CN102841155A - Method for detecting related substances in fosfomycin sodium by high-performance liquid chromatography - Google Patents
Method for detecting related substances in fosfomycin sodium by high-performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for detecting related substances in fosfomycin sodium by a high-performance liquid chromatography applied to the field of substance detection of a compound. The method comprises the following steps: taking a reference solution 1 to inject into a high performance liquid chromatograph; recording a spectrogram, and computing the separation degree of a fosfomycin peak and an impurity A peak, wherein the separation degree is greater than or equal to 1.5; taking the reference solution, sampling in parallel, and injecting into the high performance liquid chromatograph; recording the spectrogram, and computing relative standard deviation of the fosfomycin peak area, wherein the standard deviation is smaller than or equal to 0.85%; taking a test solution and the reference solution 2 to respectively inject into the high performance liquid chromatograph; recording the spectrogram; and computing the peak area of the impurities in the test solution. The method is good in reproducibility of related substance detection in the fosfomycin sodium, high in sensitivity, strong in specificity, accurate in detection, simple to operate, mild in detection condition, wide in application of detection device and apparatus, wide in source of a mobile phase orthophosphate aqueous solution and simple to prepare.
Description
Technical field
The present invention relates to a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium in the compound material detection range.
Background technology
Phosphonomycin (Fosfomycin) be 1967 by a kind of broad-spectrum antibiotic of finding in Merck company and the Spain CEPA company Streptothrix from Spain's soil; Its molecular weight is very little, is a brand-new microbiotic that is different from other any microbiotic structures.In China, Europe and Japan and other countries and regional widespread use, it is applied to clinical kind and mainly contains 3 phosphonomycin, is respectively fosfomycin sodium, FOM-Ca and fosfomycin trometamol at present, and wherein, China's application kind more widely is a fosfomycin sodium.Fosfomycin sodium has a broad antifungal spectrum, bad reaction are little, are prone to get in the infected tissue, are that treatment is light, one of a line medication of grade and moderate infection, are the ideal medicament that treatment severe infection, drug-fast bacteria infection are united use.Safety, low toxicity, need not to try quick characteristics and offer convenience to clinical practice, comparing with other microbiotic has unique advantage.But, all do not record at present the detection method of its related substance in the legal detection method of fosfomycin sodium, be quality and the security thereof that guarantees medicine, need badly and set up detection method it is controlled.Therefore, develop a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium is the new problem that needs to be resolved hurrily always.
Summary of the invention
The object of the present invention is to provide a kind of favorable reproducibility, highly sensitive, detect accurately, specificity is strong, method of operating is easy, the widely used employing high performance liquid chromatography of test facilities and instruments detects related substance in the fosfomycin sodium method.
The objective of the invention is to realize like this: a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium, said method comprises the steps:
(1) chromatographic condition
Chromatographic column is the silicagel column of amino-propyl silane bonding, and detecting device is a differential refraction detector, and moving phase is the orthophosphate WS;
(2) preparation of reference substance solution
Get the fosfomycin sodium standard items and be mixed with the reference substance solution of fosfomycin sodium with moving phase;
(3) with reference to the preparation of solution one
(a) with water-moistened fosfomycin sodium raw material, in baking oven, heat, obtain the fosfomycin sodium residue, impure A in the residue dissolves said residue with an amount of moving phase, obtains residue solution; (b) getting the fosfomycin sodium raw material is dissolved in an amount of said residue solution and obtains with reference to solution one;
(4) preparation of need testing solution
Get the fosfomycin sodium raw material and be mixed with need testing solution with moving phase;
(5) with reference to the preparation of solution two
Need testing solution is diluted with moving phase, obtain with reference to solution two;
(6) assay method
Get with reference to solution one and inject high performance liquid chromatograph, the record spectrogram, the degree of separation of calculating phosphonomycin peak and impurity A peak should be more than or equal to 1.5; Get reference substance solution, parallel sample introduction injects high performance liquid chromatograph, the record spectrogram, and the relative standard deviation of calculating the phosphonomycin peak area should be less than or equal to 0.85%; Get need testing solution, inject high performance liquid chromatograph respectively, record spectrogram, the peak area of impurity in the calculating need testing solution with reference to solution two;
In step (1), the silicagel column of described amino-propyl silane bonding is the Zorbax-NH2 post; The temperature of said detecting device is 30-40 ℃, and preferred temperature is 35 ℃; Said orthophosphate is selected from one or more in sodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, the potassium dihydrogen phosphate, the preferably phosphoric acid potassium dihydrogen; The concentration of the said orthophosphate WS is 0.05-0.12mol/l, preferred 0.08mol/l; In step (2), the concentration of fosfomycin sodium is 0.06-0.24g/ml in the said reference substance solution, preferred 0.12g/ml; In the step (a) in step (3), the temperature of said baking oven for heating is 50-70 ℃, 60 ℃ of preferred temperature; The time of said heating is 16-30 hour, and the preferred time is 24 hours; Said residue solution is that every 100ml contains the residue obtained residue solution of the said fosfomycin sodium raw material heating of 1-2g, and preferred every 100ml contains the residue obtained residue solution of the said fosfomycin sodium raw material heating of 1.5g; In the step (b) in step (3), saidly contain the fosfomycin sodium raw material 0.6-2.4g that is dissolved in the said residue solution with reference to solution one for every 10ml, preferred every 10ml contains the fosfomycin sodium raw material 1.2g that is dissolved in the said residue solution; In step (4), the concentration of fosfomycin sodium is 0.06-0.24g/ml in the said need testing solution, and the concentration of preferred fosfomycin sodium is 0.12g/ml; In step (5), every 200ml is said to contain the said need testing solution of 0.5-1.5ml with reference to solution two, and preferred every 200ml is said to contain the said need testing solution of 1ml with reference to solution two; In step (6), said is 3-20 μ l with reference to solution one, reference substance solution, need testing solution, with reference to the sampling volume of solution two, and preferred sampling volume is 10 μ l; Said impurity is impurity A, nonspecific impurity and total impurities; The compound method of the described orthophosphate WS is filtered for to place water to dissolve an amount of said orthophosphate, and is ultrasonic; Described impurity A is (1, the 2-dihydroxypropyl) phosphonic acids.
