1755 1 2 1 1 3 1* 1 1. 1000502. 0710513. 110164 UPLC UPLC ACQUITY UPLC BEH C 18 50 mm 2. 1 mm 1. 7 μm A- 0. 1% B 0 ~ 10 min 4% A 12% A10 ~ 22 min 12% A 16% A22 ~ 35 min 16% A 25% A 0. 2 ml min - 1 230 nm 40 / Chempattern TM PCA Score Loading UPLC 8 A 4 PCA UPLC UPLC A R917 A 0254-1793201410 - 1755-08 UPLC fingerprint analysis of Sanqishangyao tablets HU Xiao - ru 1 YANG Mei - li 1 2 SUN Lei 1 WANG Ming - juan 1 LI Ai - hua 3 DAI Zhong 1* MA Shuang - cheng 1 1. National Institutes for Food and Drug Control Beijing 100050 China2. Baoding Institute for Food and Drug Control Baoding 071051 China 3. Shenyang Green Pharmaceutical Co. Ltd. Shenyang 110164 China Abstract ObjectiveTo establish a UPLC chromatographic fingerprint analysis method of Sanqishangyao tablets. MethodsThe characteristic fingerprint was performed by HPLC and characteristic peaks were identified by LC - MS. The samples were separated on the ACQUITY UPLC BEH - C 18 column50 mm 2. 1 mm 1. 7 μm and e- luted with a gradient elution0-10 min 4% A 12% A10-22 min 12% A 16% A22-35 min 16% A 25% Aof acetonitrile as the mobile phase A and the solution containing 0. 1% formic acid as the mobile phase B at a flow rate of 0. 2 ml min - 1. The detection wavelength was 230 nm and the column temperature was 40. According to the chempattern TM advanced chemometrics solution a common pattern was built and statistically analyzed by principal components analysispca. The Score plot and the Loading plot were obtained with the retention time and the peak area for each main chromatographic peak as the variable. Four characteristic peaks in the fingerprints were identified with applying electrospray ionizationesisource in the positive /negative ion mode. ResultsThe characteristic fingerprint of Sanqishangyao tablets was established with naringin as the reference peak. Four characteristic peaks were identified as hydroxysafflor yellow A neoeriocitrin naringin paeoniflorin. Eight co - possessing peaks were designated as characteristic common peaks. The PCA showed the chemical compositions in the 27 samples were obviously different. The main difference of the chemical compositions was the content of neoeriocitrin naringin and paeoniflorin. ConclusionThe fingerprint method can reflect the actual quality with good stability and reproducibility and be applied in the quality control of Sanqishangyao tablets. * Tel 01067095376E - maildaizhong@ nicpbp. org. cn Tel 01067095376E - mailhuxru2008@ 126. com
1756 Key wordssanqishangyao tabletsuplccharacteristic chromatogramprincipal components analysishydroxysafflor yellow Apaeoniflorinneoeriocitrinnaringin UPLC 1 Agilent 6320 Ion Trap LC - MS Agilent 1200 DAD ACQUITY UPLC 1-2 Waters Empower2 Mettler XS105DU Millipore 2010 A 111637-201308 3 96. 5% 110736-201438 2010 96. 4% 110722-201111 93. 2% 2000ZFB0115 4 Fisher Millipore 27 4-9 27 1 Tab 1 1 Sources of Sanqishangyao tablets sample No. source batch sample No. source batch sample No. source batch 1 Anhui 130101 10 Inner Mongolia 20130104 19 Shanghai 130104 2 Changchun 20120403 11 Shandong 130213 20 Sichuan 130410 3 Gansu 20130101 12 Shandong 130102 21 Sichuan 120801 4 Heilongjiang 20121001 13 Shanxi 20130109 22 Sichuan 12120032 5 Heilongjiang 130402 14 Shanxi 121201 23 Shaanxi 131540 6 Jilin 20120703 15 Shaanxi 012211011 24 Shaanxi 121201 7 Jilin 20120604 16 Shaanxi 20121208 25 Shaanxi 20120201 8 Jilin 130201 17 Shaanxi 20130102 26 Yunnan 120502 9 Heilongjiang 20111004 18 Shanghai 121206 27 Yunnan 20130106 2 0. 22 μm 2. 1 2. 2 2. 1. 1 ACQUITY UPLC BEH C 18 50 mm 1 ml 0. 4 mg 2. 1 mm 1. 7 μm A- 0. 1% 2. 1. 2 10 B 0 ~ 10 min 4% A 12% A10 ~ 2 g 22 min 12% A 16% A22 ~35 min 16% A 25% A 20 ml 300 0. 2 ml min -1 230 nm 40 W 40 khz 30 min 1 μl UPLC 1
