 The column had a length of 250mm, an inner diameter of 4.6mm, a filler of octadecylsilane chemically bonded silica gel and a particle diameter of 5. Mu.m.
The adoption of the method has the further beneficial effects that: the chromatographic column adopting the parameters can achieve good separation effect in a short time, has excellent peak shape, has good operation consistency, and ensures that good column efficiency is achieved when the chromatographic column is used at low pressure.
Further, in step 7, the specific method of gradient elution is as follows: according to the volume ratio, 0min-7min, the mobile phase A is 50%, and the mobile phase B is 50%;7min-12min, 50% to 40% of mobile phase A and 50% to 60% of mobile phase B; 12min to 22min, mobile phase A40% and mobile phase B60%; 22-23 min, 40-50% of mobile phase A and 60-50% of mobile phase B; 23min-30min, mobile phase A is 50%, mobile phase B is 50%.
The adoption of the method has the further beneficial effects that: according to the invention, the gradient elution adopting the parameters is found, the target peak symmetry factor and the separation degree are good, the mixture can achieve a good separation effect, the excellent peak shape is possessed, and the purpose of accurate quantification is achieved.
Further, in step 7, the column temperature was 25℃and the flow rate was 1.0ml/min.
The adoption of the method has the further beneficial effects that: according to the invention, the research shows that the peak height of the Wen Mubiao peak of the column at the temperature of 25 ℃ is reduced, the column pressure is increased, the retention time of the Wen Mubiao peak of the column at the temperature of 30 ℃ is advanced, the separation degree is reduced, and the peak response and the separation degree can be better considered at the temperature of 25 ℃ compared with the column temperature of 20 ℃ and the column temperature of 30 ℃. Therefore, although all of these 3 column temperatures can meet the detection requirements, 25℃is more suitable as the column temperature.
According to the invention, the research shows that when the flow rate is 0.8ml/min, 1.0ml/min and 1.2ml/min, the peak shape and the separation degree of the target peak are not obviously different, but the flow rate of 0.8ml/min is late, and the flow rate column pressure of 1.2ml/min is higher. Thus, 1.0ml/min was chosen as the flow rate.
Further, in step 8, the isopropyl isothiocyanate content in the cefathiamidine test sample is calculated according to formula 1:
in which W is S Weighing the reference substances in mg; a is that S Is used as reference substancePeak area of the liquid; d (D) S Dilution volume of control solution, ml; p is the content of the reference substance; a is that T Peak area of the sample solution; d (D) S Is the dilution volume of the sample solution, ml; w (W) T Is the sample weighing amount of the test sample, mg.
The adoption of the method has the further beneficial effects that: the external standard method is adopted for quantitative analysis, all components in the sample do not need to be subjected to peak emission and separation, and only the target object is subjected to peak emission, so that the quantitative analysis can be carried out on the target peak components. The external standard method is the most accurate method for quantifying the reference substance because of the comparison of homogeneous components.
Term interpretation:
the term "symmetry" refers to a parameter used to examine the symmetry of the peak pattern in HPLC detection, reflecting the performance of a chromatographic column; in liquid chromatography, the peak form is considered to be preferable when the symmetry is in the range of 0.8 to 1.2.
The term "degree of separation", which is equal to the ratio of the difference between the retention times of adjacent chromatographic peaks to the peak-to-peak width-to-average value of the two chromatographic peaks, is used to examine the degree of separation between the component to be measured and adjacent coexisting or difficult-to-separate substances, and is a key indicator for measuring the performance of a chromatographic system. In general, when the separation degree of the chromatographic peaks corresponding to the peak heights of the two peaks is 1.5 or more, the separation of the two peaks is considered to be 99% or more. The greater the separation value, the better the separation.
Drawings
FIG. 1 is a high performance liquid chromatogram obtained by the first method in step 1.1 of Experimental example 1 of the present invention.
FIG. 2 is a high performance liquid chromatogram obtained by the second method in step 1.1 of Experimental example 1 of the present invention.
