A method for detecting ultra-trace residual ethylene oxide

Abstract

The present application provides a method for detecting ultra-trace residual ethylene oxide, which belongs to the field of drug analysis technology. After dissolving ciprofloxacin / ciprofloxacin hydrochloride in water, add a derivatization reagent containing pentafluorobenzenethiol, heat and shake in a water bath to complete the reaction, cool to room temperature, add cyclohexane to extract the derivatization reaction product, take the upper cyclohexane solution as the test liquid, inject it into the gas chromatograph in a liquid injection mode, use ECD as the detector, and calculate the content of ethylene oxide in the test sample according to the peak area of ​​the reaction product by the external standard method. This method has good performance in specificity, stability, accuracy, precision and durability, with high detection sensitivity and the detection amount can reach ultra-trace level.

Description

Technical Field

[0001] The present application relates to a method for detecting ultra-trace residual ethylene oxide, and belongs to the technical field of drug analysis. Background Art

[0002] Ethylene oxide (ECH) is a simple heterocyclic compound, which is classified as a Class I human carcinogen by the International Agency for Research on Cancer (IARC); the European Chemicals Agency (ECHA) classifies the carcinogenicity, mutagenicity and reproductive toxicity of ethylene oxide as Class 1B, and the acute toxicity as Class 3; the U.S. National Institutes of Health (NIH) classifies ethylene oxide as a "known human carcinogen."

[0003] According to the production process of ciprofloxacin, this product and its hydrochloride may have the risk of residual ethylene oxide impurities. Summary of the invention

[0004] In view of this, the present application provides a method for detecting ultra-trace residual ethylene oxide, which has good performance in specificity, stability, accuracy, precision and durability. The method completes the detection by liquid sampling, has high detection sensitivity, and the detection amount can reach the ultra-trace level (1ppb).

[0005] Specifically, the present application is implemented through the following scheme:

[0006] A method for detecting ultra-trace residual ethylene oxide comprises the following steps: dissolving ciprofloxacin / ciprofloxacin hydrochloride in water, adding a derivatization reagent containing pentafluorobenzenethiol, heating and shaking in a water bath to complete the reaction, cooling to room temperature, adding cyclohexane, shaking and standing to separate layers, taking the upper clear liquid, injecting it into a gas chromatograph by liquid injection mode, and determining the content of ethylene oxide.

[0007] The above method establishes a derivatization-gas chromatography determination system to determine the ethylene oxide in ciprofloxacin and ciprofloxacin hydrochloride raw materials. During the test, ethylene oxide undergoes a ring-opening addition reaction with pentafluorobenzenethiol (see Formula 1), and the content of ethylene oxide is calculated by measuring the response value of the derivatization product. The halogenated hydrocarbons are determined using an ECD detector, which greatly improves the detection sensitivity and realizes the determination of ultra-trace ethylene oxide residues in ciprofloxacin and ciprofloxacin hydrochloride.

[0008]

[0009] Further, as a preference:

[0010] The preparation method of the derivatization reagent is as follows: dissolving sodium carbonate in water, adding pentafluorothiophenol and ultrasonically shaking, and then fixing the pentafluorothiophenol to a concentration of 5 μl / ml, wherein the role of sodium carbonate is to increase the solubility of pentafluorothiophenol, and the concentration is 0.2 g / ml.

[0011] The water bath temperature is 40-45°C and the shaking is performed for 0.5-1.5h.

[0012] The chromatographic conditions in the gas chromatograph are: using a capillary column with polyethylene glycol as the stationary liquid, nitrogen as the carrier gas, flow rate: 1.3-1.7 ml / min; injection port temperature 210-230°C, detector (ECD) temperature 300°C; split injection, split ratio 5: 1. More preferably, the starting temperature is 100°C, maintained for 2 minutes, and the temperature is increased to 200°C at a rate of 10°C per minute and maintained for 5 minutes.

[0013] When conducting the test, a reference solution needs to be prepared. The reference solution is a solution formed by dissolving ethylene oxide in water, and the concentration of the solution is 20ng / ml.

