Method for detecting residual quantity of micro or trace fosfomycin tromethamine

Abstract

The invention provides a method for detecting the residual quantity of micro or trace fosfomycin tromethamine, and belongs to the field of pharmaceutical analysis. According to the method, a low-capacity hydroxyl system cathode protection column and a high-capacity anion exchange separation column are used as chromatographic columns, an anion electrolysis regeneration suppressor is adopted, a sodium hydroxide or potassium hydroxide solution with the current of 36 mA to 44 mA and the current of 15 mmol / L to 17 mmol / L is used as leacheate, and the residual quantity of fosfomycin tromethamine is calculated according to the peak area by an external standard method. Compared with the prior art, the method has the advantages that the sensitivity is high, the quantitation limit of fosfomycin trometamol is as low as 0.0475 mu g / ml, the reproducibility is good, the operation is simple, the detection condition is mild, the application of detection equipment and instruments is wide, the source of leacheate is wide, the preparation is simple, and the like.

Description

Technical Field

[0001] The invention relates to a method for detecting micro or trace amounts of fosfomycin tromethamine residues in the field of drug analysis. Background Art

[0002] Fosfomycin Trometamol, also known as Fosfomycin Trometamol in English, is a salt of fosfomycin and trometamol. It is an antibiotic drug. The antibacterial activity in the body is produced by fosfomycin, which can effectively inhibit the action of pyruvate transferase necessary for bacterial cell wall synthesis. It has a killing effect on most Gram-positive and Gram-negative bacteria. It is mainly used to treat respiratory tract infections and lower urinary tract infections, such as cystitis, urethritis and intestinal infections, as well as skin and soft tissue infections caused by pathogenic bacteria that are sensitive to this product.

[0003] In the production of fosfomycin tromethamine raw materials or preparations (such as fosfomycin tromethamine powder, etc.), the residue of fosfomycin tromethamine will pollute other co-products, so the surface residue limit of the stainless steel wiping method of this product needs to be controlled to 0.019mg / 20ml, etc. The quality standards of fosfomycin tromethamine in the pharmacopoeias of various countries are found. Both the Chinese Pharmacopoeia and the European Pharmacopoeia use high performance liquid chromatography (differential detector) to control its content, but the differential detector has low sensitivity and cannot accurately quantify the content of trace or trace amounts of fosfomycin tromethamine in the production equipment. Therefore, the development of a method for detecting the trace or trace amount of fosfomycin tromethamine residues in fosfomycin tromethamine raw materials or preparations production equipment is a new issue that needs to be solved urgently. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for detecting the micro or trace amount of fosfomycin tromethamine residue in fosfomycin tromethamine raw materials or preparation product production equipment by ion chromatography. The method adopts ion chromatography for detection. The method has the advantages of high sensitivity, good reproducibility, simple operation, mild detection conditions, wide application of detection equipment and instruments, wide sources of eluent, simple preparation, etc.

[0005] The object of the present invention is achieved in this way: a method for detecting a trace or trace amount of fosfomycin tromethamine residue, characterized in that the method adopts an ion chromatograph and comprises the following steps:

[0006] (1) Chromatographic conditions:

[0007] Chromatographic columns: low capacity hydroxide system cathodic protection column and high capacity anion exchange separation column

[0008] Suppressor: Adopt anion electrolysis regeneration suppressor, current 36mA~44mA

[0009] Detector: Conductivity detector, cell temperature 31℃~39℃

[0010] Column temperature: 27℃~33℃

[0011] Temperature box: 15℃~40℃

[0012] Flow rate: 0.5ml / min~1.5ml / min

[0013] Injection volume: 20μl~150μl

[0014] Eluent: 15mmol / L~17mmol / L sodium hydroxide or potassium hydroxide solution

[0015] (2) Preparation of reference solution: prepare a reference solution of fosfomycin tromethamine reference substance with ultrapure water;

[0016] (3) Preparation of test solution: Place the test sample in ultrapure water, dissolve it by ultrasonication, and filter it to obtain the test solution;

[0017] (4) Determination method: Accurately measure the reference solution and the test solution, inject them into the ion chromatograph respectively, record the chromatogram, and calculate the residual amount of fosfomycin tromethamine by the peak area according to the external standard method.