FIG. 3 is a UV chromatogram of a full wavelength scan using an ultraviolet detector (DAD) in step 1.2 of Experimental example 1 of the present invention.
FIG. 4 is a high performance liquid chromatogram obtained in step 1.3 of Experimental example 1 of the present invention, wherein the column temperature was 20 ℃.
FIG. 5 is a high performance liquid chromatogram obtained in step 1.3 of Experimental example 1 of the present invention, wherein the column temperature was 25 ℃.
FIG. 6 is a high performance liquid chromatogram obtained in step 1.3 of Experimental example 1 of the present invention, wherein the column temperature was 30 ℃.
FIG. 7 is a high performance liquid chromatogram obtained in step 1.4 of Experimental example 1 of the present invention at a flow rate of 0.8 ml/min.
FIG. 8 is a high performance liquid chromatogram obtained in step 1.4 of Experimental example 1 of the present invention at a flow rate of 1.0ml/min.
FIG. 9 is a high performance liquid chromatogram obtained in step 1.4 of Experimental example 1 of the present invention at a flow rate of 1.2 ml/min.
FIG. 10 is a high performance liquid chromatogram of a blank solution in step 1.5 of Experimental example 1 of the present invention.
FIG. 11 is a high performance liquid chromatogram of the sample solution in step 1.5 of Experimental example 1 of the present invention.
FIG. 12 is a high performance liquid chromatogram of the control solution in step 1.5 of Experimental example 1 of the present invention.
FIG. 13 is a standard graph of isopropyl isothiocyanate in experimental example 3 of the present invention.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples being provided for the purpose of illustrating the invention and not for the purpose of limiting the same.
Example 1: chromatographic condition and System applicability test
Step 1.1: elution gradient determination
Octadecyl bonded silica gel column is used as filler; the detection wavelength is 240nm; phosphate buffer is used as a mobile phase A, and acetonitrile is used as a mobile phase B. The same test solution was tested under the following chromatographic conditions. The preparation method of the phosphate buffer solution comprises the following steps: 6.95g of disodium hydrogen phosphate dodecahydrate and 1.29g of citric acid monohydrate were weighed, dissolved in ultrapure water and diluted to 1000ml.
The method comprises the following steps: the flow rate is 1.0ml/min, the column temperature is 25 ℃, the sample injection amount is 20 μl, and gradient elution is carried out, specifically: according to the volume ratio, 0min-7min, the mobile phase A is 55%, and the mobile phase B is 45%;7min-12min, 55% to 40% of mobile phase A and 45% to 60% of mobile phase B; 12min to 22min, mobile phase A40% and mobile phase B60%; 22-23 min, 40-55% mobile phase A and 60-45% mobile phase B; 23min-30min, mobile phase A is 55%, mobile phase B is 45%.
The second method is as follows: the flow rate is 1.0ml/min, the column temperature is 25 ℃, the sample injection amount is 20 μl, and gradient elution is carried out, specifically: according to the volume ratio, 0min-7min, the mobile phase A is 50%, and the mobile phase B is 50%;7min-12min, 50% to 40% of mobile phase A and 50% to 60% of mobile phase B; 12min to 22min, mobile phase A40% and mobile phase B60%; 22-23 min, 40-50% of mobile phase A and 60-50% of mobile phase B; 23min-30min, mobile phase A is 50%, mobile phase B is 50%.
Preparation of sample solution: precisely weighing 200 mg+/-0.5 mg of cefathiamidine to be measured, placing in a 10ml volumetric flask, precisely transferring 1ml of the reference substance stock solution in the step 1, placing in the same volumetric flask, adding 1ml of the derivatization reaction solution, carrying out water bath at 25 ℃ for 30-40 min, then using a diluent to fix the volume to a scale, and shaking uniformly to obtain a sample-adding sample solution.
The preparation method of the reference substance stock solution comprises the following steps: the isopropyl isothiocyanate reference substance was weighed, and acetone was added to prepare a solution containing 2.4. Mu.g of isopropyl isothiocyanate per 1ml as a reference stock solution. The preparation method of the derivatization reaction solution comprises the following steps: the mixed solution of diethylamine and acetone in a volume ratio of 1:9 is used as a derivatization reaction solution. The preparation method of the derivatization reaction solution comprises the following steps: the mixed solution of phosphate buffer solution and acetonitrile in the volume ratio of 1:1 is used as a diluent.
