measurement for the determination of ribavirin by HPLC [J]. Chin J Pharm Anal(), 2007, 27(11): 1803-1805. [9] LIU Y L, LI D M, FEN L, et al. Uncertainty of measurement in the HPLC determination of Fufangdanshen tablets [J]. Chin J Pharm Anal(), 2007, 27(11): 1800-1802. [10] QIN L, CHEN S H, FAN Y G. Evaluation of for the content determination of vitamin B12 injection by UV [J]. J Yunnan Univ Tradit Chin Med(), 2013, 36(6): 19-21. [11] QIN L, XIAO Y, CHEN S H, et al. Uncertainty evaluation for determination of pentoxyverine citrate tablets by HPLC [J]. China Pharm(), 2014, 17(3): 505-507. [12] QUINTELA M, BÁGUENA J, GOTOR G, et al. Estimation of the uncertainty associated with the results based on the validation of chromatographic analysis procedures: Application to the determination of chlorides by high performance liquid chromatography and of fatty acids by high resolution gas chromatography [J]. J Chromatogr A, 2012(1223): 107-117. [13] KONIECZKA P, NAMIE SNIK J. Estimating uncertainty in analytical procedures based on chromatographic techniques [J]. J Chromatogr A, 2010(1217): 882-891. [14] JJG196-2006 [S]. 2006: 5-8. 2014-05-09 HPLC 7 1 2 2 (1. 317500 2. 318000) 5-() B A 7 Hypersil C 18 (4.6 mm 250 mm 5 µm) 0.7 ml min 1 A -(1 3) B 0.1%5-284 nm () 320 nm 220 nm B A 270 nm 210 nm 25 5-() B A 10.08~100.80 µg ml 1 (r=0.999 8) 3.55~35.50 µg ml 1 (r=0.999 3) 5.80~58.00 µg ml 1 (r=0.999 1) 4.20~42.00 µg ml 1 (r=0.999 9) 40.50~405.00 µg ml 1 (r=1.000) 12.28~122.80 µg ml 1 (r=0.999 5) 7.90~79.00 µg ml 1 (r=0.999 9) 5-() B A 98.56% 96.94% 99.61% 97.54% 100.04% 98.59% 99.81% RSD 0.97% 1.73% 1.03% 1.35% 0.86% 1.70% 1.43%(n=6) 5-() B A R917.101 B 1007-7693(2015)02-0194-05 DOI: 10.13748/j.cnki.issn1007-7693.2015.02.016 Simultaneous Determination of Seven Active Components in Mian anning Mixture by HPLC Under Multiple UV Wavelengths ZHANG Xiaojing 1, WANG Yingying 2, LI Jun 2 (1.Zhejiang Wenling Food and Drug Inspection Center, Wenling 317500, China; 2.Zhejiang Taizhou Institute for Food and Drug Control, Taizhou 318000, China) ABSTRACT: OBJECTIVE To develop an HPLC method with multiple wavelength ultraviolet for the simultaneous determination the content of 5-hydroxymethyl-2-furaldehyde, polygalaxanthone, atractylenolide, atractylenolide, salvianolic acid B, tanshinone A and catalpol in Mian anning mixture. METHODS The sample was separated on a Hypersil C 18 column(4.6 mm 250 mm, 5 µm) with a mobile phase consisting of methanol-acetonitrile(1 3) and 0.1% phosphoric acid solution. The flow rate was 0.7 ml min 1. The detection wavelength was set at 284 nm for 5-hydroxymethyl-2-furaldehyde, 320 nm for polygalaxanthone, 220 nm for atractylenolide and atractylenolide, 270 nm for salvianolic acid B and tanshinone A, 210 nm for catalpol. The column temperature was 25. RESULTS The calibration curves of 5-hydroxymethyl-2-furaldehyde, polygalaxanthone, atractylenolide, atractylenolide, salvianolic acid B, tanshinone A and catalpol were linear in the range of 10.08 100.80 µg ml 1 (r=0.999 8), 3.55 35.50 µg ml 1 (r=0.999 3), 5.80 58.00 µg ml 1 Tel: (0576)89930618 E-mail: zhangxiaojinglw@163.com 194 Chin J Mod Appl Pharm, 2015 February, Vol.32 No.2 2015 2 32 2