Main points of the present invention are to adopt high performance liquid chromatography to detect the method for related substance in the fosfomycin sodium.Its principle is: utilize the different of fosfomycin sodium and impurity design feature; Pass through high performance liquid chromatography; The silicagel column that adopts amino-propyl silane bonding is as chromatographic column, and the orthophosphate WS makes fosfomycin sodium and impurity obtain good separating as moving phase; Detect through differential refraction detector again, thereby and calculate the content that obtains related substance in the fosfomycin sodium raw material.
A kind ofly adopt method that high performance liquid chromatography detects related substance in the fosfomycin sodium compared with prior art; Have the favorable reproducibility that detects for related substance in the fosfomycin sodium, highly sensitive, specificity strong, detections is accurate, easy and simple to handle, testing conditions is gentle, test facilities and instruments is widely used, moving phase orthophosphate aqueous feed solution source is wide, prepare advantages such as simple, will be widely used in the compound material detection range.
Below in conjunction with embodiment the present invention is elaborated.
Fig. 1 is with reference to the synoptic diagram of solution one HPLC collection of illustrative plates among the embodiment one.
Fig. 2 is the synoptic diagram of reference substance solution HPLC collection of illustrative plates among the embodiment one.
Fig. 3 is the synoptic diagram of need testing solution HPLC collection of illustrative plates among the embodiment one.
Fig. 4 is with reference to the synoptic diagram of solution two HPLC collection of illustrative plates among the embodiment one.
Fig. 5 is the structural formula figure of impurity A among the present invention.
Embodiment
Following examples will help understanding of the present invention, but these embodiment have been merely and the present invention are explained the present invention is not limited to these contents.
The implication of " test sample " and " fosfomycin sodium standard items " is following in following examples:
Test sample: be the fosfomycin sodium raw material of related substance to be determined.
Fosfomycin sodium standard items: be the phosphonomycin sodium pure product behind the purified removal impurity of fosfomycin sodium raw material.
Embodiment one
(1) chromatographic condition
Chromatographic column: Zorbax-NH2 (4.6 * 250mm, 5 μ m)
Detecting device: RID (differential refraction detector)
Detector temperature: 35 ℃
Wherein, chromatographic column Zorbax-NH2 is the silicagel column of the amino-propyl silane bonding of Agilent company.
The preparation of moving phase: precision takes by weighing the 10.89g potassium dihydrogen phosphate, places the 1000ml purified water to dissolve, and filters, and is ultrasonic, obtains the potassium dihydrogen phosphate of 0.08mol/l.
(2) preparation of reference substance solution
Dissolve 0.6g fosfomycin sodium standard items with moving phase, and be diluted to 5.0ml, obtain the reference substance solution that every 1ml contains 0.12g fosfomycin sodium standard items with moving phase.
(3) with reference to the preparation of solution one
(a) with 60 μ l water-wet 0.3g test samples, heating is 24 hours in 60 ℃ of baking ovens.Use the moving phase dissolved residue, and be diluted to 20.0ml with moving phase and obtain residue solution, obtain every 100ml and contain, contain impurity A in this solution the residue obtained residue solution of 1.5g fosfomycin sodium heating; (b) dissolving 0.6g test sample is in residue solution, and with the residue solution dilution to 5.0ml, obtain every 10ml contain the 1.2g test sample with reference to solution one, contain impurity A in this solution.
(4) preparation of need testing solution
Dissolve the 0.6g test sample with moving phase, and be diluted to 5.0ml, obtain the need testing solution that every 1ml contains the 0.12g test sample with moving phase.