[0018] The low-capacity hydroxide system cathodic protection column is selected from Dionex IonPac TM AG11-HC, 4×50 mm; the high capacity anion exchange separation column is selected from Dionex IonPac TM AS11-HC, 4×250mm; the anion electrolysis regeneration suppressor is selected from Dionex ASRS 300, 4mm; the current is 38-41mA; the cell temperature is 33℃~36℃; the column temperature is 28℃~31℃; the temperature of the incubator is 16℃~20℃; the flow rate is 0.8ml / min~1.2ml / min; the sodium hydroxide or potassium hydroxide is selected from chromatographic pure sodium hydroxide or potassium hydroxide; the injection volume is 90μl~115μl, preferably, the injection volume is 90μl~100μl; the eluent is selected from 15mmol / L~17mmol / L potassium hydroxide solution.

[0019] In the step (2), the specific method of preparing the reference substance solution with ultrapure water by taking the fosfomycin tromethamine reference substance is as follows: taking an appropriate amount of the fosfomycin tromethamine reference substance, diluting it with ultrapure water to prepare a solution containing 0.05 μg to 2 μg of fosfomycin tromethamine in 1 ml, preferably, diluting it with ultrapure water to prepare a solution containing 0.65 μg to 1.15 μg of fosfomycin tromethamine in 1 ml.

[0020] In the step (3), the specific method of placing the test sample in ultrapure water, dissolving it by ultrasound, and filtering it to obtain the test sample solution is to place the test sample in 20 ml of ultrapure water, dissolving it by ultrasound, and filtering it to obtain the test sample solution.

[0021] The quantitative limit of the residual amount of fosfomycin tromethamine is 0.0475 μg / ml.

[0022] The calculation formula for calculating the residual amount of fosfomycin tromethamine by peak area according to the external standard method is:

[0023]

[0024] Where:

[0025] A 对 : Main peak area of ​​reference solution;

[0026] A 供 : Main peak area of ​​test solution;

[0027] W 对 : Weight of reference substance, mg;

[0028] P: Purity of reference substance, %.

[0029] The test sample is selected from a wipe; the wipe is selected from a wiping cotton swab; the wipe is obtained by wiping the surface of an object; the surface of the object is selected from one or more of a stainless steel object surface and a polyfluoroethylene object surface; the surface of the object exists on production equipment; the product produced by the production equipment is a fosfomycin tromethamine raw material or preparation; the preparation is selected from one or more of fosfomycin tromethamine powder and fosfomycin tromethamine granules.

[0030] The gist of the present invention is to provide a method for detecting trace or trace amounts of fosfomycin tromethamine residues in fosfomycin tromethamine raw materials or preparation product production equipment by using ion chromatography. The pharmaceutical principle is: through the overall synergistic effect of various technical features in the technical solution of the present application, the technical effect of detecting trace or trace amounts of fosfomycin tromethamine residues in fosfomycin tromethamine raw materials or preparation product production equipment is achieved, and the quantitative limit of fosfomycin tromethamine is as low as 0.0475 μg / ml.

[0031] Compared with the prior art, the method for detecting trace or micro amounts of fosfomycin tromethamine residues in fosfomycin tromethamine raw materials or preparation product production equipment by using ion chromatography has the advantages of high sensitivity (the quantitative limit of fosfomycin tromethamine is as low as 0.0475 μg / ml), good reproducibility, simple operation, mild detection conditions, wide application of detection equipment and instruments, wide sources of eluent, simple preparation, etc., and will be widely used in the field of fosfomycin tromethamine drug analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0033] Figure 1 This is the ultrapure water color spectrum of Example 1 of the present invention.

[0034] Figure 2 This is the chromatogram of the reference solution of Example 1 of the present invention.

[0035] Figure 3 It is the chromatogram of the test solution of Example 1 of the present invention.

[0036] Figure 4 This is a diagram of the wiping sampling method of an embodiment of the present invention DETAILED DESCRIPTION

[0037] The following examples will help to understand the present invention, but these examples are only for illustrating the present invention, and the present invention is not limited to these contents.

