猪胆粉HPLC-ELSD全轮廓谱图主要胆汁酸测定及化学计量学分析

目的：建立测定猪胆粉中主要胆汁酸类成分的HPLS-ELSD法，并对样品的全轮廓色谱图进行化学计量学分析。方法：采用SB-C₁₈色谱柱（4.6 mm×250 mm，5 μm），柱温为40℃，流动相为0.2%甲酸水溶液-乙腈，梯度洗脱，流速1.0 mL·min^-1；ELSD漂移管温度为110℃，氮气流量为2.8 L·h^-1，增益为1。使用COW法校正样品的色谱峰，并对数据进行聚类分析和主成分分析。结果：有2批样品未检出甘氨猪去氧胆酸和甘氨鹅去氧胆酸，另外20批样品的甘氨猪去氧胆酸和甘氨鹅去氧胆酸的含量分别在6.4%~36.5%和4.5%~27.0%之间。化学计量学分析结果表明，自制样品与对照药材图谱更接近，而与市售样品差异较大。结论：该法适合从定性和定量的角度对猪胆粉进行质量评价与控制。

Objective: To establish a method for determination of the major bile acids in pig bile powder and its chemometric analysis of the whole chromatographic profiles.Methods: This analysis was performed at 40℃ on a SBC₁₈ column(4.6 mm×250 mm, 5 μm). The mobile phase consisted of 0.2% formic acid solution and acetonitrile as a linear gradient elution and the flow rate was 1.0 mL·min^-1. An ELSD was used with a nitrogen flow-rate of 2.8 L·h^-1 at a drift tube temperature of 110℃ and the gain was 1. The data of these chromatograms were processed by COW method and analyzed by hierarchical clustering analysis(HCA)and principle component analysis(PCA)Results: Glycohyodeoxycholic acid and glycochenodeoxycholic acid were not detected in two batches samples from market. The content ranges of glycohyodeoxycholic acid and glycochenodeoxycholic acid in the other samples were 6.4%-36.5% and 4.5%-27.0%, respectively. The chemometrics showed that self-prepared samples were closer with reference medicinal material than market samples.Conclusion: Simultaneous qualitative and quantitative methods are suitable for the quality evaluation and the quality control of pig bile powder.