(5) with reference to the preparation of solution two
The accurate need testing solution 1.0ml that draws is added in the 100ml volumetric flask, and is diluted to scale with moving phase, shakes up; The accurate above-mentioned liquid of 5.0ml of drawing is diluted to 10.0ml with moving phase, obtain every 200ml contain the 1ml need testing solution with reference to solution two.
(6) assay method
Get with reference to solution one, treat sample introduction behind the instrument stabilizer, sample size 10.0 μ l, the record spectrogram, calculating phosphonomycin peak 1 should be more than or equal to 1.5 with the degree of separation (R) at impurity A peak 2.
Present embodiment is 2.9 with reference to the degree of separation (R) at phosphonomycin peak in the solution one 1 and impurity A peak 2, sees Fig. 1 with reference to the HPLC collection of illustrative plates of solution one.
Get reference substance solution, treat sample introduction behind the instrument stabilizer, parallel sample introduction 6 pins, sample size 10 μ l, the record spectrogram, the relative standard deviation (RSD) who calculates the phosphonomycin peak area should be smaller or equal to 0.85%.
Present embodiment, the peak area of parallel sample introduction 6 pins of reference substance solution, the mean value and the RSD situation of peak area see the following form 1:
The peak area information slip of parallel sample introduction 6 pins of table 1 reference substance solution
| Project | Peak area A1 | Peak area A2 | Peak area A3 | Peak area A4 | Peak area A5 | Peak area A6 | A mean value | RSD |
| Numerical value | 6090931 | 6016712 | 6029446 | 6057545 | 6023670 | 6028969 | 6041212 | 0.47% |
The HPLC collection of illustrative plates of reference substance solution is seen Fig. 2.
Get 3 batches of fosfomycin sodium bulk drugs, prepare, obtain the need testing solution of 3 lot numbers and with reference to solution two according to the preparation method of need testing solution and with reference to the preparation method of solution two; Advance need testing solution respectively and with reference to solution two; Sample size 10 μ l, the record collection of illustrative plates respectively repeats once.Need testing solution and see Fig. 3 and Fig. 4 respectively with reference to the HPLC collection of illustrative plates of solution two.Horizontal ordinate among Fig. 1 to Fig. 4 " MIN " representative " minute ", ordinate " MV ", representative " millivolt ".
The chromatographic peak separation case: the separation case of phosphonomycin peak 1, impurity A peak 2 and nonspecific impurity peaks 3 is better in the collection of illustrative plates of need testing solution.
Computing formula:
Impurity A and nonspecific impurity calculate by single impurity formula:
Total impurities:
Wherein ri, rb, r always be respectively single impurity, with reference to the peak area of solution two, total impurities.
Result of calculation: fosfomycin sodium related substance testing result is seen table 2.
Table 2 fosfomycin sodium related substance testing result
Embodiment two
(1) chromatographic condition
Chromatographic column: Zorbax-NH2 (4.6 * 250mm, 5 μ m)
Detecting device: RID (differential refraction detector)
Detector temperature: 30 ℃
Wherein, chromatographic column Zorbax-NH2 is the silicagel column of the amino-propyl silane bonding of Agilent company.
The preparation of moving phase: precision takes by weighing the 6.80g potassium dihydrogen phosphate, places the 1000ml purified water to dissolve, and filters, and is ultrasonic, obtains the potassium dihydrogen phosphate of 0.05mol/l.
(2) preparation of reference substance solution
Dissolve 0.3g fosfomycin sodium standard items with moving phase, and be diluted to 5.0ml, obtain the reference substance solution that every 1ml contains 0.06g fosfomycin sodium standard items with moving phase.
(3) with reference to the preparation of solution one
(a) with 30 μ l water-wet 0.2g test samples, heating is 30 hours in 50 ℃ of baking ovens.Use the moving phase dissolved residue, and be diluted to 20.0ml with moving phase and obtain residue solution, obtain every 100ml and contain, contain impurity A in this solution the residue obtained residue solution of 1.0g fosfomycin sodium heating; (b) dissolving 0.3g test sample is in residue solution, and with the residue solution dilution to 5.0ml, obtain every 10ml contain the 0.6g test sample with reference to solution one, contain impurity A in this solution.
(4) preparation of need testing solution
Dissolve the 0.3g test sample with moving phase, and be diluted to 5.0ml, obtain the need testing solution that every 1ml contains the 0.06g test sample with moving phase.
(5) with reference to the preparation of solution two
The accurate need testing solution 1ml that draws is added in the 100ml volumetric flask, and is diluted to scale with moving phase, shakes up; The accurate above-mentioned liquid of 5.0ml of drawing is diluted to 10.0ml with moving phase, obtain every 200ml contain the 1ml need testing solution with reference to solution two.
(6) assay method
Get with reference to solution one, treat sample introduction behind the instrument stabilizer, sample size 20.0 μ l, the record spectrogram, calculating phosphonomycin peak 1 should be more than or equal to 1.5 with the degree of separation (R) at impurity A peak 2.