Assay: accurately sucking 20 μl of sample solution, injecting into high performance liquid chromatograph, and recording chromatogram. The results are shown in FIGS. 1-2.
As can be seen from FIGS. 1-2, the method II has good symmetry factor and separation degree of the target peak in the sample solution, so that the method II is selected as the elution gradient for measuring the content of isopropyl isothiocyanate in cefathiamidine.
Step 1.2: wavelength determination
And (3) carrying out full-wavelength scanning by using an ultraviolet detector (DAD) according to the finally determined chromatographic conditions of gradient investigation, detecting a sample solution, and investigating the absorption wavelength of a target peak, wherein the full-wavelength scanning result is shown in figure 3.
As can be seen from FIG. 3, the target peak has a maximum absorption at 240nm, and the maximum absorption wavelength is usually selected as the detection wavelength, so that the wavelength of the method for measuring the content of isopropyl isothiocyanate in cefathiamidine is selected to be 240nm.
Step 1.3: column temperature determination
And detecting the same sample solution according to the chromatographic conditions finally determined by the investigation, and selecting different column temperatures for the investigation, wherein the results are shown in figures 4-6.
By comparing the peak shapes and the separation degree of target peaks in the test sample at different column temperatures of 20 ℃,25 ℃ and 30 ℃, the peak heights of the Wen Mubiao peaks of the 20 ℃ column are reduced, the column pressure is increased, the retention time of the Wen Mubiao peaks of the 30 ℃ column is advanced, the separation degree is reduced, and the peak response and the separation degree can be better considered at the column temperature of 25 ℃ compared with the column temperature of 20 ℃ and the column temperature of 30 ℃ at the column temperatures of 3. Therefore, although all of these 3 column temperatures can meet the detection requirements, 25℃is more suitable as the column temperature.
Step 1.4: flow rate determination
And detecting the same sample solution according to the chromatographic conditions finally determined by the investigation, and selecting different flow rates for the investigation, wherein the results are shown in figures 7-9.
By comparing the peak shape and the separation degree of the target peak in the sample at different flow rates of 0.8ml/min, 1.0ml/min and 1.2ml/min, the peak shape and the separation degree of the target peak have no obvious difference at the 3 different flow rates, but the flow rate of 0.8ml/min is late in peak outlet, and the flow rate of 1.2ml/min is high in column pressure. Thus, 1.0ml/min was chosen as the flow rate.
Step 1.5: specificity test
And precisely sucking 20 mu l of each of the blank solution, the reference solution and the sample solution according to chromatographic conditions finally determined by elution gradient investigation and wavelength investigation results, injecting the solution into a high performance liquid chromatograph, and recording a chromatogram. The results are shown in FIGS. 10-12.
The detection result shows that the blank solution is free from interference under the chromatographic condition, the symmetry factor and the separation degree of each chromatographic peak in the reference substance solution and the sample solution are good, and the theoretical plate number is not lower than 2000, which indicates that the chromatographic condition is good and meets the requirements.
The chromatographic conditions were finally determined: the chromatographic column is Kromasil 100-5-C 18 The column had a length of 250mm, an inner diameter of 4.6mm, a filler of octadecylsilane chemically bonded silica gel and a particle diameter of 5. Mu.m. Phosphate buffer is used as a mobile phase A, and acetonitrile is used as a mobile phase B. An ultraviolet detector with a detection wavelength of 240nm; the column temperature is 25 ℃; the flow rate is 1.0ml/min; the specific method for gradient elution comprises the following steps: according to the volume ratio, 0min-7min, the mobile phase A is 50%, and the mobile phase B is 50%;7min-12min, 50% to 40% of mobile phase A and 50% to 60% of mobile phase B; 12min to 22min, mobile phase A40% and mobile phase B60%; 22-23 min, 40-50% of mobile phase A and 60-50% of mobile phase B; 23min-30min, mobile phase A is 50%, mobile phase B is 50%.
