CN112710773B - Method for simultaneously detecting fosfomycin and diol thereof by adopting ion chromatography - Google Patents
Method for simultaneously detecting fosfomycin and diol thereof by adopting ion chromatography Download PDFInfo
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Abstract
The invention belongs to the technical field of chemical substance analysis and detection, and discloses a method for simultaneously detecting fosfomycin and diol thereof by adopting an ion chromatography. The method for simultaneously detecting the fosfomycin and the diol thereof by adopting the ion chromatography comprises the following steps: (1) selection of ion chromatography instrument and chromatography test conditions: the chromatographic column is a low-capacity hydroxide system cathode protection column, the suppressor adopts an electrolytic membrane suppressor, and the leacheate is NaOH or KOH solution; (2) drawing a fosfomycin standard curve by taking fosfomycin sodium as a standard substance; (3) drawing a fosfomycin diol standard curve by taking fosfomycin trometamol EP impurity A as a standard substance; (4) and testing and obtaining peak areas of the fosfomycin and the diol in the sample, and substituting the peak areas into a standard curve to obtain corresponding concentrations. The method has the advantages of good reproducibility, low detection limit, high sensitivity, simple operation, wide application of used ion chromatography equipment and the like, and can simultaneously test the fosfomycin and the diol thereof.
Description
Technical Field
The invention relates to the technical field of chemical substance analysis and detection, in particular to a method for simultaneously detecting fosfomycin and diol thereof by adopting an ion chromatography.
Background
In recent years, infection of multiple drug-resistant bacteria, pan-resistant bacteria and even full-resistant bacteria continuously appears, and a serious challenge is brought to diagnosis and treatment of clinical infectious diseases. Researches prove that the fosfomycin has good antibacterial activity on drug-resistant bacteria due to the unique antibacterial mechanism, so that the fosfomycin is very important in clinical anti-infection treatment. However, in different links of fosfomycin production, fosfomycin wastewater with different concentrations can be generated, and fosfomycin serving as an antibiotic is discharged into natural water, so that drug resistance of bacteria in the environment can be caused to threaten the ecological environment and human health. Therefore, it is necessary to determine the content of fosfomycin in fosfomycin wastewater generated in different production links to select the optimal treatment method.
In the Chinese pharmacopoeia, an antibiotic microorganism assay method is adopted to detect the fosfomycin, the method can measure the fosfomycin content in batches, but the operation process is relatively complex, the time consumption is long, the method is easily interfered by other antibiotics, and the specificity and the accuracy are lacked. The ultraviolet-visible spectrophotometry has the advantages of simple operation and relatively high sensitivity, but the fosfomycin has no ultraviolet-visible absorption at the position of more than 220nm, so the conventional ultraviolet-visible spectrophotometry cannot detect the fosfomycin. After alizarin is used as a color developing agent, the fosfomycin can be tested by adopting an ultraviolet-visible spectrophotometry, after the fosfomycin and the alizarin in an ethanol solution are compounded, a strong absorption peak is formed at a 545nm position, and the detection limit of the fosfomycin can reach about 1.4 mg/L. The high performance liquid chromatography can also measure the concentration of the fosfomycin, but the detection limit is high, and the method is not suitable for testing low-concentration fosfomycin wastewater.
In order to overcome the defects of the method, Chinese patent document CN103728385A proposes a method for detecting fosfomycin by using an ion chromatography, the method can quickly and accurately detect the content of fosfomycin in fosfomycin wastewater, and the detection limit can reach 0.06 mg/L. However, fosfomycin contains an epoxy group in its chemical structure, which is less stable and is susceptible to hydrolysis to form fosfomycin diol. Therefore, the fosfomycin wastewater contains not only fosfomycin but also fosfomycin glycol. The method for detecting fosfomycin by using ion chromatography proposed in Chinese patent document CN103728385A cannot distinguish fosfomycin from fosfomycin glycol (fosfomycin and fosfomycin glycol have very close peak time), so when fosfomycin and fosfomycin glycol coexist in fosfomycin wastewater or other samples, the method cannot detect fosfomycin and fosfomycin glycol simultaneously, and can affect the accuracy of fosfomycin test. Therefore, it is necessary to develop a rapid and accurate method with low detection limit for simultaneously determining the contents of fosfomycin and fosfomycin diol.