Present embodiment is 2.2 with reference to the degree of separation (R) at phosphonomycin peak in the solution one 1 and impurity A peak 2.
Get reference substance solution, treat sample introduction behind the instrument stabilizer, parallel sample introduction 6 pins, sample size 20 μ l, the record spectrogram, the relative standard deviation (RSD) who calculates the phosphonomycin peak area should be smaller or equal to 0.85%.
Present embodiment, the RSD of parallel sample introduction 6 pins of reference substance solution is through being calculated as 0.51%.
Get 3 batches of fosfomycin sodium bulk drugs, prepare, obtain the need testing solution of 3 lot numbers and with reference to solution two according to the preparation method of need testing solution and with reference to the preparation method of solution two; Advance need testing solution respectively and with reference to solution two; Sample size 20 μ l, the record collection of illustrative plates respectively repeats once.
The chromatographic peak separation case: the separation case of phosphonomycin peak 1, impurity A peak 2 and nonspecific impurity peaks 3 is general in the collection of illustrative plates of need testing solution.
Computing formula: with embodiment one.
Result of calculation: fosfomycin sodium related substance testing result is seen table 3.
Table 3 fosfomycin sodium related substance testing result
Embodiment three
(1) chromatographic condition
Chromatographic column: Zorbax-NH2 (4.6 * 250mm, 5 μ m)
Detecting device: RID (differential refraction detector)
Detector temperature: 40 ℃
Wherein, chromatographic column Zorbax-NH2 is the silicagel column of the amino-propyl silane bonding of Agilent company.
The preparation of moving phase: precision takes by weighing the 16.33g potassium dihydrogen phosphate, places the 1000ml purified water to dissolve, and filters, and is ultrasonic, obtains the potassium dihydrogen phosphate of 0.12mol/l.
(2) preparation of reference substance solution
Dissolve 1.2g fosfomycin sodium standard items with moving phase, and be diluted to 5.0ml, obtain the reference substance solution that every 1ml contains 0.24g fosfomycin sodium standard items with moving phase.
(3) with reference to the preparation of solution one
(a) with 90 μ l water-wet 0.4g test samples, heating is 16 hours in 70 ℃ of baking ovens.Use the moving phase dissolved residue, and be diluted to 20.0ml with moving phase and obtain residue solution, obtain every 100ml and contain, contain impurity A in this solution the residue obtained residue solution of 2.0g fosfomycin sodium heating; (b) dissolving 1.2g test sample is in residue solution, and with the residue solution dilution to 5.0ml, obtain every 10ml contain the 2.4g test sample with reference to solution one, contain impurity A in this solution.
(4) preparation of need testing solution
Dissolve the 1.2g test sample with moving phase, and be diluted to 5.0ml, obtain the need testing solution that every 1ml contains the 0.24g test sample with moving phase.
(5) with reference to the preparation of solution two
The accurate need testing solution 1ml that draws is added in the 100ml volumetric flask, and is diluted to scale with moving phase, shakes up; The accurate above-mentioned liquid of 5.0ml of drawing is diluted to 10.0ml with moving phase, obtain every 200ml contain the 1ml need testing solution with reference to solution two.
(6) assay method
Get with reference to solution one, treat sample introduction behind the instrument stabilizer, sample size 5.0 μ l, the record spectrogram, calculating phosphonomycin peak 1 should be more than or equal to 1.5 with the degree of separation (R) at impurity A peak 2.
Present embodiment is 2.3 with reference to the degree of separation (R) at phosphonomycin peak in the solution one 1 and impurity A peak 2.
Get reference substance solution, treat sample introduction behind the instrument stabilizer, parallel sample introduction 6 pins, sample size 5 μ l, the record spectrogram, the relative standard deviation (RSD) who calculates phosphonomycin peak 1 area should be smaller or equal to 0.85%.
Present embodiment, the RSD of parallel sample introduction 6 pins of reference substance solution is through being calculated as 0.53%.
Get 3 batches of fosfomycin sodium bulk drugs, prepare, obtain the need testing solution of 3 lot numbers and with reference to solution two according to the preparation method of need testing solution and with reference to the preparation method of solution two; Advance need testing solution respectively and with reference to solution two; Sample size 5 μ l, the record collection of illustrative plates respectively repeats once.
The chromatographic peak separation case: the separation case of phosphonomycin peak 1, impurity A peak 2 and nonspecific impurity peaks 3 is general in the collection of illustrative plates of need testing solution.
Computing formula: with embodiment one.
Result of calculation: fosfomycin sodium related substance testing result is seen table 4.
Table 4 fosfomycin sodium related substance testing result
Embodiment four
(1) chromatographic condition
Chromatographic column: Zorbax-NH2 (4.6 * 250mm, 5 μ m)
Detecting device: RID (differential refraction detector)
Detector temperature: 35 ℃
Wherein, chromatographic column Zorbax-NH2 is the silicagel column of the amino-propyl silane bonding of Agilent company.