[0038] Embodiment 1

[0039] 1. Chromatographic conditions

[0040] Column: Dionex IonPac TM AG11-HC (4×50 mm) guard column and Dionex IonPac TM AS11-HC (4×250 mm) anion exchange chromatography column;

[0041] Column temperature: 30°C;

[0042] Suppressor: Dionex ASRS 300 (4mm) anion electrolytic regeneration suppressor, current 40mA;

[0043] Detector: conductivity detector, cell temperature 35°C;

[0044] Incubator: 18°C;

[0045] Eluent: 15.8mmol / L potassium hydroxide (chromatographic grade) aqueous solution;

[0046] Flow rate: 1.0ml / min.

[0047] 2. Reference solution: Take an appropriate amount of fosfomycin tromethamine reference substance and dilute it with ultrapure water to make a solution containing 0.95 μg of fosfomycin tromethamine in 1 ml.

[0048] 3. For the test solution, place the cotton swab in 20 ml of ultrapure water, dissolve it by ultrasonication, and filter it to prepare the test solution.

[0049] 4. Determination Method

[0050] Accurately measure 100 μl of the reference solution and the test solution, inject them into the ion chromatograph, record the chromatogram, and calculate the residual amount of fosfomycin tromethamine by the peak area according to the external standard method.

[0051] 5. Calculation formula

[0052]

[0053] Where:

[0054] A 对 : Main peak area of ​​reference solution;

[0055] A 供 : Main peak area of ​​test solution;

[0056] W 对 : Weight of reference substance, mg;

[0057] P: Purity of reference substance, %.

[0058] 6. Calculation results

[0059] The peak area of ​​5 consecutive injections of the reference solution is:

[0060] A1=0.0690A2=0.0704A3=0.0694A4=0.0700A5=0.0682

[0061] A 对 Average = 0.0694RSD = 1.3%

[0062] The peak area of ​​the sample at the outlet of the screening machine is: A 供1 =0.0363

[0063] The peak area of ​​the sample on the sieve of the screening machine is: A 供2 =0.0075

[0064] The peak area of ​​the test sample on the inner wall of the mixing tank is: A 供3 =0.0172

[0065] The peak area of ​​the sample at the outlet of the mixing tank is: A 供4 =0.0562

[0067] Residual amount at the screening machine outlet (mg / 20ml) = ×20=0.0099mg / 20ml

[0070] Residue on sieve of sieving machine (mg / 20ml) = ×20=0.00205mg / 20ml

[0073] Residue on the inner wall of the mixing tank (mg / 20ml) = ×20=0.0047mg / 20ml

[0076] Residual amount at the mixing tank outlet (mg / 20ml) = ×20=0.0154mg / 20ml

[0078] Embodiment 2

[0079] This study systematically investigated the specificity, linearity, limit of quantification and detection limit, repeatability, accuracy, stability and wipe recovery of this method.

[0080] 1. Exclusivity

[0081] Solvent: ultrapure water;

[0082] Mixed blank excipient stock solution: weigh the prescribed amount of sucrose 30 mg, stevioside 1.9 mg, saccharin sodium 3.3 mg and orange flavor (tangerine flavor) liquid flavor 10 mg, add ultrapure water to dissolve and dilute to 200 ml, filter, and prepare a mixed blank excipient stock solution.

[0083] Reference solution: accurately weigh 10 mg of fosfomycin tromethamine reference substance, place in a 100 ml volumetric flask, dissolve and dilute to scale with ultrapure water, and shake well;

[0084] Preparation of 2-fold blank excipient solution: Accurately pipette 2.0 ml of the mixed blank excipient stock solution into a 20 ml volumetric flask, dilute to the mark with ultrapure water, shake well, then accurately measure 1.0 ml into a 100 ml volumetric flask, dilute to the mark with ultrapure water, shake well, and use this as the 2-fold blank excipient solution.

[0085] Take the reference solution, solvent, 2 times blank auxiliary material solution, 20ml blank absorbent cotton water ultrasonic solution, and 20ml blank absorbent cotton water ultrasonic solution after wiping stainless steel, respectively, filter, accurately measure 100μl of each, inject into ion chromatograph, record the chromatogram, and the results show that the solvent, blank auxiliary material solution, 20ml blank absorbent cotton water ultrasonic solution, and 20ml blank absorbent cotton water ultrasonic solution after wiping stainless steel do not interfere with the determination of fosfomycin tromethamine.