(r=0.999 1), 4.20 42.00 µg ml 1 (r=0.999 9), 40.50 405.00 µg ml 1 (r=1.000), 12.28 122.80 µg ml 1 (r=0.999 5), 7.90 79.00 µg ml 1 (r=0.999 9), and the average recoveries(n=6) were 98.56%(RSD=0.97%), 96.94%(RSD=1.73%), 99.61% (RSD=1.03%), 97.54%(RSD=1.35%), 100.04%(RSD=0.86%), 98.59%(RSD=1.70%), 99.81%(RSD=1.43%), respectively. CONCLUSION The method is feasible, accurate and stable, which can control the quatity of Mian anning Mixture. KEY WORDS: Mian anning mixture; 5-hydroxymethyl-2-furaldehyde; polygalaxanthone ; atractylenolide ; atractylenolide ; salvianolic acid B; tanshinone A; catalpol ( ) () 7 [1-2] HPLC 5- ( )() ( ) B A 5-() B A 1 1.1 Agilent 1200 G1311A G1330B G1322A Chemstation G1315B () 5-( 111626-201308 98.3%) () ( 111850-20120395.7%) B( 111562-20131397.0%) A( 110766-200619100% 12 h ) ( 110808-20121098.1% 12 h ) ( 73069-13-3 98.0%)( 73030-71-4 98.0%) (120 ml 1 130401 130801 131001)( ) ( ) 1.2 Hypersil C 18 (4.6 mm 250 mm 5 µm) A -(1 3) B 0.1% [2-4] 0~12 min 45.0%A 12~18 min 45.0% 52.0%A 18~45 min 52.0% 70.0%A 45~69 min 70.0%A 69~80 min 70.0% 45.0%A [3-5] 0~18 min λ 1 =284 nm [6] 18~26 min λ 2 =320 nm [2] 26~39 min λ 3 =220 nm [7-9] 39~58 min λ 4 =270 nm [10] 58~80 min λ 5 =210 nm [11-12] 0.7 ml min 1 25 20 µl ( ) <3 500 >2.0 1.3 7 5-( ) B A 10 20 20 20 10 10 10 ml 5-() B A 1.008 0.355 0 0.580 0 0.420 0 4.050 1.228 0.790 0 2.5 2.0 1.0 2.0 4.0 2.5 2.0 ml 50 ml 5-() B A 0.050 4 0.014 2 0.011 6 0.016 8 0.324 0 0.061 4 0.031 6 1.4 5 ml 50 ml 2015 2 32 2 Chin J Mod Appl Pharm, 2015 February, Vol.32 No.2 195
1.5 ( ) () 1 1.4 2 2.1 20 µl 1.2 5-() B A 5- () B A 1 1 A B C D E F 1 5-2 () 3 4 5 B 6 A 7 Fig. 1 HPLC chromatograms A reference substance; B sample solution; C sample without Rehmanniae Radix Praeparata; D sample without Polygalae Radix; E sample without Atractylodis Macrocephalae Rhizoma; F sample without Salviae Miltiorrhizae Radix et Rhizoma; 1 5-hydroxymethyl-2-furaldehyde; 2 polygalaxanthone ; 3 atractylenolide ; 4 atractylenolide I; 5 salvianolic acid B; 6 tanshinone IIA; 7 catalpol. 2.2 1.3 0.1 0.2 0.5 0.8 1.0 ml 5 10 ml 1.2 (Y) (X) 5- () B A 1 196 Chin J Mod Appl Pharm, 2015 February, Vol.32 No.2 2015 2 32 2
1 7 Tab. 1 Regression equation, correlation coefficient and linearity range of the seven components / µg ml 1 r 5-10.08~100.80 Y=8.168 4 10 3 X 16.9 0.999 8 () 3.55~35.50 Y=6.354 9 10 3 X+59.4 0.999 3 5.80~58.00 Y=4.628 7 10 3 X 29.8 0.999 1 4.20~42.00 Y=9.684 9 10 3 X+62.7 0.999 9 B 40.50~405.00 Y=2.187 3 10 4 X+104.8 1.000 A 12.28~122.80 Y=8.046 1 10 3 X 33.4 0.999 5 7.90~79.00 Y=4.142 2 10 3 X+51.2 0.999 9 2.3 1.31.2 20 µl 6 7 (RSD) 5- () B A RSD 0.86% 1.13% 1.64% 0.59% 0.37% 1.02% 1.23% 2.4 1.4 6 1.2 RSD 5-() B A RSD 0.97% 1.43% 1.57% 0.84% 0.76% 1.16% 1.29% 2.5 0 2 4 6 8 12 h 20 µl 5- () B A RSD 1.02% 1.24% 1.39% 0.64% 0.59% 1.11% 0.93% 12 h 2.6 1.5 9 1.0 ml 10 ml 0.4 0.4 0.4 0.5 0.5 0.5 0.6 0.6 0.6 ml 1.2 2 2 (n=9) Tab. 2 Analytical results of recoveries(n=9) 5- () B A / /mg /% mg 1 2 3 1 2 3 0.403 2 0.398 2 0.399 1 0.389 5 98.76 98.98 96.60 0.504 0 0.496 9 0.498 3 0.492 1 98.59 