The preparation of moving phase: precision takes by weighing 28.65g sodium hydrogen phosphate (Na
2HPO
412H
2O), place the 1000ml purified water to dissolve, filter, ultrasonic, obtain the disodium phosphate soln of 0.08mol/l.
(2) preparation of reference substance solution
Dissolve 0.6g fosfomycin sodium standard items with moving phase, and be diluted to 5.0ml, obtain the reference substance solution that every 1ml contains 0.12g fosfomycin sodium standard items with moving phase.
(3) with reference to the preparation of solution one
(a) with 60 μ l water-wet 0.3g test samples, heating is 24 hours in 60 ℃ of baking ovens.Use the moving phase dissolved residue, and be diluted to 20.0ml with moving phase and obtain residue solution, obtain every 100ml and contain, contain impurity A in this solution the residue obtained residue solution of 1.5g fosfomycin sodium heating; (b) dissolving 0.6g test sample is in residue solution, and with the residue solution dilution to 5.0ml, obtain every 10ml contain the 1.2g test sample with reference to solution one, contain impurity A in this solution.
(4) preparation of need testing solution
Dissolve the 0.6g test sample with moving phase, and be diluted to 5.0ml, obtain the need testing solution that every 1ml contains the 0.12g test sample with moving phase.
(5) with reference to the preparation of solution two
The accurate need testing solution 1.0ml that draws is added in the 100ml volumetric flask, and is diluted to scale with moving phase, shakes up; The accurate above-mentioned liquid of 5.0ml of drawing is diluted to 10.0ml with moving phase, obtain every 200ml contain the 1ml need testing solution with reference to solution two.
(6) assay method
Get with reference to solution one, treat sample introduction behind the instrument stabilizer, sample size 3 μ l, the record spectrogram, calculating phosphonomycin peak 1 should be more than or equal to 1.5 with the degree of separation (R) at impurity A peak 2.
Present embodiment is 2.3 with reference to the degree of separation (R) at phosphonomycin peak in the solution one 1 and impurity A peak 2.
Get reference substance solution, treat sample introduction behind the instrument stabilizer, parallel sample introduction 6 pins, sample size 3 μ l, the record spectrogram, the relative standard deviation (RSD) who calculates the phosphonomycin peak area should be smaller or equal to 0.85%.
Present embodiment, the relative standard deviation (RSD) of the phosphonomycin peak area of parallel sample introduction 6 pins of reference substance solution is 0.51%.
Get 3 batches of fosfomycin sodium bulk drugs, prepare, obtain the need testing solution of 3 lot numbers and with reference to solution two according to the preparation method of need testing solution and with reference to the preparation method of solution two; Advance need testing solution respectively and with reference to solution two; Sample size 3 μ l, the record collection of illustrative plates respectively repeats once.The chromatographic peak separation case: the separation case of phosphonomycin peak 1, impurity A peak 2 and nonspecific impurity peaks 3 is better in the collection of illustrative plates of need testing solution.
Computing formula: with embodiment one.
Result of calculation: fosfomycin sodium related substance testing result is seen table 5.
Table 5 fosfomycin sodium related substance testing result
Embodiment five
(1) chromatographic condition
Chromatographic column: Zorbax-NH2 (4.6 * 250mm, 5 μ m)
Detecting device: RID (differential refraction detector)
Detector temperature: 35 ℃
Wherein, chromatographic column Zorbax-NH2 is the silicagel column of the amino-propyl silane bonding of Agilent company.
The preparation of moving phase: precision takes by weighing the 13.93g dipotassium hydrogen phosphate, places the 1000ml purified water to dissolve, and filters, and is ultrasonic, obtains the dipotassium hydrogen phosphate solution of 0.08mol/l.
(2) preparation of reference substance solution
Dissolve 0.6g fosfomycin sodium standard items with moving phase, and be diluted to 5.0ml, obtain the reference substance solution that every 1ml contains 0.12g fosfomycin sodium standard items with moving phase.
(3) with reference to the preparation of solution one
(a) with 60 μ l water-wet 0.3g test samples, heating is 24 hours in 60 ℃ of baking ovens.Use the moving phase dissolved residue, and be diluted to 20.0ml with moving phase and obtain residue solution, obtain every 100ml and contain, contain impurity A in this solution the residue obtained residue solution of 1.5g fosfomycin sodium heating; (b) dissolving 0.6g test sample is in residue solution, and with the residue solution dilution to 5.0ml, obtain every 10ml contain the 1.2g test sample with reference to solution one, contain impurity A in this solution.
(4) preparation of need testing solution
Dissolve the 0.6g test sample with moving phase, and be diluted to 5.0ml, obtain the need testing solution that every 1ml contains the 0.12g test sample with moving phase.