[1] 中国药典2015年版.一部[S]. 2015:319 ChP 2015. Vol Ⅰ[S]. 2015:319
[2] 南京中医药大学.中药大辞典[M].第二版.上海:上海科学技术出版社, 2006:3064 Nanjing University of Chinese Medicine. Great Dictionary of Chinese Medicine[M]. 2nd Ed. Shanghai:Shanghai Science and Technology Publishers,2006:3064
[3] 邓启华,姚颖,刘婷,等.猪胆汁中主要胆汁酸的提取分离[J].中国生化药物杂志, 2012, 33(4):405 DENG QH, YAO Y, LIU T, et al. The extraction and purification of three kinds of bile acids from pig bile[J]. Chin J Biochem Pharm, 2012, 33(4):405
[4] KANDRAC J,KEVRESAN S,GU JK,et al. Isolation and determination of bile acids[J]. Eur J Drug Metab Pharmacokinet, 2006,31(3):157
[5] HE J, LI J, SUN WJ, et al. Preparative isolation and purification of three glycine conjugated cholic acids from PULVIS FELLIS SUIS by high-speed countercurrent chromatography coupled with ELSD detection[J]. J Liq Chromatogr Relat Technol, 2012, 35(5):737
[6] 陈志维,黎玉翠,暴梅佳,等.柱前衍生化高效液相色谱法测定猪胆粉结合胆酸中甘氨酸和牛磺酸的含量[J].药物分析杂志, 2010,30(2):288 CHEN ZW, LI YC, BAO MJ, et al. Determination of glycine and tqurine in Pulvis Fellis Suis conjugated cholic acid by pre-column derivation on HPLC[J]. Chin J Pharm Anal, 2010, 30(2):288
[7] 宋占军,邓振华,韩纠缦.薄层扫描法测定熊胆中结合胆汁酸含量[J].药物分析杂志, 1993,13(3):194 SONG ZJ,DENG ZH, HAN JM. Determination of conjugated cholic acids in bear bile by TLC-scan[J]. Chin J Pharm Anal, 1993, 13(3):194
[8] 何姣,李静,朱砂,等. RP-HPLC法同时测定猪胆粉药材中3种牛磺结合型胆酸的含量[J].药物分析杂志, 2012,32(2):229 HE J,LI J, ZHU S, et al. RP-HPLC simultaneous determination of three tauro-conjugated cholic acids in Pulvis Fellis Suis[J]. Chin J Pharm Anal,2012, 32(2):229
[9] KONG WJ, JIN C, LIU W, et al. Development and validation of a UPLC-ELSD method for fast simultaneous determination of five bile acid derivatives in Calculus Bovis and its medicinal preparations[J]. Food Chem, 2010, 120(4):1193
[10] KONG WJ,JIN C,XIAO XH,et al. Determination of multicomponent contents in Calculus Bovis by ultra-performance liquid chromatography-evaporative light scattering detection and its application for quality control[J]. J Sep Sci,2010, 33(10):1518
[11] ROCCALDO S, ANTIMO G, FEDERICA I, et al. HPLC-ELSD analysis of amidated bile acids:an effective and rapid way to assist continuous flow chemistry processes[J]. Talanta, 2012, 100(10):364
[12] GEORG K, GUSTAV P. Analysis of bile acids in serum and bile by capillary gas-liquid chromatography[J]. J Lipid Res, 1978, 19(6):771
[13] GOWDA GAN, SHANAIAH N, COOPER A, et al. Bile acids conjugation in human bile is not random:new insights from ¹H-NMR spectroscopy at 800 MHz[J]. Lipids, 2009, 44(6):527
[14] OMKAR BI,TEDROS B,NILS A. et al. Simultaneous quantification of glycine-and taurine-conjugated bile acids, total bile acids, and choline-containing phospholipids in human bile using ¹H NMR spectroscopy[J]. J Pharm Biomed Anal, 2010, 53(3):667
[15] HU Z, HE LC,ZHANG J, et al. Determination of three bile acids in artificial Calculus Bovis and its medicinal preparations by micellar electrokinetic capillary electrophoresis[J]. J Chromatogr B, 2006, 837(1-2):11
[16] HE J, ZHANG YM, ITO Y, et al. Semi-preparative isolation and purification of three tauro-conjugated cholic acids from Pulvis Fellis Suis by HSCCC coupled with ELSD detection[J]. Chromatographia, 2011,73(3-4):361
[17] TSAI S, ZHONG YS, WENG JF, et al. Determination of bile acids in pig liver, pig kidney and bovine liver by gas chromatographychemical ionization tandem mass spectrometry with total ion chromatograms and extraction ion chromatograms[J]. J Chromatogr A, 2011, 1218(3):524
[18] KELLER S,JAHREIS G. Determination of underivatised sterols and bile acid trimethyl silyl ether methyl esters by gas chromatographymass spectrometry-single ion monitoring in faeces[J]. J Chromatogr B, 2004, 813(1-2):199
[19] GAUTROT J, ZHU XX. Molar mass of main-chain bile acid-based oligo-esters measured by SEC,MALDI-TOF spectrometry and NMR spectroscopy:a comparative study[J]. Anal Chim Acta, 2007, 581(2):281
[20] MIMS D,HERCULES D. Quantification of bile acids directly from urine by MALDI-TOF-MS[J]. Anal Bioanal Chem,2003,375(5):609
[21] MIMS D, HERCULES D. Quantification of bile acids directly from plasma by MALDI-TOF-MS[J]. Anal Bioanal Chem, 2004, 378(5):1322
[22] CRISTINA LQ,LINDSAY MD,HECTOR VM,et al. Bile UPLC-MS fingerprinting and bile acid fluxes during human liver transplantation[J]. Electrophoresis, 2011,32(15):2063
[23] XIANG X, HAN Y, NEUVONEN M, et al. High performance liquid chromatography-tandem mass spectrometry for the determination of bile acid concentrations in human plasma[J]. J Chromatogr B, 2010,878(1):51
[24] HAGIO M, MATSUMOTO M, FUKUSHIMA M, et al. Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS[J]. J Lipid Res, 2008, 50(1):173
[25] LI K,WANG H,BRANT C,et al. Multiplex quantification of lamprey specific bile acid derivatives in environmental water using UHPLCMS/MS[J]. J Chromatogr B, 2011, 879(32):3879
[26] WANG G, STACEY N, EARL J. Determination of individual bile acids in serum by high performance liquid chromatography[J]. Biomed Chromatogr, 1990, 4(4):136
[27] CAVRINI V, GATTI R, RODA A, et al. HPLC-fluorescence determination of bile acids in pharmaceuticals and bile after derivatization with 2-bromoacetyl-6-methoxynaphthalene[J]. J Pharm Biomed Anal, 1993, 11(8):761
[28] GATTI R, RODA A, CERRE C, et al. HPLC-fluorescence determination of individual free and conjugated bile acids in human serum[J]. Biomed Chromatogr, 1997,11(1):11
[29] MILAN N, MILAN P, JIří K, et al. High-performance liquid chromatographic determination of ursodeoxycholic acid after solid phase extraction of blood serum and detection-oriented derivatization[J]. J Pharm Biomed Anal, 2001, 24(s5-6):937