Experimental example 2: investigation of derivatization reaction conditions
Precisely transferring 4 parts of 1ml of reference substance stock solution, respectively placing in 10ml volumetric flasks, adding 1ml of derivatization reaction solution, respectively performing water bath at 25deg.C for 10min, 20min, 30min and 40min, and diluting with diluent to volume to scale.
The response of isopropyl isothiocyanate derivative was measured according to the above-described measurement method, and the results are shown in Table 1.
TABLE 1 comparison of the influence of different reaction times on the results of derivatization reactions
| Reaction time | 10min | 20min | 30min | 40min |
| Target response peak area | 45.98522 | 50.08335 | 52.21424 | 51.04218 |
The result shows that when the reaction time is 10min, the reaction is obviously not complete; the reaction time is more than 20min, and the target peak responses are not quite different. Thus, the reaction time was chosen to be 30min-40min.
Experimental example 3: method verification
Step 3.1: linearity and range
Accurately weighing a proper amount of isopropyl isothiocyanate reference substance, adding acetone for dilution, and adding a derivatization reaction solution for reaction to obtain a series of reference substance solutions. 20. Mu.l of the sample was precisely sucked, injected into a liquid chromatograph, measured under the above-defined chromatographic conditions, the chromatogram was recorded, the concentration was taken as an abscissa (X), the peak area was taken as an ordinate (Y), and linear regression analysis was performed to calculate the correlation coefficient (r) of the regression line. The results are detailed in Table 2. Standard plots of isopropyl isothiocyanate are shown in fig. 13.
Table 2 summary of the results of the linear experiments
Conclusion: according to the linear test result, the quantitative limiting concentration is 0.01194 mug/ml, the minimum quantification (about 0.00006%) and the minimum detection amount (about 0.00002%) of the main component and the isopropyl isothiocyanate derivative compound content are calculated according to the sample injection concentration of the sample of 20mg/ml (calculated by cefathiamidine), the sample injection concentration of the sample is determined to be 20mg/ml, the correlation coefficient r of the regression equation is greater than 0.990, and the linear relation is good.
Step 3.2: limit of detection and limit of quantification
Precisely weighing a proper amount of isopropyl isothiocyanate reference substance, gradually diluting with acetone, adding a derivatization reaction solution for reaction, and taking the solution when the peak height of the isopropyl isothiocyanate derivative is about 10 times of the baseline noise as a quantitative limiting solution; the solution up to the point where the peak heights of the components were about 3 times the baseline noise was used as the detection limit solution.
And (3) precisely taking 20 mu l of each of the quantitative limit solution and the detection limit solution, injecting the solution into a high performance liquid chromatograph, recording a chromatogram, and detailing sample injection results shown in tables 3 and 4.
TABLE 3 limit of detection and limit of quantification results
TABLE 4 quantitative limit reproducibility peak area test results
| 1 | 2 | 3 | 4 | 5 | 6 | RSD(％) |
| 1.61705 | 1.82894 | 1.91321 | 1.60999 | 1.79939 | 1.78232 | 6.88 |
Conclusion: the RSD of the peak area of the solution with the quantitative limit of 6 parts is not more than 10.0%, and the signal-to-noise ratio meets the detection requirement; the signal to noise ratio of the solution with the detection limit of 3 parts meets the detection requirement.
Step 3.3 accuracy (sample recovery)
200 mg+/-0.5 mg of cefathiamidine is weighed by a sample adding recovery method, 9 parts are precisely weighed, the weighed cefathiamidine is placed in different 10ml volumetric flasks, 3 parts of isopropyl isothiocyanate reference substances 12 mug, 24 mug and 36 mug are precisely added respectively, 1ml of derivatization reaction solution is added respectively, after water bath at 25 ℃ for 30min, the sample is diluted with a diluent to volume to scale, and shaking is carried out uniformly, so that three sample adding recovery test solutions with different concentrations of 50%, 100% and 150% are obtained. Precisely sucking 20 μl, and injecting into high performance liquid chromatograph for measurement. The results are shown in Table 5.