(5) with reference to the preparation of solution two
The accurate need testing solution 1.0ml that draws is added in the 100ml volumetric flask, and is diluted to scale with moving phase, shakes up; The accurate above-mentioned liquid of 5.0ml of drawing is diluted to 10.0ml with moving phase, obtain every 200ml contain the 1ml need testing solution with reference to solution two.
(6) assay method
Get with reference to solution one, treat sample introduction behind the instrument stabilizer, sample size 3 μ l, the record spectrogram, calculating phosphonomycin peak 1 should be more than or equal to 1.5 with the degree of separation (R) at impurity A peak 2.
Present embodiment is 2.2 with reference to the degree of separation (R) at phosphonomycin peak in the solution one 1 and impurity A peak 2.
Get reference substance solution, treat sample introduction behind the instrument stabilizer, parallel sample introduction 6 pins, sample size 3 μ l, the record spectrogram, the relative standard deviation (RSD) who calculates the phosphonomycin peak area should be smaller or equal to 0.85%.
Present embodiment, the relative standard deviation (RSD) of the phosphonomycin peak area of parallel sample introduction 6 pins of reference substance solution is 0.61%.
Get 3 batches of fosfomycin sodium bulk drugs, prepare, obtain the need testing solution of 3 lot numbers and with reference to solution two according to the preparation method of need testing solution and with reference to the preparation method of solution two; Advance need testing solution respectively and with reference to solution two; Sample size 3 μ l, the record collection of illustrative plates respectively repeats once.The chromatographic peak separation case: the separation case of phosphonomycin peak 1, impurity A peak 2 and nonspecific impurity peaks 3 is general in the collection of illustrative plates of need testing solution.
Computing formula: with embodiment one.
Result of calculation: fosfomycin sodium related substance testing result is seen table 6.
Table 6 fosfomycin sodium related substance testing result
Embodiment six
(1) chromatographic condition
Chromatographic column: Zorbax-NH2 (4.6 * 250mm, 5 μ m)
Detecting device: RID (differential refraction detector)
Detector temperature: 35 ℃
Wherein, chromatographic column Zorbax-NH2 is the silicagel column of the amino-propyl silane bonding of Agilent company.
The preparation of moving phase: precision takes by weighing the 9.6g sodium dihydrogen phosphate, places the 1000ml purified water to dissolve, and filters, and is ultrasonic, obtains the sodium dihydrogen phosphate of 0.08mol/l.
(2) preparation of reference substance solution
Dissolve 0.6g fosfomycin sodium standard items with moving phase, and be diluted to 5.0ml, obtain the reference substance solution that every 1ml contains 0.12g fosfomycin sodium standard items with moving phase.
(3) with reference to the preparation of solution one
(a) with 60 μ l water-wet 0.3g test samples, heating is 24 hours in 60 ℃ of baking ovens.Use the moving phase dissolved residue, and be diluted to 20.0ml with moving phase and obtain residue solution, obtain every 100ml and contain, contain impurity A in this solution the residue obtained residue solution of 1.5g fosfomycin sodium heating; (b) dissolving 0.6g test sample is in residue solution, and with the residue solution dilution to 5.0ml, obtain every 10ml contain the 1.2g test sample with reference to solution one, contain impurity A in this solution.
(4) preparation of need testing solution
Dissolve the 0.6g test sample with moving phase, and be diluted to 5.0ml, obtain the need testing solution that every 1ml contains the 0.12g test sample with moving phase.
(5) with reference to the preparation of solution two
The accurate need testing solution 1.0ml that draws is added in the 100ml volumetric flask, and is diluted to scale with moving phase, shakes up; The accurate above-mentioned liquid of 5.0ml of drawing is diluted to 10.0ml with moving phase, obtain every 200ml contain the 1ml need testing solution with reference to solution two.
(6) assay method
Get with reference to solution one, treat sample introduction behind the instrument stabilizer, sample size 10 μ l, the record spectrogram, calculating phosphonomycin peak 1 should be more than or equal to 1.5 with the degree of separation (R) at impurity A peak 2.
Present embodiment is 2.7 with reference to the degree of separation (R) at phosphonomycin peak in the solution one 1 and impurity A peak 2.
Get reference substance solution, treat sample introduction behind the instrument stabilizer, parallel sample introduction 6 pins, sample size 10 μ l, the record spectrogram, the relative standard deviation (RSD) who calculates the phosphonomycin peak area should be smaller or equal to 0.85%.
Present embodiment, the relative standard deviation (RSD) of the phosphonomycin peak area of parallel sample introduction 6 pins of reference substance solution is 0.60%.
Get 3 batches of fosfomycin sodium bulk drugs, prepare, obtain the need testing solution of 3 lot numbers and with reference to solution two according to the preparation method of need testing solution and with reference to the preparation method of solution two; Advance need testing solution respectively and with reference to solution two; Sample size 10 μ l, the record collection of illustrative plates respectively repeats once.The chromatographic peak separation case: the separation case of phosphonomycin peak 1, impurity A peak 2 and nonspecific impurity peaks 3 is better in the collection of illustrative plates of need testing solution.
