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CN-122017078-A - Method for detecting N-nitrosodiethylamine in epolamine diclofenac

CN122017078ACN 122017078 ACN122017078 ACN 122017078ACN-122017078-A

Abstract

The invention discloses a method for detecting N-nitrosodiethylamine in epothilone, which comprises the steps of dissolving an epothilone sample in methanol solution, adding aqueous solution of formic acid for mixing, precipitating and separating the epothilone in the sample, centrifugally separating, taking supernatant, and quantitatively detecting the N-nitrosodiethylamine in the supernatant by adopting a liquid chromatography-tandem mass spectrometry method. Based on the principle that diclofenac is converted into free acid to precipitate under the acidic condition, 2% formic acid is creatively selected as a precipitator, so that the matrix interference of the main component is effectively removed, the problem that the main component is separated out in a chromatographic system is solved, and a mass spectrometer instrument is protected. Proved by methodology, the method has the advantages of extremely high sensitivity, good accuracy, strong repeatability, quick analysis and good durability. The invention provides a reliable and efficient detection means for the quality control of trace N-nitrosodiethylamine in the epothilone drug substance, and has extremely high practical value.

Inventors

  • ZHANG SHUXIA
  • XING RONGHUA
  • ZHANG SHOUJIE
  • LI KE
  • ZHAO FANG
  • ZANG MINGWU
  • LEI CHUNNI
  • LIU SHENGNAN
  • Hua Xiangmei
  • XU DAN
  • CAI XIANGYU

Assignees

  • 郑州海关技术中心

Dates

Publication Date
20260512
Application Date
20260317

Claims (10)

  1. 1. The method for detecting N-nitrosodiethylamine in epolamine diclofenac comprises the following steps: S1, accurately weighing a diclofenac epolamine sample, adding a D 6 -N-nitrosodimethylamine internal standard solution, adding a solvent, shaking to dissolve, adding a volatile acid aqueous solution, shaking uniformly, standing, centrifuging and filtering to prepare a sample solution; S2, accurately weighing the diclofenac epolamine without N-nitrosodiethylamine, adding the D 6 -N-nitrosodimethylamine internal standard solution and the N-nitrosodiethylamine reference stock solution a, adding a solvent, shaking to dissolve, adding a volatile acid aqueous solution, shaking uniformly, standing, centrifuging and filtering to prepare a reference solution, wherein the concentration of the N-nitrosodiethylamine is 7 ng/mL, and the concentration of the D 6 -N-nitrosodimethylamine is 7 ng/mL; S3, precisely removing the sample solution and the reference substance solution, injecting the sample solution and the reference substance solution into a liquid chromatograph tandem mass spectrometer, and recording a chromatogram; S4, obtaining the content of the N-nitrosodiethylamine by calculating the peak area according to an internal standard method if chromatographic peaks consistent with the retention time of the N-nitrosodiethylamine exist in the chromatogram of the sample solution.
  2. 2. The method according to claim 1, wherein the liquid chromatography is carried out on a column Poroshell, a column EC-C18.7 μm, 3.0. 3.0 mm X100 mm, a mobile phase A containing 0.1% formic acid in water, and a mobile phase B containing 0.1% formic acid in methanol, the gradient of the mobile phase being: 0min mobile phase A95% mobile phase B5%; 3 min Mobile phase A95% and Mobile phase B5%; 7 min% mobile phase A5% mobile phase B95%; 9 min% mobile phase A and 95% mobile phase B; 9.1 min, mobile phase A95% and mobile phase B5%; 10min is mobile phase A95% and mobile phase B5%.
  3. 3. The detection method according to claim 2, wherein the liquid chromatography conditions further comprise a column temperature of 40 ℃, a flow rate of 0.50 mL/min, and a sample injection amount of 15. Mu.L.
  4. 4. The detection method according to claim 1, wherein the mass spectrum parameter is ion source APCI, positive ion mode, and the scanning mode is MRM multi-reaction monitoring, the quantitative ion pair of N-nitrosodiethylamine is m/z 103-75.1, and the qualitative ion pair is m/z 103-47.1.
  5. 5. The detection method according to claim 1, wherein the solvent is methanol and/or the aqueous volatile acid solution is an aqueous formic acid solution.
  6. 6. The detection method according to claim 1, wherein the internal standard solution is a methanol solution of 140 μg/L of D 6 -N-nitrosodimethylamine.
  7. 7. The detection method according to claim 1, wherein the preparation method of the N-nitrosodiethylamine reference stock solution a comprises the steps of transferring the N-nitrosodiethylamine standard solution into a volumetric flask, fixing the volume by methanol, and uniformly mixing to obtain the N-nitrosodiethylamine reference stock solution a with the concentration of 140 mug/L.
  8. 8. The detection method according to claim 1, wherein in step S1, the mixing ratio of the diclofenac epolamine sample and the formic acid aqueous solution is 1.8 mL of 2% formic acid aqueous solution per 0.2g sample, and/or in step S2, the mixing ratio of the diclofenac epolamine free of N-nitrosodiethylamine and the formic acid aqueous solution is 1.8 mL of 2% formic acid aqueous solution per 0.2g sample.
  9. 9. The detection method according to claim 1, wherein the quantitative limit of the method is 0.7 ng/mL, the detection limit is 0.35 ng/mL, the linear range is 0.7-10.5 ng/mL, and the correlation coefficient R is more than or equal to 0.999.
  10. 10. The use of the detection method according to claims 1-9 for quality control of trace amounts of N-nitrosodiethylamine in an epothilone drug substance.