[0014] The solution concentration X and the ethylene oxide content Y satisfy the linear equation: Y = 346.4286X - 112.2395

[0015] The above method realizes the determination of volatile small molecule ethylene oxide in raw materials, and realizes the high-sensitivity residual amount determination of target substances through the combination of derivatization-gas chromatography-electron capture detector. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the linear relationship diagram obtained in this application;

[0017] Figure 2 This is a typical chromatogram of the reference solution;

[0018] Figure 3 This is a typical chromatogram of a blank test;

[0019] Figure 4 This is a typical chromatogram of the detection limit solution;

[0020] Figure 5 This is a typical chromatogram of the quantitation limit solution;

[0021] Figure 6 This is a typical chromatogram of a 100% concentration level spiked solution in ciprofloxacin hydrochloride;

[0022] Figure 7 This is a typical chromatogram of a 100% concentration level spiked solution in ciprofloxacin;

[0023] Figure 8 This is a typical chromatogram of the reference solution at a durable flow rate of 1.3 ml / min;

[0024] Fig. 9 This is a typical chromatogram of the reference solution at a durable flow rate of 1.7 ml / min;

[0025] Fig.10 This is a typical chromatogram of the reference solution when the injection port temperature is 210°C for durability;

[0026] Fig.11 This is a typical chromatogram of the reference solution when the injection port temperature is 230°C for durability;

[0027] Fig.12 This is a typical chromatogram of a ciprofloxacin test solution;

[0028] Fig.13 This is a typical chromatogram of ciprofloxacin hydrochloride test solution. DETAILED DESCRIPTION

[0029] This example refers to the 2020 edition of the Chinese Pharmacopoeia, Part IV, and the quality section of the International Technical Requirements for Drug Registration of ICH, and other relevant materials to determine the impurity ethylene oxide in ciprofloxacin and ciprofloxacin hydrochloride provided by Shangyu Jingxin Pharmaceutical Co., Ltd., and conduct a comprehensive methodological study on the determination method. The method specificity, linearity and range, detection limit and quantification limit, accuracy, precision, solution stability and durability were verified, and the test results met the relevant requirements for verification.

[0030] 1. Raw materials and instruments

[0031] In this case, the reference material information is shown in Table 1, the test material information is shown in Table 2, the instruments and equipment used are shown in Table 3, and the reagent materials are shown in Table 4.

[0032] Table 1: Reference Material Information Table

[0033] Name / Code batch number content source Ethylene oxide B2221014 99.5％ Aladdin

[0034] Table 2: Sample information table

[0035]

[0036] Table 3: Instrument and equipment information

[0037]

[0038] Table 4: Reagent material information table

[0039]

[0040] 2. Experimental part

[0041] The specificity, solution stability, linear correlation, quantitative limit and detection limit, accuracy at different limit concentrations, precision, durability and other items of the impurity ethylene oxide were tested. The specific process is as follows.

[0042] 1. Specificity experiment

[0043] 1) Detection object:

[0044] In the detection process, ciprofloxacin was used as the test solution with a concentration of 20 mg / ml; the detected impurity was ethylene oxide with a proposed limit of 1.0 ppm and a concentration of 20 ng / ml at the 100% limit.

[0045] 2) Preparation of solution:

[0046] It mainly includes derivatization reagent, reference solution, test solution and blank solution. The preparation method of each solution is as follows:

[0047] (1) Derivatization reagents

[0048] Take 20g of sodium carbonate, put it into a 100ml volumetric flask, add purified water, and dissolve it by ultrasonication. Add 500μl of pentafluorobenzenethiol, and disperse it by ultrasonication. Add water to dilute to the scale, mix well, and prepare it before use (if it is turbid, shake it well before use).

[0049] (2) Reference substance stock solution and reference substance solution

[0050] Take an appropriate amount of ethylene oxide reference substance, dissolve it in water and dilute it to make a solution containing about 200 ng per 1 ml as the reference substance stock solution;

[0051] Take 10.0ml of the impurity reference stock solution, place it in a 100ml volumetric flask, dilute it to the mark with water, shake it well, accurately measure 1ml, place it in a 20ml headspace bottle, accurately add 1ml of the derivatization reagent, seal it, heat and shake it in a 40℃ water bath for 1 hour, cool it to room temperature, accurately add 2ml of cyclohexane, shake it repeatedly upside down for 30 seconds, let it stand to separate, take the upper clear liquid to obtain the impurity ethylene oxide reference solution, and prepare it before use.