Table 5 summary of accuracy results
Conclusion: the sample adding recovery rate of the sample to be tested is 99.16% -107.82%, the recovery rate average value is 103.92%, the RSD is not more than 6%, and the method meets the requirements, and has good accuracy.
Step 3.4 precision
3.4.1 repeatability
According to the preparation method of the 100% sample-adding recovery test solution under the 'step 3.3 accuracy', 6 parts are prepared in parallel, 20 μl is precisely sucked, and the mixture is injected into a liquid chromatograph for measurement. The results are shown in Table 6.
TABLE 6 summary of the repeatability results (analyst A)
Conclusion: the recovery rate of 6 parts of 100% sample-adding recovery test solution is 101.17% -104.65%, the recovery rate average value is 103.20%, RSD is not more than 6%, the requirements are met, and the repeatability is good.
3.4.2 intermediate precision
Different analysts prepare 6 parts of 100% sample recovery test solution in parallel according to the same method under the repeatability item, and respectively measure the recovery rate of 6 parts of 100% sample recovery test solution on different instruments at different times by using different chromatographic columns, and calculate RSD values, and the results are shown in tables 7 and 8 in detail.
TABLE 7 intermediate precision-repeatability results summary (Analyzer B)
Conclusion: different personnel and different dates are respectively tested by using different instruments, and the recovery rate of 6 parts of 100% sample-adding recovery test solution is 98.27% -102.29%, the average value of the recovery rate is 99.65%, the RSD is less than 6%, the requirements are met, and the repeatability in the middle precision is good.
Table 8 summary of intermediate precision results
Note that: repeatability: personnel A; date: 2020.12.11; instrument: a1-LC807; chromatographic column: c-201901.
Intermediate precision: personnel B; date: 2020.12.14; instrument: A1-LC806; chromatographic column: c-116.
Conclusion: two analysts use different instruments and systems to test, and the recovery rate RSD of 12 parts of 100% sample-adding recovery test solution is not more than 10%, so that the requirements are met, and the intermediate precision is good.
In summary, the repeatability and the intermediate precision of the method for detecting isopropyl isothiocyanate by adopting the established detection method meet the verification requirement, and the method has good precision.
Step 3.5 stability
Preparing a sample solution and a reference substance solution, and respectively standing the sample solution at room temperature for 6h, 16h and 30h; placing the reference substance solution at room temperature for 9h, 19h and 33h respectively; and (3) precisely removing 20 μl of each stable solution, injecting into a high performance liquid chromatograph, recording a chromatogram, and detailing sample injection results in tables 9 and 10.
TABLE 9 summary of test solution stability results
| Time of placement | Peak area (mAU) |
| 0h | Not detected |
| 6h | Not detected |
| 16h | Not detected |
| 30h | Not detected |
| RSD％ | / |
Table 10 summary of stability results for control solutions
| Time of placement | Peak area (mAU)) |
| 0h | 34.09429 |
| 9h | 33.27932 |
| 19h | 33.39987 |
| 33h | 32.72381 |
| RSD％ | 1.69 |
Conclusion: under the condition of room temperature, the solution of the sample is not detected within 30 hours, the solution stability is good, and the measurement requirement is met; the peak area RSD of the reference substance solution is less than or equal to 5% within 33h, the solution stability is good, and the measurement requirement is met.
Experimental example 4: method for detecting content of isopropyl isothiocyanate in cefathiamidine
Through a great deal of experimental investigation by the inventor of the application, the method for detecting the content of isopropyl isothiocyanate in the final cefathiamidine is determined as follows:
step 1: preparing reference stock solution
The isopropyl isothiocyanate reference substance (24 mg.+ -. 0.5 mg) is weighed, placed in a 100ml volumetric flask, and acetone is added to prepare a solution containing 2.4 mug isopropyl isothiocyanate per 1ml as a reference substance stock solution.
