目的 基于二維液相色譜-高分辨串聯(lián)質譜（2D-LC-HR- MS/MS）法分析頭孢丙烯原料藥雜質譜。方法 一維色譜條件進行樣品色譜圖采集，確認各雜質的出峰位置，采用 YMC Hydrosphere 色譜柱（250 mm×4.6 mm，5 μm），以 11.5 g·L^-1磷酸二氫銨（用磷酸調節(jié) pH 為 4.4）為流動相 A，乙腈-流動相 A（50∶50）為流動項 B，梯度洗脫；柱溫 40 ℃；體積流量1.0 mL·min-1；檢測波長 230 nm；進樣量 4 μL。二維液相脫鹽后切進高分辨串聯(lián)質譜進行分析，根據結果推斷雜質結構及生成機制，采用 Waters BEH C₁₈色譜柱（50 mm×2.1 mm，1.7 μm），以 0.01% 甲酸水溶液為流動相 A，乙腈為流動相 B，切峰后開始 A 相由 98% 到 1%，柱溫 40 ℃，體積流量 0.3 mL·min-1，質譜采用 Xevo G2-XS QTof MS 系統(tǒng)，離子源為 ESI 源，毛細管電壓 3.0 kV，霧化器溫度 450 ℃，掃描范圍 m/z 100～2 000。結果 頭孢丙烯樣品中存在 9 個雜質色譜峰，其中 5 個雜質為已知雜質，峰 3 為雜質 B（頭孢羥氨芐）、峰 5 為雜質 D、峰 6 為雜質 F、峰 7 為雜質 G、峰 9 為雜質 I；對其中 3 個未知雜質可能的結構式進行了初步推測以及探討了可能的生成途徑，峰 2 分子式為 C₁₈H₁₉N₃O₆S，該化合物比頭孢丙烯多一個氧，分析其為頭孢丙烯的氧化雜質；峰 4 分子式為 C₁₆H₁₅N₃O₆S，與雜質 B 相比增加了 1 個氧原子，減少了 2 個氫原子，判斷其為 7-ACA 內酯與對羥基苯甲甘氨酸甲酯（HPGM）反應產物中的硫原子繼續(xù)發(fā)生了氧化生成的；峰 8 分子式為 C₈H₉NO₂S，該組分為頭孢丙烯分子結構的一部分。峰 1 有待進一步研究。結論 該方法有效解決了頭孢丙烯流動相中含不揮發(fā)性磷酸鹽的色譜體系與色譜-質譜快速鑒定雜質不兼容的難題，可以簡單、快速地對頭孢丙烯有關物質進行定性分析及雜質譜研究。

Objective To analyze the impurity profile of cefprozil API using 2D-LC-HR-MS/MS. Methods The sample chromatogram was acquired by one-dimensional chromatography conditions to confirm the peak positions of each impurity. The YMC Hydrosphere column (250 mm×4.6 mm, 5 μm) was used, with 11.5 g·L^-1 ammonium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid) as solvent A, a mixture of acetonitrile and solvent A (50:50) as solvent B, and gradient elution; column temperature was 40℃; flow rate was 1.0 mL·min-1; detection wavelength was 230 nm; injection volume was 4 μL. The impurity profile was analyzed by 2D-LC desalting and high-resolution tandem mass spectrometry (HR-MS/MS) after cutting into the mass spectrometer. Based on the results, the structures and formation mechanisms of the impurities were inferred. The Waters BEH C₁₈ column (50 mm×2.1 mm, 1.7 μm) was used, with 0.01% formic acid aqueous solution as solvent A and acetonitrile as solvent B. The peaks were cut and the A phase was gradually increased from 98% to 1% starting from the cut. The column temperature was 40° C, the flow rate was 0.3 mL·min-1, and the mass spectrometer used was the Waters Xevo G2-XS QTof MS system with an ESI ion source, a capillary voltage of 3 kV, a vaporizer temperature of 450 ℃, and a scanning range of m/z 100—2 000. Results Nine impurity peaks were identified in the cefprozil sample, of which five were known impurities. Peak 3 was impurity B (cefhydroxime), peak 5 was impurity D, peak 6 was impurity F, peak 7 was impurity G, and peak 9 was impurity I. Preliminary structural hypotheses were made for the three unknown impurities and the possible generation routes were discussed. Peak 2 has a molecular formula of C₁₈H₁₉N₃O₆S, which was one oxygen atom more than cefprozil. It was analyzed as an oxidation impurity of cefprozil. Peak 4 has a molecular formula of C₁₆H₁₅N₃O₆S, which was one oxygen atom and two hydrogen atoms less than impurity B. It was judged that it was the sulfur atom of the reaction product between 7-ACA lactam and HPGM (hydroxyphenylglycine methyl ester) continuing to undergo oxidation. Peak 8 has a molecular formula of C₈H₉NO₂S, which is part of the molecular structure of cefprozil. Peak 1 needs further study. Conclusion This method effectively solved the problem of the chromatographic system with non-volatile phosphate in the mobile phase of cefprozil flowing phase and the incompatibility of chromatography-mass spectrometry rapid identification of impurities. It can simply and quickly determine the qualitative analysis and impurity spectrum of cefprozil-related substances.

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廣東省重點領域研發(fā)計劃項目（2022B1111070004）；國家自然科學基金重點項目（61633006）