Computing formula: with embodiment one.
Result of calculation: fosfomycin sodium related substance testing result is seen table 7.
Table 7 fosfomycin sodium related substance testing result
Embodiment seven
(1) chromatographic condition
Chromatographic column: Zorbax-NH2 (4.6 * 250mm, 5 μ m)
Detecting device: RID (differential refraction detector)
Detector temperature: 35 ℃
Wherein, chromatographic column Zorbax-NH2 is the silicagel column of the amino-propyl silane bonding of Agilent company.
The preparation of moving phase: precision takes by weighing 5.44g potassium dihydrogen phosphate and 4.8g sodium dihydrogen phosphate; Place the 1000ml purified water to dissolve; Filter; Ultrasonic, obtain the potassium dihydrogen phosphate of total concentration 0.08mol/l and the mixed solution of sodium dihydrogen phosphate, wherein potassium dihydrogen phosphate and sodium dihydrogen phosphate divide other concentration to be 0.04mol/l.
(2) preparation of reference substance solution
Dissolve 0.6g fosfomycin sodium standard items with moving phase, and be diluted to 5.0ml, obtain the reference substance solution that every 1ml contains 0.12g fosfomycin sodium standard items with moving phase.
(3) with reference to the preparation of solution one
(a) with 60 μ l water-wet 0.3g test samples, heating is 24 hours in 60 ℃ of baking ovens.Use the moving phase dissolved residue, and be diluted to 20.0ml with moving phase and obtain residue solution, obtain every 100ml and contain, contain impurity A in this solution the residue obtained residue solution of 1.5g fosfomycin sodium heating; (b) dissolving 0.6g test sample is in residue solution, and with the residue solution dilution to 5.0ml, obtain every 10ml contain the 1.2g test sample with reference to solution one, contain impurity A in this solution.
(4) preparation of need testing solution
Dissolve the 0.6g test sample with moving phase, and be diluted to 5.0ml, obtain the need testing solution that every 1ml contains the 0.12g test sample with moving phase.
(5) with reference to the preparation of solution two
The accurate need testing solution 1.0ml that draws is added in the 100ml volumetric flask, and is diluted to scale with moving phase, shakes up; The accurate above-mentioned liquid of 5.0ml of drawing is diluted to 10.0ml with moving phase, obtain every 200ml contain the 1ml need testing solution with reference to solution two.
(6) assay method
Get with reference to solution one, treat sample introduction behind the instrument stabilizer, sample size 10 μ l, the record spectrogram, calculating phosphonomycin peak 1 should be more than or equal to 1.5 with the degree of separation (R) at impurity A peak 2.
Present embodiment is 2.0 with reference to the degree of separation (R) at phosphonomycin peak in the solution one 1 and impurity A peak 2.
Get reference substance solution, treat sample introduction behind the instrument stabilizer, parallel sample introduction 6 pins, sample size 10 μ l, the record spectrogram, the relative standard deviation (RSD) who calculates the phosphonomycin peak area should be smaller or equal to 0.85%.
Present embodiment, the relative standard deviation (RSD) of the phosphonomycin peak area of parallel sample introduction 6 pins of reference substance solution is 0.66%.
Get 3 batches of fosfomycin sodium bulk drugs, prepare, obtain the need testing solution of 3 lot numbers and with reference to solution two according to the preparation method of need testing solution and with reference to the preparation method of solution two; Advance need testing solution respectively and with reference to solution two; Sample size 10 μ l, the record collection of illustrative plates respectively repeats once.The chromatographic peak separation case: the separation case of phosphonomycin peak 1, impurity A peak 2 and nonspecific impurity peaks 3 is general in the collection of illustrative plates of need testing solution.
Computing formula: with embodiment one.
Result of calculation: fosfomycin sodium related substance testing result is seen table 8.
Table 8 fosfomycin sodium related substance testing result
Fig. 5 is that impurity A is the structural formula figure of (1, the 2-dihydroxypropyl) phosphonic acids among the present invention in the accompanying drawing, and its English being called (1,2-dihydroxypropyl) phosphonic acid.The make a comment or criticism various salt of phosphoric acid (H3PO4) of " orthophosphate " mentioned among the present invention.
Among Fig. 1 to Fig. 4, each symbolic representation: 1 phosphonomycin peak; 2 impurity A peaks; 3 nonspecific impurity peaks.
