柱后衍生阳离子交换色谱法测定甘肃紫斑牡丹花粉氨基酸含量

目的：建立柱后衍生阳离子交换色谱方法，测定甘肃紫斑牡丹花粉氨基酸的含量。方法：样品加6 mol·L^-1盐酸于110℃水解22 h。采用柱后衍生阳离子交换色谱法，对甘肃产紫斑牡丹花粉氨基酸的含量进行分析；色谱柱：LCAK06/Na型磺酸基强酸性阳离子交换树脂色谱柱（4.6 mm×150 mm，5 μm），梯度洗脱，其中A泵（溶液泵）流速为0.45 mL·min^-1，M泵（茚三酮泵）流速为0.25 mL·min^-1，检测波长为440 nm（脯氨酸）和570 nm（其余氨基酸），进样量为50 μL。结果：该测定方法中天冬氨酸、苏氨酸、丝氨酸、谷氨酸、脯氨酸、甘氨酸、丙氨酸、胱氨酸、缬氨酸、蛋氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、组氨酸、赖氨酸、精氨酸的线性关系良好；精密度RSD范围为0.10%~1.6%，重复性RSD范围为0.20%~1.5%；平均回收率（n=6）在89.5%~98.2%之间，RSD在0.81%~2.9%之间。样品中所含氨基酸的范围为4.13~44.23 mg·g^-1，破壁前后所测得氨基酸含量无显著差异。结论：所建立的分析方法可作为紫斑牡丹花粉中氨基酸的定量分析。

Objective: To establish a method for detection of amino acids in Paeonia rockii pollen.Methods: The sample was hydrolyzed with 6 mol·L^-1 hydrochloric acid for 22 hours at 110℃.The content of amino acids in Paeonia rockii pollen were detected by cation-exchange chromatography with post-column derivatization.The cation exchange resin LCAK06/Na column(4.6 mm×150 mm, 5 μm)with gradient elution was adopted, where A pump(solution pump)and M pump(ninhydrin pump)were at a flow rate of 0.45 mL·min^-1 and 0.25 mL·min^-1, respectively.The detection wavelength were set at 440 nm(proline)and 570 nm(the other amino acids), and injection volume was 50 μL.Results: The method showed a good linear relationship for the determination of aspartate acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine; the RSDs of precision were in the range of 0.10%-1.6%, the RSDs of repeatability were in the range of 0.20%-1.5%, the average recoveries and RSDs were between 89.5%-98.2% and 0.81%-2.9%(n=6),respectively.The contents of amino acids ranged from 4.13 to 44.23 mg·g^-1 in the samples,and their contents had no significant difference between wall-unbroken and broken pollen.Conclusion: The developed method can be applied to the analysis of amino acids in Paeonia rockii pollen.