98.87 97.64 0.604 8 0.595 1 0.600 8 0.603 9 98.40 99.34 99.85 0.142 0 0.136 4 0.136 5 0.136 4 96.06 96.13 96.06 0.177 5 0.177 4 0.170 6 0.170 8 99.94 96.11 96.23 0.213 0 0.204 5 0.212 7 0.204 7 96.01 99.86 96.10 0.232 0 0.232 2 0.231 5 0.224 8 100.09 99.78 96.90 0.290 0 0.289 9 0.289 6 0.290 2 99.97 99.86 100.07 0.348 0 0.347 9 0.347 4 0.348 1 99.97 99.83 100.03 0.168 0 0.166 3 0.161 9 0.167 4 98.99 96.37 99.64 0.210 0 0.204 8 0.201 6 0.203 8 97.52 96.00 97.05 0.252 0 0.245 9 0.242 1 0.248 6 97.58 96.07 98.65 1.620 1.619 1.641 1.602 99.94 101.30 98.89 2.025 2.021 2.054 2.007 99.80 101.43 99.11 2.430 2.422 2.436 2.429 99.67 100.25 99.96 0.491 2 0.488 7 0.489 8 0.473 2 99.49 99.71 96.34 0.614 0 0.591 6 0.613 4 0.609 6 96.35 99.90 99.28 0.736 8 0.735 5 0.736 4 0.710 4 99.82 99.95 96.42 0.316 0 0.318 9 0.303 9 0.315 8 100.92 96.17 99.94 0.395 0 0.399 2 0.394 9 0.394 8 101.06 99.97 99.95 0.474 0 0.473 9 0.475 5 0.473 8 99.98 100.32 99.96 / % RSD/ % 98.56 0.97 96.94 1.73 99.61 1.03 97.54 1.35 100.04 0.86 98.59 1.70 99.81 1.43 2015 2 32 2 Chin J Mod Appl Pharm, 2015 February, Vol.32 No.2 197
2.7 3 1.4 1.2 5- () B A 3 3 (n=3, x ± s ) Tab. 3 Results of content determination(n=3, x ± s ) 5- / ()/ / / B/ A/ / 130401 0.496±0.003 0.138±0.001 0.122±0.001 0.176±0.001 3.15±0.02 0.626±0.006 0.332±0.001 130801 0.507±0.005 0.132±0.001 0.126±0.001 0.182±0.002 3.01±0.03 0.615±0.004 0.341±0.003 131001 0.510±0.002 0.151±0.002 0.139±0.001 0.165±0.001 3.42±0.03 0.647±0.004 0.357±0.002 3 3.1 25 30 35 25 3.2-0.1% -0.1% -0.1% () -0.1% 5- -(1 3) 0.1% >2 7 REFERENCES [1] [S]. 1998: 166. [2]. [S]. 2010: 70-71, 95-96, 116-117, 146-147, 30, 36. [3]. B A [J]., 2007, 26(10): 1215-1216. [4],,,. [J]., 2010, 29(5): 662-663. [5] CAI Y Y, SHI T T, HUANG Q L. Determination of 5-hydroxymethylfurfural in Liuweidihuang pills by HPLC [J]. [J]. Chin J Mod Appl Pharm(), 2012, 29(04): 356-358. [6] QIN X Y, ZHOU J, LI X Y, et al. RP-HPLC determination of 5-hydroxymethylfurfural in prepared rehmannia root [J]. J Fourth Mil Med Univ(), 2006, 27(06): 510-511. [7] LIU Y Q, CAI Q. HPLC determination of atractylenolide and atractylenolide in 50 batches crude drugs and slices of Atractylodes macrocephala Koidz. from different sources [J]. Chin J Pharm Anal(), 2012, 32(7): 1249-1252. [8] CHEN H P, ZHANG J H, WANG X Y, et al. Contents comparision of atractylode I,, in Rhizoma Atractylodis Macrocephalae and the processed with soils [J]. J Chin Med Mater( ), 2011, 34(03): 354-357. [9] DUAN Q, LI C P, ZHAO Z D, et al. Quantitative determination of atractylenolides I,, in simple parching Atractylodes macrocephala by HPLC [J]. Cent South Pharm(), 2009, 7(5): 321-323. [10] YU Y M, MOU N, ZHANG P, et al. Determination of cryptotanshinone and tanshinone A in Shenqi Shengji granule by HPLC [J]. [J]. Chin J Mod Appl Pharm( ), 2013, 301(4): 425-428. [11],,,. HPLC [J]., 2010, 16(1): 23-25. [12] MEN J Y, DONG Z M, CUI Y Q, et al. RP-HPLC simultaneous determination of catalpol and gastrodin in Tianma Toufengling capsules [J]. Chin J Pharm Anal( ), 2009, 29(6): 964-967. 2014-04-29 198 Chin J Mod Appl Pharm, 2015 February, Vol.32 No.2 2015 2 32 2