[0086] 2. Linear

[0087] Accurately weigh 19.0 mg of fosfomycin tromethamine reference substance, place it in a 50 ml volumetric flask, dissolve and dilute to the scale with ultrapure water, and shake well; accurately measure 2.5 ml, place it in a 100 ml volumetric flask, dilute to the scale with ultrapure water, and shake well as the stock solution. Prepare the test solution according to Table 1 below.

[0088] Table 1

[0089] serial number Remove volume from stock solution Final volume after dilution with water Final sample concentration 1 —— —— Limit of Quantitation 2 5.0ml 100ml 0.475μg / ml 3 10.0ml 100ml 0.944μg / ml 4 15.0ml 100ml 1.416μg / ml 5 20.0ml 100ml 1.888μg / ml

[0090] Precisely prepare, accurately measure 100 μl of each of the above solutions 1 to 5 and inject them into the ion chromatograph, record the chromatogram, inject each sample once, draw the curve equation with concentration as the horizontal axis and peak area as the vertical axis, the measurement results are shown in Table 2:

[0091] Table 2 Linear determination results of fosfomycin tromethamine

[0092]

[0093] The results showed that in the range of 0.0475 μg / ml to 1.906 μg / ml, the concentration of fosfomycin tromethamine in the test solution and the peak area of ​​the test result showed a good linear relationship.

[0094] 3. Limit of detection and limit of quantification

[0095] Accurately weigh 19.0 mg of fosfomycin tromethamine reference substance, place it in a 50 ml volumetric flask, dissolve it with ultrapure water and dilute it to the mark, shake well; accurately measure 5.0 ml, place it in a 20 ml volumetric flask, dilute it to the mark with ultrapure water, shake well; accurately measure 1.0 ml, place it in a 100 ml volumetric flask, dilute it to the mark with ultrapure water, shake well, as a stock solution. Then dilute it with ultrapure water to make a series of dilute solutions of different concentrations (in the low concentration area). Take 100 μl of each solution and inject it into the ion chromatograph, record the chromatogram, calculate the corresponding concentration when the signal-to-noise ratio S / N≈10 as the value of the quantitative limit; calculate the corresponding concentration when the signal-to-noise ratio S / N≈3 as the value of the detection limit. The measurement results are shown in Table 3:

[0096] Table 3 Results of the quantitative limit and detection limit of fosfomycin tromethamine

[0097] name Limit of quantitation (ug / ml) Detection limit (ug / ml) Fosfomycin tromethamine 0.0475 0.0142

[0098] 4. Repeatability test

[0099] Reference solution: Accurately weigh 19.0 mg of fosfomycin tromethamine reference substance, place in a 50 ml volumetric flask, dissolve and dilute to the mark with ultrapure water, and shake well; accurately measure 5 ml, place in a 20 ml volumetric flask, dilute to the mark with ultrapure water, and shake well; accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0100] Test solution: accurately weigh six portions of 19.0 mg of fosfomycin tromethamine, place them in 50 ml volumetric flasks, dissolve them in ultrapure water and dilute to the mark, and shake well; accurately measure 5 ml each, place them in 6 20 ml volumetric flasks, add 1.0 ml of the mixed blank excipient stock solution to each, dilute to the mark with ultrapure water, and shake well; accurately measure 1 ml again, place it in a 100 ml volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0101] The sample was injected and analyzed according to the chromatographic conditions in Example 1, and the RSD value of fosfomycin tromethamine was calculated based on the determined content results (see Table 4). The results showed that the method had good repeatability.

[0102] Table 4 Fosfomycin tromethamine repeatability determination results

[0103]

[0104] 5. Accuracy test

[0105] Accurately weigh 19.0 mg of fosfomycin tromethamine reference substance, place it in a 50 ml volumetric flask, dissolve and dilute to the scale with ultrapure water, and shake well; accurately measure 5 ml, place it in a 20 ml volumetric flask, dilute to the scale with ultrapure water, and shake well; accurately measure 1 ml again, place it in a 100 ml volumetric flask, dilute to the scale with ultrapure water, and shake well as the reference substance solution.