Claims (9)
1. method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium, it is characterized in that: said method comprises the steps:
(1) chromatographic condition
Chromatographic column is the silicagel column of amino-propyl silane bonding, and detecting device is a differential refraction detector, and moving phase is the orthophosphate WS;
(2) preparation of reference substance solution
Get the fosfomycin sodium standard items and be mixed with the reference substance solution of fosfomycin sodium with moving phase;
(3) with reference to the preparation of solution one
(a) with water-moistened fosfomycin sodium raw material, in baking oven, heat, obtain the fosfomycin sodium residue, impure A in the residue dissolves said residue with an amount of moving phase, obtains residue solution; (b) getting the fosfomycin sodium raw material is dissolved in an amount of said residue solution and obtains with reference to solution one;
(4) preparation of need testing solution
Get the fosfomycin sodium raw material and be mixed with need testing solution with moving phase;
(5) with reference to the preparation of solution two
Need testing solution is diluted with moving phase, obtain with reference to solution two;
(6) assay method
Get with reference to solution one and inject high performance liquid chromatograph, the record spectrogram, the degree of separation of calculating phosphonomycin peak and impurity A peak should be more than or equal to 1.5; Get reference substance solution, parallel sample introduction injects high performance liquid chromatograph, the record spectrogram, and the relative standard deviation of calculating the phosphonomycin peak area should be less than or equal to 0.85%; Get need testing solution, inject high performance liquid chromatograph respectively, record spectrogram, the peak area of impurity in the calculating need testing solution with reference to solution two.
2. a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium according to claim 1, it is characterized in that: in step (1), the silicagel column of described amino-propyl silane bonding is the Zorbax-NH2 post; The temperature of said detecting device is 30-40 ℃, and preferred temperature is 35 ℃; Said orthophosphate is selected from one or more in sodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, the potassium dihydrogen phosphate, the preferably phosphoric acid potassium dihydrogen; The concentration of the said orthophosphate WS is 0.05-0.12mol/l, preferred 0.08mol/l.
3. a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium according to claim 1, it is characterized in that: in step (2), the concentration of fosfomycin sodium is 0.06-0.24g/ml in the said reference substance solution, preferred 0.12g/ml.
4. a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium according to claim 1, it is characterized in that: in the step (a) in step (3), the temperature of said baking oven for heating is 50-70 ℃, 60 ℃ of preferred temperature; The time of said heating is 16-30 hour, and the preferred time is 24 hours; Said residue solution is that every 100ml contains the residue obtained residue solution of the said fosfomycin sodium raw material heating of 1-2g, and preferred every 100ml contains the residue obtained residue solution of the said fosfomycin sodium raw material heating of 1.5g; In the step (b) in step (3), saidly contain the fosfomycin sodium raw material 0.6-2.4g that is dissolved in the said residue solution with reference to solution one for every 10ml, preferred every 10ml contains the fosfomycin sodium raw material 1.2g that is dissolved in the said residue solution.
5. a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium according to claim 1; It is characterized in that: in step (4); The concentration of fosfomycin sodium is 0.06-0.24g/ml in the said need testing solution, and the concentration of preferred fosfomycin sodium is 0.12g/ml.
6. a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium according to claim 1; It is characterized in that: in step (5); Every 200ml is said to contain the said need testing solution of 0.5-1.5ml with reference to solution two, and preferred every 200ml is said to contain the said need testing solution of 1ml with reference to solution two.
7. a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium according to claim 1; It is characterized in that: in step (6); Said is 3-20 μ l with reference to solution one, reference substance solution, need testing solution, with reference to the sampling volume of solution two, and preferred sampling volume is 10 μ l; Said impurity is impurity A, nonspecific impurity and total impurities.
8. a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium according to claim 1 is characterized in that: the compound method of the described orthophosphate WS is filtered for to place water to dissolve an amount of said orthophosphate, and is ultrasonic.
9. a kind of method that adopts high performance liquid chromatography to detect related substance in the fosfomycin sodium according to claim 1, it is characterized in that: described impurity A is (1, the 2-dihydroxypropyl) phosphonic acids.
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| CN103728385A (en) * | 2013-12-24 | 2014-04-16 | 中国环境科学研究院 | Method for detecting fosfomycin sodium in pharmaceutical wastewater by adopting ion chromatography |
| CN115097051A (en) * | 2022-07-27 | 2022-09-23 | 广州艾奇西新药研究有限公司 | Method for separating and detecting fosfomycin sodium and related substances in preparation thereof by using high performance liquid chromatography |
| CN115166083A (en) * | 2022-07-04 | 2022-10-11 | 国药集团致君（深圳）制药有限公司 | Detection method and application of fosfomycin trometamol related substances |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103728385A (en) * | 2013-12-24 | 2014-04-16 | 中国环境科学研究院 | Method for detecting fosfomycin sodium in pharmaceutical wastewater by adopting ion chromatography |
| CN115166083A (en) * | 2022-07-04 | 2022-10-11 | 国药集团致君（深圳）制药有限公司 | Detection method and application of fosfomycin trometamol related substances |
| CN115097051A (en) * | 2022-07-27 | 2022-09-23 | 广州艾奇西新药研究有限公司 | Method for separating and detecting fosfomycin sodium and related substances in preparation thereof by using high performance liquid chromatography |
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