Disclosure of Invention
The invention aims to overcome the defects of the background technology and provide a method for simultaneously detecting fosfomycin and diol thereof by adopting ion chromatography.
In order to achieve the purpose of the invention, the method for simultaneously detecting the fosfomycin and the diol thereof by adopting the ion chromatography comprises the following steps:
(1) selection of ion chromatography instrument and chromatography test conditions: the chromatographic column is a low-capacity hydroxide system cathode protection column, the suppressor adopts an electrolytic membrane suppressor, and the leacheate is NaOH or KOH solution;
(2) drawing a fosfomycin standard curve: preparing fosfomycin sodium (CAS number: 25162-71-4) standard solutions with different concentrations, injecting samples by using an automatic sample injector, carrying out quantitative analysis by an external standard method, and carrying out linear regression analysis on the concentration of the fosfomycin and the peak area of the measured fosfomycin;
(3) drawing a standard curve of the fosfomycin diol: preparing standard solutions of fosfomycin trometamol EP impurity A (CAS number: 84954-80-3) with different concentrations, namely fosfomycin diol solutions with different concentrations, injecting the solutions by using an automatic sample injector, carrying out quantitative analysis by an external standard method, and carrying out linear regression analysis on the concentration of the fosfomycin diol and the peak area of the measured fosfomycin diol;
(4) determination of fosfomycin and diol contents in the sample: and (3) for a water sample, adjusting the dilution times to enable the concentration of the water sample to be in the range of a standard curve, detecting to obtain the peak area of fosfomycin and the peak area of a diol of the fosfomycin, quantifying by adopting an external standard method, substituting the measured peak areas into a unitary linear regression equation, and calculating the concentrations of fosfomycin and fosfomycin diol in the sample to be detected.
Further, the chromatographic column is Dionex IonPacTMAS11-HC anion column (preferably 250X 4mm, Dionex, USA).
Further, the suppressor is ASRS 300.
Further, the concentration of the NaOH or KOH solution is 8-10 mmol/L.
Further, the parameters in the ion chromatography are set as follows: the sample introduction amount is 25 mu L, the column temperature is 25-35 ℃, the flow rate is 0.8-1.0mL/min, the suppressor current is 20-25mA, and the analysis time is more than 13 min.
Further, the suppressor current value (in mA) was 2.5 times the leacheate concentration value (in mmol/L).
Further, the concentration of the fosfomycin sodium in the step (2) is 0.0025mmol/L to 1.0 mmol/L.
Further, the concentration of the fosfomycin diol in the step (3) is 0.02mmol/L-1.0 mmol/L.
In the invention, the limit of the quantification of the fosfomycin is 0.0025mmol/L, and the limit of the quantification of the fosfomycin diol is 0.02 mmol/L; the detection limit of the fosfomycin is 0.0005mmol/L, and the detection limit of the fosfomycin diol is 0.0004 mmol/L; the fosfomycin peak-out time is 9.6-9.7minmin, and the fosfomycin diol peak-out time is 8.4-8.5 min.
Compared with the prior art, the method for simultaneously detecting the fosfomycin and the diol thereof by adopting the ion chromatography has the advantages of good reproducibility, low detection limit, high sensitivity, simple operation, wide application of used ion chromatography equipment and the like.