[0106] Accurately weigh three portions of 15.2 mg, 19.0 mg, and 22.8 mg of fosfomycin tromethamine, respectively, and place them in 50 ml volumetric flasks, add ultrapure water to dissolve and dilute to the scale, and shake well as the stock solution; accurately measure 5 ml of the stock solution, place them in 20 ml volumetric flasks, add 1.0 ml of the mixed blank excipient stock solution, dilute to the scale with ultrapure water, and shake well; accurately measure 1 ml again, place it in a 100 ml volumetric flask, dilute to the scale with ultrapure water, and shake well as the test solution.

[0107] The sample was injected and analyzed according to the chromatographic conditions in Example 1, and the recovery was calculated according to the formula "recovery rate = recovery amount / added amount × 100%" (see Table 5). The results showed that the average recovery rate of fosfomycin tromethamine was 100.2% and the RSD value was 1.2%, indicating that the recovery rate of the sample was good.

[0108] Table 5 Fosfomycin tromethamine accuracy determination results

[0109]

[0110] 6. Stability test

[0111] Accurately weigh 19.0 mg of fosfomycin tromethamine reference substance, place it in a 50 ml volumetric flask, dissolve and dilute to the scale with ultrapure water, and shake well; accurately measure 5 ml, place it in a 20 ml volumetric flask, dilute to the scale with ultrapure water, and shake well; accurately measure 1 ml again, place it in a 100 ml volumetric flask, dilute to the scale with ultrapure water, and shake well as the reference substance solution.

[0112] Accurately weigh 19.0 mg of fosfomycin tromethamine, place in 50 ml volumetric flasks, dissolve and dilute to the scale with ultrapure water, and shake well; accurately measure 5 ml, place in 20 ml volumetric flasks, add 1.0 ml of mixed blank excipient stock solution, dilute to the scale with ultrapure water, and shake well; accurately measure 1 ml again, place in a 100 ml volumetric flask, dilute to the scale with ultrapure water, and shake well as the test solution.

[0113] According to the chromatographic conditions in Example 1, the samples were injected at 0h, 2h, 4h, 6h, 8h, 12h, 18h, and 24h, and the corresponding contents were calculated (see Table 6). The RSD of the fosfomycin tromethamine content was 2.1%, and the maximum absolute deviation from the initial value was 4.9%, indicating that the sample had good stability within 24 hours.

[0114] Table 6 Fosfomycin tromethamine stability test results

[0115] Component name 0h 2h 4h 6h 8h 12h 18h 24h content(%) 97.5 100.5 102.0 102.4 102.0 102.4 101.0 97.4

[0116] 7. Stainless steel wiping recovery rate

[0117] Weigh 15.2mg, 19.0mg, and 22.8mg of fosfomycin tromethamine in three portions, place in a 50ml volumetric flask, dissolve and dilute to scale with water, shake well, and use as stock solution. Accurately measure 5.0ml of the stock solution, place in a 20ml volumetric flask, add 1.0ml of the mixed blank excipient solution, dilute to scale with water, and shake well; then accurately measure 2.0ml and place in a 10ml volumetric flask, dilute to scale with water, shake well, and use as the stock solution; draw 1.0ml of the stock solution and evenly inject it onto the prepared stainless steel, air dry, and press the attached Figure 4 Wipe sampling, cut off the cotton swab after sampling, dissolve in 20ml water respectively, dissolve by ultrasound, put to room temperature (20℃), filter, and use as the tested solution. Accurately weigh 19.0mg of fosfomycin tromethamine reference substance, place in a 50ml volumetric bottle, dissolve with water and dilute to scale, shake well; accurately measure 5.0ml, place in a 20ml volumetric bottle, dilute with water to scale, shake well; accurately measure 1.0ml, place in a 100ml volumetric bottle, dilute with water to scale, shake well, and use as the reference solution. Accurately measure 100μl of the reference solution and the test solution respectively, inject into the ion chromatograph, and record the chromatogram; calculate the recovery rate, the results are shown in Table 7, the recovery rate is 84.3%, and the relative standard deviation of the recovery rate is 1.6%.