1757 1 Fig 1 HPLC chromatograms of methanol extract of Sanqishangyao tablets and medical materials A. Sanqishangyao tablets B. naringin 2. 3 9. 0 ml min - 1 325 0. 6 V ESI + ESI - 230 nm m /z 50 ~ 1200 0. 17 MPa 2 2 ESI + A ESI - B ESI + CHPLC - UV230 nm D Fig2 Total ion chromatorgramtic ESI + A TICESI - B /MS ion chromatogramesi + Cand HPLC - UV230 nmcharacteristic fingerprintdof Sanqishangyao tablets 2. 4 1 5 6 8 9 10 11 12 2. 4. 1 RSD 1. 1% ~ 2. 2% 6 12 2. 4. 2
1758 0 1 2 4 8 12 h 12 1 5 6 8 6 12 9 10 11 12 RSD 1. 2% ~ 1 5 6 8 9 10 11 12 2. 8% RSD 1. 7% ~ 3. 5% 2. 5 12 h 2. 1. 2 2. 2. 4. 3 6 27 27 2. 1. 2 3 3 27 UPLC Fig 3 UPLC chromatograms of 27 batches of Sanqishangyao tablets A. 1 ~ 9 samples 1-9 B. 10 ~ 18 samples 10-18 C. 19 ~ 27 samples 19-27
1759 8 5 9 12 A LC - MS 10 11 2 Tab 2 2 Retention time and identified compounds of the characteristic peaks in HPLC fingerprints M r peak No. retention time/min identified compound precursor ion/m /z fragment ionsm /z λ max /nm Ahydro 5 7. 45 * 612 - xysafflor yellow A 595. 2M - H 2 O + H - 577. 3M - 2H 2 O + 613. 1 M + H + H - 451. 1M - glu + H - 433. 1M - glu - H 611. 2M + H - 2 O + H - 223 402 9 11. 45 * paeoniflorin 480 358. 8M - Ar - COO - 319. 0M - glu + H + 481. 1 M + H + 196. 9M - 2glu + H + 178. 9M - H 2 O - 2glu + 479. 2M - H - H + 232 569. 1M - CO + H + 561. 1M - 2H 2 O + 11 17. 72 * neoeriocitrin 596 597. 2M + H + H + 541. 2M - CO - CO + H + 451. 1 M - 595. 3M - H - rha + H + 435. 1M - Orha + H + 331. 0M - 227 284 rha - C 7 H 6 O 2 + H + 289. 0M - rha - glu + H + 12 22. 38 * naringin 580 563. 1M - H 2 O + H + 545. 1M - 2H 2 O + 581. 1M + H + H + 527. 1M - 3H 2 O + H + 459. 1M - C 7 H 6 603. 2 M + Na + O 579. 3M - H - 2 + 435. 1M - rha + H + 417. 2M - rha - H 2 O + H + 383. 1 273. 0M - rha - glu + H + 227 283 * compound structures were identified by comparison with authentic standards 3 10-11 27 UPLC 3 4 Chempattern TM 4. 1 4 PCA 27 Score PCA Loading 4 Score 27 10 24 8 15 ~ 10 24 8 25 min 10 24 8 5 10 24 8 5 ~ 10 min 10 24 8 4. 2 UPLC Loading UPLC 3 12 11 9
1760 4 27 Fig4 PCA analysis of the fingerprints of Sanqishangyao tablets A. common fingerprint pattern B. 1 2 Score 1 ~ 27 Score plot of 1-2 principal components based on the raw datasamples 1-27 C. 1 2 Loading 1-12 Loading plot of 1-2 principal components based on the raw datapeaks 1-12 5. Ahydroxysafflor yellow A 9. paeoniflorin 11. neoeriocitrin 12. naringin UPLC HPLC UPLC 3 5 1 μl A UPLC 2
1761 5 8 24 10 Fig 5 Comparison between chromatograms of outlier sample 8 24 and 10 and common patterns for Sanqishangyao tablets A. 8 sample 8upperand fingerprint common patternb. 24 sample 24upperand fingerprint common patternc. 10 sample 10upperand fingerprint common pattern
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