Drawings
FIG. 1 is the time to peak for fosfomycin in example 1;
FIG. 2 is the time to peak of the fosfomycin diol product of example 1;
FIG. 3 is a standard curve for fosfomycin in example 1;
FIG. 4 is a standard curve for the fosfomycin diol of example 1;
FIG. 5 is an ion chromatogram of fosfomycin wastewater after dilution 2000 times in example 1;
FIG. 6 is an ion chromatogram of a mixed solution of fosfomycin and its glycol under 10mmol KOH elution;
FIG. 7 is an ion chromatogram of actual waste water of fosfomycin under 30mmol NaOH elution;
FIG. 8 is an ion chromatogram of a mixed solution of fosfomycin and its diol under 20mmol of NaOH.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention. The following description is only for the purpose of explanation and is not intended to limit the invention.
The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
Example 1
Detection of fosfomycin and its glycols in fosfomycin pharmaceutical wastewater:
the chromatographic column used was Dionex IonPacTMAS11-HC anion column (250X 4mm, Dionex, USA), the suppressor used was ASRS300, and the ion chromatography conditions used were: the sample introduction amount is 25 mu L, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the leacheate is 10mmol/L NaOH solution, the suppressor current is 25mA, and the analysis time of each sample is 60 min.
Preparing solutions with different concentrations (0.0025mmol/L-1.0mmol/L) by fosfomycin sodium for obtaining a standard curve of fosfomycin; solutions of various concentrations were prepared with fosfomycin trometamol EP impurity A fosfomycin diol (0.02mmol/L-1.0mmol/L) to obtain a standard curve of fosfomycin diol.
Taking the fosfomycin wastewater, diluting the fosfomycin wastewater by 2000 times after passing through a 0.22 mu m filter membrane, taking 35 mL ion chromatography sample bottles, and respectively adding 5mL diluted samples to serve as three parallel samples.
And operating the ion chromatograph, injecting samples by using an automatic sample injector after the baseline is stable, and testing the standard substance solution and the fosfomycin wastewater water samples with different concentrations. The time of the fosfomycin peak is shown in figure 1, and the time of the fosfomycin diol peak is shown in figure 2.
And (5) drawing a standard curve according to the concentration of the standard solution and the measured peak area to obtain a linear regression equation. The standard line of fosfomycin is shown in Table 1 and FIG. 3, and its limit of quantitation is 0.0025 mmol/L.
TABLE 1 Standard Curve for Phosphomycin from example 1
The line marked for the fosfomycin diol is shown in Table 2 and FIG. 4, with a limit of quantitation of 0.02 mmol/L.
TABLE 2 Standard Curve for Phosphomycin diol of example 1
| Peak area, [ mu ] S ] min | Phosphomycin concentration, mmol/L |
| 6.3698 | 1 |
| 3.0128 | 0.5 |
| 1.0964 | 0.2 |
| 0.5021 | 0.1 |
| 0.2376 | 0.05 |
| 0.0926 | 0.02 |
The ion chromatogram for fosfomycin wastewater test is shown in figure 5, and the measured peak areas of fosfomycin and fosfomycin glycol in fosfomycin wastewater are respectively substituted into the respective standard curves to calculate the concentrations of fosfomycin and fosfomycin glycol in fosfomycin wastewater, and the results are shown in table 3.
TABLE 3 concentrations of fosfomycin and fosfomycin diol in fosfomycin wastewater of example 1
| Phosphomycin concentration, mmol/L | Fosfomycin diol concentration, mmol/ | |
| Sample | ||
| 1 | 112.6 | 967.8 |
| |
114.6 | 940.4 |
| |
111.0 | 953.7 |
| Mean value of | 112.8 | 953.9 |
| Standard deviation of | 1.8 | 13.7 |
Example 2
When the mixed solution containing both a fosfomycin diol and fosfomycin was examined under the same test conditions as in example 1 using a 10mmol/L KOH solution as a eluent, it was found from FIG. 6 that the peaks of both substances did not affect each other, and the concentrations of both substances could be calculated from the peak areas.