[0052] (3) Test solution

[0053] Take about 20 mg of this product, place it in a 20 ml headspace bottle, accurately add 1 ml of purified water, shake to dissolve, accurately add 1 ml of derivatization reagent, seal, heat in a 40°C water bath and shake for 1 hour, cool to room temperature, accurately add 2 ml of cyclohexane, shake repeatedly inverted for 30 seconds, stand to separate, take the upper clear liquid and prepare it before use.

[0054] (4) Blank solution

[0055] Accurately measure 1 ml of purified water and place it in a 20 ml headspace bottle. Accurately add 1 ml of derivatization reagent, seal it, heat and shake it in a 40°C water bath for 1 hour, cool it to room temperature, accurately add 2 ml of cyclohexane, invert and shake it repeatedly for 30 seconds, let it stand to separate into layers, take the upper clear liquid and prepare it before use.

[0056] 3) Chromatographic conditions:

[0057] A capillary column with polyethylene glycol as the stationary phase (HP-INNOWAX, 30m*0.32mm, 0.5μm, or with similar polarity) was used; nitrogen was used as the carrier gas, with a flow rate of 1.5ml / min; the initial temperature was 100°C, maintained for 2 minutes, then increased to 200°C at a rate of 10°C per minute, and maintained for 5 minutes; the injection port temperature was 220°C, and the detector (ECD) temperature was 300°C; split injection, split ratio 5:1; direct injection, injection volume 2μl.

[0058] 4) Processing of measurement results:

[0059] During the determination, take blank solution, reference solution and test solution for analysis respectively, record the chromatogram, and calculate the content of ethylene oxide impurity by peak area according to the external standard method. The ethylene oxide impurity shall not exceed 1.0 ppm.

[0060]

[0061] A 样 is the peak area of ​​the impurity in the chromatogram of the test solution;

[0062] m 对 is the sample weight of the reference substance, mg;

[0063] C 对 is the content of the reference substance, %;

[0064] S 样 is the dilution factor of the test solution;

[0065] A 对 is the impurity peak area in the chromatogram of the reference solution;

[0066] m 样 is the sample weight, mg;

[0067] S 对 is the dilution factor of the reference solution.

[0068] After the system is stable, take the reference solution for injection. It is required that the RSD of the main peak area in at least 6 samples of the reference solution should not be greater than 10%, and the separation degree between the target peak and other adjacent peaks should be greater than 1.5. The test results are shown in Table 5.

[0069] Table 5: System specificity test results of reference solution

[0070]

[0071] As can be seen from Table 5: the reference solution was injected 6 times in a row, the RSD of the peak area of ​​ethylene oxide derivatives was less than 10.0%, the separation degree of the ethylene oxide derivative peak and other adjacent peaks was 1.86, and the blank solution had no background interference at the retention time of the ethylene oxide derivative peak. The experimental results all met the verification requirements.

[0072] 2. Solution stability test

[0073] The reference stock solution (preparation method in the "Specificity Experiment" section) was taken and sampled and analyzed after derivatization reaction at 0, 24, and 48 hours, and the chromatogram was recorded to examine the stability of the solution. Among them, the stability is measured by the relative change in the peak area at each time point when the reference stock solution increases over time. The solution stability results are shown in Table 6.

[0074] The relative change in peak area was calculated using the following formula:

[0075]

[0076] Wherein, RD% is the absolute value of the relative change (%) with respect to 0HR;

[0077] X n is the peak area (A) at the time of injection at the nth hour;

[0078] X 0 is the peak area (A) at 0 hour injection.

[0079] Table 6: Solution stability test results

[0080]

[0081] As can be seen from Table 6: the reference solution was placed at room temperature for 0, 24, and 48 hours before derivatization. The RD% of the peak area of ​​the solution at 24 hours was 2.4, and the RD% of the peak area of ​​the solution at 48 hours was 2.6. The relative changes in the peak area of ​​the target substance in the reference solution at each time point compared with that at 0 hours were all less than 10.0%.

