Separation and identification of folic acid and its related substances by liquid chromatography-mass spectrometry
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摘要:
用ODS色谱柱和10 mmol/L乙酸铵缓冲溶液-甲醇流动相(pH 6.3)将叶酸有关物质进行线性梯度洗脱分离,电喷雾正离子化-四极杆-飞行时间质谱(ESI-Q-TOF/MS)高分辨测定各有关物质母离子及其子离子的准确质荷比和元素组成,通过质谱解析、有机反应机制分析、或对照品对照鉴定它们的结构。在所建立的LC-ESI-Q-TOF/MS条件下,叶酸及其有关物质分离良好,检测并鉴定叶酸及其强制降解试验样品中23个主要有关物质,其中2个为《欧洲药典》规定的已知杂质,4个与文献报道已知杂质一致,其余17个为新鉴定的未知杂质。鉴定结果可为叶酸质量控制提供参考依据。
Abstract:The related substances in folic acid were separated on an ODS column by linear gradient elution using the mixture of 10 mmol/L ammonium acetate buffer solution and methanol (pH 6.3) as the mobile phase, and their accurate mass-charge ratio and elemental composition of parent and product ions were detected by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF/MS). The structures of the related substances were then determined by MS spectra elucidation, organic reaction mechanism analysis, or reference substances confirmation. Under the established LC-ESI-Q-TOF/MS conditions, folic acid and its related substances were adequately separated. Among the 23 main related substances found, 2 were known impurities listed in European Pharmacopoeia, 4 were consistent with the literature reports, and the remaining 17 were newly identified unknown related substances in this study. The results of separation and identification can provide some useful reference for the quality control of folic acid.
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Keywords:
- folic acid /
- related substances /
- degradation products /
- structural identification /
- LC-MS
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Table 1 Mass balance of Folic Acid and its stressed test solutions
Sample c/(mg/mL) AreaImp AreaTotal AreaTotal/c Calibration Test sample 1.003 888198 35558327 35451971 1.00 Dry photolysis 1.032 1036933 34141675 33083018 0.98 Alkaline 1.030 4612861 36378124 35318567 1.00 Oxidation 1.022 4700314 35609756 34843205 0.98 Wet photolysis 1.020 2252711 35383000 34689216 0.93 Dry heat 1.024 3906760 34478456 33670367 0.99 Wet heat 1.012 2997786 35357191 34937936 0.95 Table 2 Related substances identified in folic acid by LC-Q-TOF/MS
No. [M+H]+(m/z) Ion formula Diff.(×10-6) Product ions(m/z) UV λmax/nm Origins API 442.1480 [C19H20N7O6]+ −2.25 313.1045 ,295.0941 281, 346 / 1(Glu) 148.0603 [C5H10NO4]+ 0.91 130.0497 ,102.0547 ,84.0447 281, 347 Dr 2(EP-A) 267.1000 [C12H15N2O5]+ −9.21 130.0508 ,120.0452 273 Dr/Pr 3 324.1223 [C14H18N3O6]+ −9.18 195.0781 ,177.0671 287 Dr 4 193.0848 [C7H9N6O]+ −8.15 176.0580 ,108.0562 267, 349 Dr 5(CPT) 208.0483 [C7H6N5O3]+ −8.62 190.0372 ,180.0530 ,164.0524 ,162.0424 286, 345 Dr 6 343.1294 [C18H19N2O5]+ −1.61 299.1390 ,281.1277 ,253.1336 ,214.0865 ,169.0887 265 Dr 7 295.0934 [C13H15N2O6]+ −3.19 295.0934 ,176.0609 ,148.0391 ,130.0497 ,84.0449 266 Dr 8 313.1036 [C13H17N2O7]+ −1.83 166.0498 ,148.0391 279 Dr 9 265.0675 [C9H9N6O4]+ 1.81 221.0780 ,193.0828 ,178.0721 ,164.0560 275, 346 Dr 10 196.0469 [C6H6N5O3]+ −1.97 168.0519 ,140.0568 ,98.0346 279 Dr 11 279.0918 [C10H11N6O4]+ −6.12 261.0753 ,235.0968 ,193.0848 ,176.0580 274, 345 Dr 12 152.0579 [C5H6N5O]+ −8.04 135.0311 ,107.0364 ,110.0355 ,82.0402 245, 273 Dr 13 152.0580 [C5H6N5O]+ −8.70 135.0311 ,107.0364 ,110.0353 ,82.0402 284 Dr 14 313.0663 [C12H13N2O8]+ 1.12 295.0559 ,267.0611 ,249.0503 ,166.0130 ,84.0438 246, 278 Dr 15 297.0744 [C12H13N2O7]+ −9.03 279.0640 ,251.0683 ,150.0196 ,84.0446 246, 274 Dr 16(HPT) 194.0689 [C7H8N5O2]+ −8.54 176.0580 ,134.0358 ,106.0400 ,81.0480 274, 346 Dr 17 137.0709 [C7H9N2O]+ 0.29 120.0441 ,94.0647 275 Dr 18 456.1309 [C19H17N7O7]+ −7.34 438.1198 ,327.0870 ,309.0760 ,281.0808 287, 376 Dr 19(FPT) 192.0533 [C7H6N5O]+ −8.89 175.0255 ,149.0455 ,147.0311 ,122.0345 ,94.0400 280, 346 Dr 20 440.1317 [C19H18N7O6]+ −0.89 311.0885 ,293.0782 281, 347 Dr 21 443.1324 [C19H19N6O7]+ −3.23 296.0779 280 Dr 22 442.1480 [C19H20N7O6]+ −2.36 313.1045 ,295.0941 279, 346 Pr 23(EP-D) 313.1072 [C14H13N6O3]+ −9.08 295.0965 ,269.1169 ,176.0581 ,120.0453 278, 345 Dr Pr: Process related substance;Dr: Degradation related substance -
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