Comparative example 1
FIG. 7 is an ion chromatogram of actual waste water of fosfomycin using a KOH eluent of 30mmol/L and a suppressor current of 75mA, the remaining conditions being the same as in example 1. As can be seen from FIG. 7, the off-peak time of the fosfomycin glycol and fosfomycin is close, and the fosfomycin glycol and fosfomycin cannot be separated well, so that the concentrations of the fosfomycin glycol and fosfomycin in the actual waste water of fosfomycin cannot be accurately detected.
Comparative example 2
FIG. 8 is an ion chromatogram of a mixed solution containing both fosfomycin and fosfomycin diol prepared under the same conditions as in example 1 except that the concentration of NaOH leacheate was changed to 20mmol/L and the suppression current was 50 mA. As can be seen from FIG. 8, the appearance of fosfomycin is affected by chloride ion, which is a common inorganic anion in practical wastewater. Therefore, under these conditions, the concentrations of fosfomycin glycol and fosfomycin cannot be simultaneously and accurately detected.
It will be understood by those skilled in the art that the foregoing is illustrative only and is not to be construed as limiting the invention, and that various modifications, equivalents and improvements made within the spirit and scope of the invention are intended to be included therein.
Claims (6)
1. A method for simultaneously detecting fosfomycin and diol thereof by ion chromatography, which is characterized by comprising the following steps:
(1) selection of ion chromatography instrument and chromatography test conditions: the chromatographic column is a low-capacity hydroxide system cathode protection column and is a Dionex ion PacTMAS11-HC anion column, wherein the suppressor is electrolytic membrane suppressor, the leacheate is NaOH or KOH solution, and the concentration of the NaOH or KOH solution is 8-10 mmol/L;
(2) drawing a fosfomycin standard curve: preparing fosfomycin sodium standard solutions with different concentrations, injecting samples by using an automatic sample injector, carrying out quantitative analysis by an external standard method, and carrying out linear regression analysis on the concentration of fosfomycin and the peak area of the measured fosfomycin;
(3) drawing a standard curve of the fosfomycin diol: preparing fosfomycin diol solutions with different concentrations, injecting samples by using an automatic sample injector, carrying out quantitative analysis by an external standard method, and carrying out linear regression analysis on the concentration of the fosfomycin diol and the peak area of the measured fosfomycin diol;
(4) determination of fosfomycin and diol contents in the sample: and (3) for a water sample, adjusting the dilution times to enable the concentration of the water sample to be in the range of a standard curve, detecting to obtain the peak area of fosfomycin and the peak area of a diol of the fosfomycin, quantifying by adopting an external standard method, substituting the measured peak areas into a unitary linear regression equation, and calculating the concentrations of fosfomycin and fosfomycin diol in the sample to be detected.
2. The method for simultaneous detection of fosfomycin and glycols thereof by ion chromatography as claimed in claim 1, wherein the suppressor is ASRS 300.
3. The method for simultaneously detecting fosfomycin and the glycol thereof by using the ion chromatography as claimed in claim 1, wherein the parameters in the ion chromatography are set as follows: the sample introduction amount is 25 mu L, the column temperature is 25-35 ℃, the flow rate is 0.8-1.0mL/min, the suppressor current is 20-25mA, and the analysis time is more than 13 min.
4. The method for simultaneous detection of fosfomycin and glycols thereof by ion chromatography as claimed in claim 3, wherein the suppressor current value is 2.5 times the value of the eluent concentration.
5. The method for simultaneously detecting fosfomycin and its glycols by ion chromatography as claimed in claim 1, wherein the concentration of fosfomycin sodium in step (2) is 0.0025mmol/L to 1.0 mmol/L.
6. The method for simultaneous detection of fosfomycin and its glycols by ion chromatography as claimed in claim 1, wherein the concentration of fosfomycin glycol in step (3) is 0.02mmol/L to 1.0 mmol/L.
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