[0082] 3. Linearity and range

[0083] The linear correlation solutions include six groups of reference stock solutions and linear solutions. The preparation methods of each solution are as follows:

[0084] (1) Reference substance stock solution: see the preparation method in the “Specificity Experiment” section.

[0085] (2) Linear solution L1: Take 1.0 ml of the reference stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with purified water and shake well.

[0086] (3) Linear solution L2: Take 5.0 ml of the reference stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with purified water and shake well.

[0087] (4) Linear solution L3: Take 7.5 ml of the reference stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with purified water and shake well.

[0088] (5) Linear solution L4: Take 10.0 ml of the reference stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with purified water and shake well.

[0089] (6) Linear solution L5: Take 12.5 ml of the reference stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with purified water and shake well.

[0090] (7) Linear solution L6: Take 3.0 ml of the reference stock solution and place it in a 20 ml volumetric flask. Dilute to the mark with purified water and shake well.

[0091] Accurately measure 1 ml of each of the above linear correlation solutions, place in a 20 ml headspace bottle, accurately add 1 ml of the derivatization reagent, seal, heat and shake in a 40 ° C water bath for 1 hour, cool to room temperature, accurately add 2 ml of cyclohexane, seal, repeatedly shake for 30 seconds, stand for stratification, take the upper clear liquid, prepare before use, inject and analyze, and record the chromatogram. The peak area, linear equation, slope, intercept, and correlation coefficient of each concentration solution and each impurity are statistically analyzed. The results are shown in Table 7.

[0092] Table 7: Linearity test results

[0093]

[0094] It can be concluded from Table 7 that the solution concentration (X) and the peak area (Y) satisfy the linear equation Y=346.4286X-112.2395, and the linear correlation coefficient r>0.995; the intercept is 1.7% of the limit standard response value, which does not exceed 15.0% of the limit standard response value.

[0095] 4. Limit of Quantitation and Limit of Detection Experiments

[0096] In this experiment, the linear solution L1 in the linear correlation solution was taken as the quantitative limit solution, and 6 copies were prepared in parallel; 1.0 ml of the reference solution was placed in a 25 ml volumetric flask, diluted to the scale with purified water, and shaken to obtain the detection limit solution, which was prepared in parallel for 6 copies.

[0097] Accurately measure 1 ml of the quantitative limit solution and the detection limit solution, place them in a 20 ml headspace bottle, accurately add 1 ml of the derivatization reagent, seal, heat and shake in a 40 ° C water bath for 1 hour, cool to room temperature, accurately add 2 ml of cyclohexane, seal, repeatedly shake for 30 seconds, let stand and separate, take the upper clear liquid, prepare it before use, inject and analyze, and record the chromatogram. Report the peak area, signal-to-noise ratio, concentration, equivalent to the test solution concentration, and peak area RSD of the ethylene oxide derivatives in the detection limit and quantitative limit solutions. The results are shown in Tables 8 and 9.

[0098] From Tables 8 and 9, we can see that the signal-to-noise ratio of each peak in the detection limit solution is ≥4.1; the signal-to-noise ratio of each peak in the quantification limit solution is ≥16.6, and the peak area RSD is 1.2% (n=6), indicating that the sensitivity of this method meets the detection requirements.

[0099] Table 8: Detection limit test results

[0100]

[0101] Table 9: Quantitation limit test results

[0102]

[0103] 5. Accuracy experiment

[0104] The relevant solutions in the accuracy experiment include the following groups:

[0105] (1) 50% limit concentration spiked test solution: Take about 20 mg of the product and place it in a 20 ml headspace bottle. Accurately add 1 ml of linear solution L2 in sequence. Shake to dissolve. Accurately add 1 ml of derivatization reagent. Seal. Heat and shake in a 40 °C water bath for 1 hour. Cool to room temperature. Accurately add 2 ml of cyclohexane. Invert and shake repeatedly for 30 seconds. Let stand to separate. Take the supernatant and prepare 3 times in parallel.