[0118] Table 7 Fosfomycin tromethamine stainless steel wiping recovery determination results

[0119]

Claims

1. A method for detecting trace or trace amounts of fosfomycin tromethamine residues, characterized in that: The method uses an ion chromatograph and comprises the following steps: (1) Chromatographic conditions: Chromatographic columns: low capacity hydroxide system cathodic protection column and high capacity anion exchange separation column Suppressor: Adopt anion electrolysis regeneration suppressor, current 36mA~44mA Detector: Conductivity detector, cell temperature 31℃~39℃ Column temperature: 27℃~33℃ Temperature box: 15℃~40℃ Flow rate: 0.5ml / min~1.5ml / min Injection volume: 20μl~150μl Eluent: 15mmol / L~17mmol / L sodium hydroxide or potassium hydroxide solution (2) Preparation of reference solution: prepare a reference solution of fosfomycin tromethamine reference substance with ultrapure water; (3) Preparation of test solution: Place the test sample in ultrapure water, dissolve it by ultrasonication, and filter it to obtain the test solution; (4) Determination method: Accurately measure the reference solution and the test solution, inject them into the ion chromatograph respectively, record the chromatogram, and calculate the residual amount of fosfomycin tromethamine by the peak area according to the external standard method.

2. A method for detecting trace or trace amounts of fosfomycin tromethamine residues according to claim 1, characterized in that: The low-capacity hydroxide system cathodic protection column is selected from Dionex IonPac TM AG11-HC, 4×50 mm; the high capacity anion exchange separation column is selected from Dionex IonPac TM AS11-HC, 4×250mm; the anion electrolysis regeneration suppressor is selected from Dionex ASRS 300, 4mm; the current is 38-41mA; the cell temperature is 33℃~36℃; the column temperature is 28℃~31℃; the temperature of the incubator is 16℃~20℃; the flow rate is 0.8ml / min~1.2ml / min; the sodium hydroxide or potassium hydroxide is selected from chromatographic pure sodium hydroxide or potassium hydroxide; the injection volume is 90μl~115μl, preferably, the injection volume is 90μl~100μl; the eluent is selected from 15mmol / L~17mmol / L potassium hydroxide solution.

3. A method for detecting trace or trace amounts of fosfomycin tromethamine residues according to claim 1, characterized in that: In the step (2), the specific method of preparing the reference solution of the fosfomycin tromethamine reference substance with ultrapure water is as follows: taking an appropriate amount of the fosfomycin tromethamine reference substance, diluting it with ultrapure water to prepare a solution containing 0.05 μg to 2 μg of fosfomycin tromethamine in 1 ml, preferably, diluting it with ultrapure water to prepare a solution containing 0.65 μg to 1.15 μg of fosfomycin tromethamine in 1 ml.

4. A method for detecting trace or trace amounts of fosfomycin tromethamine residues according to claim 1, characterized in that: In the step (3), the specific method of placing the test sample in ultrapure water, dissolving it by ultrasound, and filtering it to obtain the test sample solution is to place the test sample in 20 ml of ultrapure water, dissolving it by ultrasound, and filtering it to obtain the test sample solution.

5. A method for detecting trace or trace amounts of fosfomycin tromethamine residues according to claim 1, characterized in that: The quantitative limit of the residual amount of fosfomycin tromethamine is 0.0475 μg / ml.

6. A method for detecting trace or trace amounts of fosfomycin tromethamine residues according to claim 1, characterized in that: The calculation formula for calculating the residual amount of fosfomycin tromethamine by peak area according to the external standard method is: Where: A 对 : Main peak area of ​​reference solution; A 供 : Main peak area of ​​test solution; W 对 : Weight of reference substance, mg; P: Purity of reference substance, %.

7. A method for detecting trace or trace amounts of fosfomycin tromethamine residues according to claim 1, characterized in that: The test sample is selected from a wipe; the wipe is selected from a wiping cotton swab; the wipe is obtained by wiping the surface of an object; the surface of the object is selected from one or more of a stainless steel object surface and a polyfluoroethylene object surface; the surface of the object exists on production equipment; the product produced by the production equipment is a fosfomycin tromethamine raw material or preparation; the preparation is selected from one or more of fosfomycin tromethamine powder and fosfomycin tromethamine granules.

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