Step 2: preparation of the derivatization reaction solution
The mixed solution of diethylamine and acetone in a volume ratio of 1:9 is used as a derivatization reaction solution.
Step 3: preparation of the diluent
The mixed solution of phosphate buffer solution and acetonitrile in the volume ratio of 1:1 is used as a diluent.
Step 4: preparing reference substance solution
Precisely transferring 1ml of the reference substance stock solution in the step 1, placing in a 10ml volumetric flask, adding 1ml of the derivatization reaction solution in the step 2, and diluting with the diluent in the step 3 to a fixed volume to scale after water bath at 25 ℃ for 30min to serve as a reference substance solution.
Step 5: preparation of test solutions
Precisely weighing 200 mg+/-0.5 mg of cefathiamidine to be measured, placing in a 10ml volumetric flask, adding 1ml of the derivatization reaction solution in the step 2, carrying out water bath at 25 ℃ for 30min, and then using the diluent in the step 3 to fix the volume to scale, and shaking uniformly to obtain a sample solution.
Step 6: preparation of blank solution
Precisely transferring 1ml of the derivatization reaction solution in the step 2, placing the solution in a 10ml volumetric flask, and after water bath for 30min at 25 ℃, fixing the volume to scale by using the diluent in the step 3 to serve as a blank solution.
Step 7: chromatographic conditions
The chromatographic column is Kromasil 100-5-C 18 The column had a length of 250mm, an inner diameter of 4.6mm, a filler of octadecylsilane chemically bonded silica gel and a particle diameter of 5. Mu.m. And (3) taking phosphate buffer solution as a mobile phase A and acetonitrile as a mobile phase B, performing gradient elution, wherein the column temperature is 25 ℃, the flow rate is 1.0ml/min, and the detection wavelength is 240nm. The specific method of gradient elution is as follows: according to the volume ratio, 0min-7min, the mobile phase A is 50%, and the mobile phase B is 50%;7min-12min, 50% to 40% of mobile phase A and 50% to 60% of mobile phase B; 12min to 22min, mobile phase A40% and mobile phase B60%; 22-23 min, 40-50% of mobile phase A and 60-50% of mobile phase B; 23min-30min, mobile phase A is 50%, mobile phase B is 50%.
Step 8: measurement
Respectively sucking 20 mu l of the reference substance solution in the step 4 and 20 mu l of the sample solution in the step 5, injecting into a high performance liquid chromatograph, and reading data; the peak shape of the reference substance solution is symmetrical, the theoretical plate number is more than 2000, the sample solution chromatogram has isopropyl isothiocyanate peak, and the retention time of the isopropyl isothiocyanate peak in the reference substance solution is consistent; blank solution chromatogram, no peak same as isopropyl isothiocyanate control appears, namely blank solution is free from interference;
and calculating the content of isopropyl isothiocyanate in the cefathiamidine sample according to a formula 1 by adopting an external standard method according to the concentration of the reference substance solution and the corresponding chromatographic peak area of the sample solution.
In which W is S Weighing the reference substances in mg; a is that S Peak area for control solution; d (D) S Dilution volume of control solution, ml; p is the content of the reference substance; a is that T Peak area of the sample solution; d (D) S Is the dilution volume of the sample solution, ml; w (W) T Is the sample weighing amount of the test sample, mg.
Experimental example 5: content determination of isopropyl isothiocyanate in cefathiamidine
The isopropyl isothiocyanate content in 10 cefathiamidines was detected by the detection method finally determined in experimental example 4, and the experimental results are shown in Table 11.
TABLE 11 determination of isopropyl isothiocyanate content in 10 cefathiamidines of Experimental example 4
| Sample numbering | Content (%) | Sample numbering | Content (%) |
| SJ170901 | Not detected | LMA1911019 | Not detected |
| SJ200601 | Not detected | LMA1911003 | Not detected |
| SJ200602 | Not detected | SSJ210401 | Not detected |
| SJ200603 | Not detected | SSJ210402 | Not detected |
| LMA1703002 | Not detected | SSJ210403 | Not detected |
As shown in Table 11, isopropyl isothiocyanate was not detected in 10 batches of cefathiamidine, and the product meets the requirement that the isopropyl isothiocyanate in each gram of cefathiamidine bulk drug is not more than 12. Mu.g, and can be marketed.