[0106] (2) 100% limit concentration spiked test solution: Take about 20 mg of the product and place it in a 20 ml headspace bottle. Accurately add 1 ml of linear solution L4 in sequence. Shake to dissolve. Accurately add 1 ml of derivatization reagent. Seal. Heat and shake in a 40 °C water bath for 1 hour. Cool to room temperature. Accurately add 2 ml of cyclohexane. Invert and shake repeatedly for 30 seconds. Let stand to separate. Take the supernatant and prepare it immediately before use. Prepare 3 copies in parallel.

[0107] (3) 150% limit concentration spiked test solution: Take about 20 mg of the product and place it in a 20 ml headspace bottle. Accurately add 1 ml of linear solution L6 in sequence. Shake to dissolve. Accurately add 1 ml of derivatization reagent. Seal the bottle. Heat and shake in a 40 °C water bath for 1 hour. Cool to room temperature. Accurately add 2 ml of cyclohexane. Invert and shake repeatedly for 30 seconds. Let stand to separate. Take the upper clear liquid and prepare 3 portions in parallel.

[0108] Inject and analyze the spiked test solution at each limit concentration and record the chromatogram. Calculate the content of each impurity by external standard method. The results are shown in Tables 10 and 11.

[0109]

[0110]

[0111]

[0112]

[0113] Sample impurity content (ng) = m 加样 ×C 样 (%)×1000000, where m 对 is the sample weight of the reference substance, mg;

[0114] m 样 is the sample weight of the test product, mg;

[0115] m 加样 is the weight of the spiked sample, mg;

[0116] C 对 is the content of the reference substance, %;

[0117] C 样 is the content of impurities in the test sample, %;

[0118] V 加入 is the volume of the corresponding linear solution added to the spiked test sample, ml;

[0119] A 加样 is the peak area of ​​the impurity in the chromatogram of the spiked test solution;

[0120] A 对 is the impurity peak area of ​​the reference solution;

[0121] S 线 is the dilution multiple of the corresponding linear solution;

[0122] S 对 is the dilution multiple of the reference solution;

[0123] S 样is the dilution factor of the test solution (the dilution factor for this determination is 1).

[0124] Table 10: Ciprofloxacin hydrochloride accuracy test results

[0125]

[0126] Table 11: Ciprofloxacin accuracy test results

[0127]

[0128] It can be seen from Tables 10 and 11 that for ciprofloxacin hydrochloride, the recoveries of the impurity ethylene oxide in the test solutions spiked with three different limit concentrations of 50%, 100% and 150% were between 79.5% and 88.6%, with an average recovery of 83.2% and an RSD of 3.6%; for ciprofloxacin, the recoveries of the impurity ethylene oxide in the test solutions spiked with three different limit concentrations of 50%, 100% and 150% were between 87.7% and 101.5%, with an average recovery of 92.8% and an RSD of 4.3%.

[0129] 6. Precision (repeatability) experiment

[0130] The experimental solution in this part adopts the method in the "Accuracy Experiment" to prepare six 100% limit concentration spiked test solution in parallel. Take the above spiked test solution for injection and record the chromatogram. The spiked recovery rate of impurity ethylene oxide is calculated according to the external standard method calculation formula given in the "Accuracy Experiment". The results are shown in Table 12.

[0131] Table 12: Precision test results of spiked test solution

[0132]

[0133]

[0134] The results in Table 12 show that: the average recovery rate of ethylene oxide in 6 repeatability tests of ciprofloxacin hydrochloride test solutions was 82.1%, and the RSD was 2.0%; the average recovery rate of ethylene oxide in 6 repeatability tests of ciprofloxacin test solutions was 91.9%, and the RSD was 5.5%, indicating that the solution prepared in this case meets the precision requirements.

[0135] 7. Durability test

[0136] Take blank solution, reference solution and test solution, change the chromatographic condition parameters according to Table 13, perform sample analysis, record the chromatogram, examine the suitability of the system, calculate the impurity ethylene oxide content of the test solution according to the impurity reference external standard method, and examine the durability of the analytical method.