Description

Method for detecting N-nitrosodiethylamine in epolamine diclofenac Technical Field The invention belongs to the technical field of medicine analysis, and particularly relates to a detection method of N-nitrosodiethylamine in epothilone. Background N-Nitrosodiethylamine (NDEA) is a possible impurity in the production of epothilone diclofenac. The impurity may have trace residue in the finished product of epothilone diclofenac. Since N-nitrosodiethylamine is a substance having a potentially genotoxic structure, its acceptable intake AI is 26.5 ng/day, the Maximum Daily Dose (MDD) of epothilone with reference to diclofenac is 360 mg/day, and the residual limit of N-nitrosodiethylamine is not more than 0.000007% (0.07 ppm) as determined by AI/MDD method. The existing detection technology faces two major challenges, namely, firstly, the epothilone diclofenac is easy to separate out in a conventional liquid chromatography mobile phase, a system is blocked, detection is interfered, and secondly, the main component has high background, and signals of trace N-nitrosodiethylamine can be seriously covered, so that the sensitivity is insufficient. Therefore, there is a need to develop a method that can not only effectively remove the matrix interference of the main component, but also meet the requirements of trace detection sensitivity, accuracy and reproducibility. Disclosure of Invention In order to solve the technical problems, the invention provides a method for detecting N-nitrosodiethylamine in epothilone, which uses formic acid to precipitate an epothilone sample, and combines a liquid chromatography-mass spectrometry tandem method to quantitatively analyze N-nitrosodiethylamine in supernatant, thereby reducing the background and reducing the interference of the N-nitrosodiethylamine to trace N-nitrosodiethylamine. The invention provides a method for detecting N-nitrosodiethylamine in epothilone, which comprises the following steps: S1, accurately weighing a diclofenac epolamine sample, adding a D 6 -N-nitrosodimethylamine internal standard solution, adding a solvent, shaking to dissolve, adding a volatile acid aqueous solution, shaking uniformly, standing, centrifuging and filtering to prepare a sample solution; S2, accurately weighing the diclofenac epolamine without N-nitrosodiethylamine, adding the D 6 -N-nitrosodimethylamine internal standard solution and the N-nitrosodiethylamine reference stock solution a, adding a solvent, shaking to dissolve, adding a volatile acid aqueous solution, shaking uniformly, standing, centrifuging and filtering to prepare a reference solution, wherein the concentration of the N-nitrosodiethylamine is 7 ng/mL, and the concentration of the D 6 -N-nitrosodimethylamine is 7 ng/mL; S3, precisely removing the sample solution and the reference substance solution, injecting the sample solution and the reference substance solution into a liquid chromatograph tandem mass spectrometer, and recording a chromatogram; S4, obtaining the content of the N-nitrosodiethylamine by calculating the peak area according to an internal standard method if chromatographic peaks consistent with the retention time of the N-nitrosodiethylamine exist in the chromatogram of the sample solution. In some embodiments, the liquid chromatography conditions are a chromatographic column of Poroshell 120: 120 EC-C18.7 μm, 3.0. 3.0 mm X100 mm, mobile phase of A: water containing 0.1% formic acid, B: methanol containing 0.1% formic acid, mobile phase gradient of: 0min mobile phase A95% mobile phase B5%; 3 min Mobile phase A95% and Mobile phase B5%; 7 min% mobile phase A5% mobile phase B95%; 9 min% mobile phase A and 95% mobile phase B; 9.1 min, mobile phase A95% and mobile phase B5%; 10min is mobile phase A95% and mobile phase B5%. In some embodiments, the liquid chromatography conditions further include column temperature of 40 ℃, flow rate of 0.50 mL/min, and sample loading of 15. Mu.L. In some embodiments, the mass spectral parameters are ion source APCI, positive ion mode, and scanning mode MRM multi-reaction monitoring, wherein the quantitative ion pair of N-nitrosodiethylamine is m/z 103- > 75.1, and the qualitative ion pair is m/z 103- > 47.1. In some embodiments, the solvent is methanol. In some embodiments, the aqueous volatile acid solution is an aqueous formic acid solution. The detection principle of the invention is that the diclofenac epolamine is dissolved in methanol aqueous solution, but when the diclofenac epolamine is in a mobile phase containing formic acid, the diclofenac epolamine can be gradually separated out, so that a chromatographic system is blocked and cannot be detected. In order to reduce the matrix interference and reduce the pollution of equipment, the method for extracting trace N-nitrosodiethylamine in the epothilone is needed, and simultaneously, the concentration interference of the epothilone is reduced, and the method for acid precipitation of the diclofenac is selected because