Comparative example 1
Simultaneous determination of the content of 4 isothiocyanate compounds in the Uighur medicine turnip seed by the reference GC method [1] As comparative example 1. The composition of isothiocyanate compounds is analyzed and the content thereof is determined by gas chromatography in the literature, specifically by the method: chromatographic column: ZB-FFAP (30 m. Times.0.25 mm. Times.0.25 μm); carrier gas: high-purity nitrogen; flow rate: 1mL/min; programming temperature: the initial temperature is 80 DEG CMaintaining for 12min, heating to 120deg.C at 10deg.C/min, heating to 210deg.C at 30deg.C/min, heating to 230deg.C at 5deg.C/min, and maintaining for 2min; sample inlet temperature: 200 ℃; split sample injection, split ratio: 90:1; detector temperature: 250 ℃; hydrogen flow rate: 30mL/min; air flow rate: 40mL/min; tail blow flow rate: 25mL/min. Methanol was used as a diluent.
The method of comparative example 1 was used to detect the presence of the following defects in cefathiamidine: because cefathiamidine is easy to dissolve in water and has poor solubility in organic solvents, isopropyl isothiocyanate can be degraded by hydrolysis reaction with water, and the solvents dissolved by the two cannot be taken into account, the gas chromatography is not suitable for detecting the content of isopropyl isothiocyanate in cefathiamidine.
Comparative example 2
The detection method of reference "research for determining the content of isothiocyanate in horseradish and mustard products by diethylamine titration" is taken as comparative example 2. The method principle of the document: the isothiocyanate and diethylamine are subjected to homogeneous reaction in acetone solution to generate corresponding thiourea, and excessive diethylamine is titrated by using standard hydrochloric acid, so that the isothiocyanate content is calculated.
The principle of the method shows that the method of the comparative example 2 has the following defects when applied to cefathiamidine detection: (1) The cefathiamidine is insoluble in acetone, and the solvent system is not suitable for detecting the cefathiamidine; (2) Cefathiamidine belongs to betaine, reacts with hydrochloric acid, consumes hydrochloric acid titration solution, and cannot be quantified. (3) The method is a titration method, is commonly used for constant analysis, and isopropyl isothiocyanate in cefathiamidine belongs to micro analysis, and the limit is 0.0012%, so the titration method is not suitable for detecting the cefathiamidine.
Reference is made to:
[1] sun Lian A, tian Lan A, B.sub. Dou Xuku A.sub.C. method for simultaneously determining the content of 4 isothiocyanate compounds in Wei medicine turnip seed [ J ]. Chinese pharmacy 2012 (47): 4482-4483.
[2] Research on determination of isothiocyanate content in horseradish and mustard products by diethylamine titration [ J ]. Chinese condiment, 2005 (2): 48-51.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (8)
1. The method for detecting the content of isopropyl isothiocyanate in cefathiamidine is characterized by comprising the following steps of:
step 1: preparing reference stock solution
Weighing isopropyl isothiocyanate reference substance, adding acetone to prepare a solution containing 2.4 mug isopropyl isothiocyanate per 1ml serving as reference substance stock solution;
step 2: preparation of the derivatization reaction solution
The mixed solution of diethylamine and acetone in a volume ratio of 1:9 is used as a derivatization reaction solution;
step 3: preparation of the diluent
The mixed solution of phosphate buffer solution and acetonitrile in the volume ratio of 1:1 is used as a diluent;
step 4: preparing reference substance solution
Precisely transferring 1ml of the reference substance stock solution in the step 1, placing the reference substance stock solution in a 10ml volumetric flask, adding 1ml of the derivatization reaction solution in the step 2, diluting the solution to a fixed volume to a scale by using the diluent in the step 3 after water bath, and taking the solution as a reference substance solution;
step 5: preparation of test solutions
Precisely weighing 200 mg+/-0.5 mg of cefathiamidine to be measured, placing in a 10ml volumetric flask, adding 1ml of the derivatization reaction solution in the step 2, after water bath, fixing the volume to a scale by using the diluent in the step 3, and shaking uniformly to obtain a sample solution;