[0137] Table 13: Chromatographic parameter adjustment range

[0138] parameter Specified value Changing value Carrier gas flow rate (ml / min) 1.5 1.3 and 1.7 Inlet temperature (℃) 220 210 and 230 .

[0139] Table 14: Ethylene oxide durability test results

[0140]

[0141] Note: ND means not detected.

[0142] Conclusion: By adjusting the chromatographic parameters, the RSD of the peak area of ​​the impurity ethylene oxide reference solution was no more than 10%, and the separation between the ethylene oxide derivative peak and other adjacent peaks was greater than 1.5; the difference in the impurity ethylene oxide content in the samples under various conditions was no more than 10% of the limit, indicating that the analytical method has good durability.

[0143] The above test results are summarized in Table 15.

[0144] Table 15: Summary of the validation of impurity ethylene oxide determination methodology

[0145]

[0146]

[0147] Based on the above experimental results, the reference solution and the test solution were sampled and analyzed according to the chromatographic conditions in the specificity experiment, and the impurity content was calculated according to the impurity reference external standard method. The test results of the six batches of samples are shown in Table 16.

[0148] Table 16: Sample test results

[0149]

[0150] The results in Table 16 once again confirm that the method in this case is used to detect volatile small molecule ethylene oxide in the drugs ciprofloxacin and ciprofloxacin hydrochloride. Ethylene oxide reacts with the reagent pentafluorobenzenethiol to form a new derivatization product. The ethylene oxide content is calculated by measuring the derivatization product. The target is detected by programmed temperature separation on an HP-INNOWAX chromatographic column, liquid injection is adopted, and an electron capture detector is used to detect the target. Compared with the conventional direct determination method without derivatization, it can detect ultra-trace amounts of ethylene oxide such as 0.08ng / ml, greatly improving the detection sensitivity. It can be applied to the determination of ethylene oxide residues in ciprofloxacin and ciprofloxacin hydrochloride.

Claims

1. A method for detecting ultra-trace residual ethylene oxide, Features: Ciprofloxacin or ciprofloxacin hydrochloride was used as the test sample. After the test sample was dissolved in water, a derivatization reagent containing pentafluorobenzenethiol was added. After heating and shaking in a water bath to complete the derivatization reaction, the derivatization reaction was cooled to room temperature. Cyclohexane was added to extract the derivatization reaction product. The demix was allowed to stand for stratification. The upper layer solution was taken as the test solution and injected into the gas chromatograph by liquid injection mode. The ECD was used as the detector. The preparation method of the derivatization reagent is as follows: dissolve sodium carbonate in water, add pentafluorothiophenol and ultrasonically shake it, and then adjust the volume of pentafluorothiophenol to a concentration of 5 μl / ml. The derivatization reaction satisfies the following reaction formula: ， The reaction product is the direct measurement target of the gas chromatograph. The content of ethylene oxide in the test sample is calculated according to the external standard method of the peak area of ​​the reaction product. The chromatographic conditions in the gas chromatograph are: a capillary column with polyethylene glycol as the stationary liquid, nitrogen as the carrier gas, flow rate: 1.3-1.7 ml / min; injection port temperature 210-230° C., detector temperature 200-400° C.; split injection, split ratio 1-50:1, when the gas chromatograph is tested, the starting temperature is 100° C., maintained for 2 minutes, and heated to 200° C. at a rate of 10° C. / min, maintained for 5 minutes, The concentration X of the solution obtained by dissolving the test sample in water and the ethylene oxide content Y satisfy: Y=346.4286X-112.2395, The detection amount of the above detection method reaches the ultra-trace level of 1ppb.

2. A method for detecting ultra-trace residual ethylene oxide according to claim 1, Features: The sodium carbonate concentration was 0.2 g / ml.

3. A method for detecting ultra-trace residual ethylene oxide according to claim 1, Features: The water bath temperature is 40-45°C and the shaking is performed for 0.5-1.5h.

4. A method for detecting ultra-trace residual ethylene oxide according to claim 1, Features: The water bath temperature was 40°C and the mixture was shaken for 1 h.

5. A method for detecting ultra-trace residual ethylene oxide according to claim 1, Features: Detector temperature 300°C; split injection, split ratio 5:1.

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