step 6: preparation of blank solution
Precisely transferring 1ml of the derivatization reaction solution in the step 2, placing the derivatization reaction solution in a 10ml volumetric flask, and after water bath, fixing the volume to a scale by using the diluent in the step 3 to serve as a blank solution;
step 7: chromatographic conditions
The chromatographic column is C 18 A column, wherein phosphate buffer solution is used as a mobile phase A, acetonitrile is used as a mobile phase B,gradient elution is carried out, the column temperature is 20-30 ℃, the flow rate is 0.8-1.2 ml/min, and the detection wavelength is 240nm by an ultraviolet detector; the specific method for gradient elution comprises the following steps: according to the volume ratio, 0min-7min, the mobile phase A is 50%, and the mobile phase B is 50%;7min-12min, 50% to 40% of mobile phase A and 50% to 60% of mobile phase B; 12min to 22min, mobile phase A40% and mobile phase B60%; 22-23 min, 40-50% of mobile phase A and 60-50% of mobile phase B; 23min-30min, mobile phase A of 50% and mobile phase B of 50%;
step 8: measurement
Respectively sucking 20 mu l of the reference substance solution in the step 4 and 20 mu l of the sample solution in the step 5, injecting into a high performance liquid chromatograph, and reading data; the peak shape of the reference substance solution is symmetrical, the theoretical plate number is more than 2000, the sample solution chromatogram has isopropyl isothiocyanate peak, and the retention time of the isopropyl isothiocyanate peak in the reference substance solution is consistent; blank solution chromatogram, no peak same as isopropyl isothiocyanate control appears, namely blank solution is free from interference;
and calculating the content of isopropyl isothiocyanate in the cefathiamidine sample by adopting an external standard method according to the concentration of the reference substance solution and the corresponding chromatographic peak area of the sample solution.
2. The method for detecting the content of isopropyl isothiocyanate in cefathiamidine according to claim 1, wherein in the step 1, the purity of the isopropyl isothiocyanate reference substance is 97.0%.
3. The method for detecting the content of isopropyl isothiocyanate in cefathiamidine according to claim 1, wherein in the step 3, the preparation method of the phosphate buffer solution is as follows: 6.95g of disodium hydrogen phosphate dodecahydrate and 1.29g of citric acid monohydrate were weighed, dissolved in ultrapure water and diluted to 1000ml.
4. The method for detecting the content of isopropyl isothiocyanate in cefathiamidine according to claim 1, wherein in step 4, step 5 and step 6, the temperature of the water bath is 25 ℃ and the time is 30-40 min.
5. The method for detecting the content of isopropyl isothiocyanate in cefathiamidine according to claim 1, wherein in the step 5, the preparation form of the cefathiamidine test sample is an injection.
6. The method for detecting isopropyl isothiocyanate content in cefathiamidine according to claim 1, wherein in the step 7, the chromatographic column is Kromasil 100-5-C 18 The column had a length of 250mm, an inner diameter of 4.6mm, a filler of octadecylsilane chemically bonded silica gel and a particle diameter of 5. Mu.m.
7. The method for detecting the content of isopropyl isothiocyanate in cefathiamidine according to claim 1, wherein in the step 7, the column temperature is 25 ℃, and the flow rate is 1.0ml/min.
8. The method for detecting the content of isopropyl isothiocyanate in cefathiamidine according to any one of claims 1 to 7, wherein in step 8, the content of isopropyl isothiocyanate in the cefathiamidine sample is calculated according to formula 1:
in which W is S Weighing the reference substances in mg; a is that S Peak area for control solution; d (D) S Dilution volume of control solution, ml; p is the content of the reference substance; a is that T Peak area of the sample solution; d (D) S Is the dilution volume of the sample solution, ml; w (W) T Is the sample weighing amount of the test sample, mg.
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