[1] 李熙莉,李平平,岳桦.紫斑牡丹研究进展[J].北方园艺,2007(5):129 LI XL,LI PP,YUE H. Advances of the study on Paeonia rockii[J]. Northern Hortic,2007(5):129
[2] 何登文.紫斑牡丹的研究概况[J].甘肃中医,2005, 18(3):34 HE DW. A brief review on Paeonia rockii[J]. Gansu J Tradit Chin Med, 2005, 18(3):34
[3] 李子璇,秦公伟,何建华等.紫斑牡丹种仁种皮中脂肪酸组成比较分析[J].种子,2010, 29(1):34 LI ZX, QIN GW, HE JH, et al. Comparative analysis of fatty acids in seed kernel and seed coat of Paeonia rockii[J]. Seed, 2010, 29(1):34
[4] 甘肃省卫生厅.关于颁布《第四批24种中药材质量标准(试行)》的通知[S]. 1996:58 Gansu Health Department. Notice on Promulgating Fourth Batch Quality Standard of 24 Kinds Chinese Herbal Medicine(Trial Implementation)[S]. 1996:58
[5] 杨树声,宋平顺,罗兴平,等.紫斑牡丹各部位中丹皮酚和芍药苷的含量分析[J].西北药学杂志,2001, 16(3):108 YANG SS, SONG PS, LUO XP, et al. Analysis of paeonol and paeoniflorin in different parts of Paeonia rockii(S. G. Haw et L. A. Laeuner)T. Hong et J. J. Li[J]. Northwest Pharm J,2001,16(3):108
[6] 王开发.我国常见八种花粉的功效探讨[J].蜜蜂杂志,2010, 30(12):5 WANG KF. Research on efficacy of 8 pollen in China[J]. J Bee, 2010, 30(12):5
[7] 李彬彬.卫生部公布第三批药食两宜名单[J].江苏调味副食品, 1999(3):23 LI BB. China Ministry of Health's third batch name list of medicine and food dual purposes material[J]. Jiangsu Hortic Subsidiary Food,1999,(3):23
[8] 成仿云.紫斑牡丹花粉发育的细胞形态学研究[J].园艺学报, 1998, 25(4):367 CHENG FY, Cytomorphological studies on the pollen development of blotched tree peony(Paeonia rochii)[J]. Acta Hortic Sin,1998, 25(4):367
[9] 曹小勇.濒危植物紫斑牡丹胚离体培养[J].氨基酸与生物资源, 2003, 25(2):35 CAO XY, In vitro culture of embryo of endangered plant Paeonia rochii[J]. Amino Acid Biotic Res,2003, 25(2):35
[10] 安阿莉,苏小玲,毛娟,等.紫斑牡丹幼胚离体培养试验[J].甘肃农业大学学报, 2009, 44(6):63 AN AL,SU XL, MAO J,et al. A study on immature embryo culture of Paeonia rochii in vitro[J]. J Gansu Agric Univ, 2009, 44(6):63
[11] 于玲,何丽霞,李嘉珏.甘肃紫斑牡丹与中原牡丹类群染色体的比较研究[J].园艺学报, 1997, 24(1):79 YU L, HE LX, LI JJ. Comparative studies on chromosome in varieties of Paeonia rockii and Paeonia suffruticosa[J]. Acta Hortic Sin,1997, 24(1):79
[12] 刘文兰,唐红,张亮,等.甘肃紫斑牡丹茎扦插繁殖技术初探[J]. 东北林业大学学报, 2012, 40(11):19 LIU WL,TANG H,ZHANG L,et al. Propagation techniques of stem cuttings of Paeonia suffruticosa var. papaveracea in Gansu[J]. J Northeast Forest Univ,2012,40(11):19
[13] 杨新,梅兴国,汪爱顺,等.高效毛细管电泳法测定红豆杉悬浮培养细胞中游离芳香氨基酸[J].氨基酸和生物资源,2002, 24(1):50 YANG X, MEI XG, WANG AS, et al. Direct determination of free aromatic amino acid in suspension of Taxus chinensis cell by high performance capillary electrophoresis[J]. Amino Acids Biotic Resour,2002,24(1):50
[14] 石岩,王钢力,林瑞超.近红外技术测定冬虫夏草中氨基酸含量[J].药物分析杂志, 2007,27(1):90 SHI Y,WANG GL,LIN RC. NIR determination of amino acid from Cordyceps sinensis(Berk.)Sacc.[J]. Chin J Pharm Anal, 2007, 27(1):90
[15] 王棘,潘雪妍,杨宏伟. HPLC法和氨基酸分析仪(AAA)法测定肠外营养注射液(25)中18种氨基酸的含量的比较[J].药物分析杂志, 2012, 32(6):1085 WANG J, PAN XY, YANG HW. Comparison of HPLC and amino acid analyzer methods in analyzing 18 different amino acids in total parenteral nutrition injection(25)[J]. Chin J Pharm Anal, 2012, 32(6):1085
[16] 赵岩,侯莹莹,唐国胜,等.柱前衍生RP-HPLC测定淫羊藿中氨基酸含量[J].药物分析杂志, 2014, 34(8):1412 ZHAO Y,HOU YY, TANG GS,et al. Determination of amino acids in Epimedium brevicornu Maxim. by RP-HPLC[J]. Chin J Pharm Anal, 2014, 34(8):1412
[17] 潘葳,宋永康,黄星.自动凯氏定氮仪测定灵芝中蛋白质的不确定度评定[J].现代科学仪器, 2009(2):92 PAN W,SONG YK,HUANG X. Evaluation of uncertainty measuring protein content in Ganoderma with Kjeltec 2300 protein analyzer[J]. Mod Sci Instr,2009(2):92
[18] 程显隆,肖新月,邹秦文,等.柱前衍生化HPLC法同时测定阿胶中4种主要氨基酸的含量[J].药物分析杂志, 2008, 28(12):1997 CHENG XL,XIAO XY, ZOU QW,et al. Pre-column derivatization HPLC simultaneous determination of 4 main amino acids in donkeyhide glue[J]. Chin J Pharm Anal,2008, 28(12):1997
[19] 艾则孜·莫合买提,沙丽娜,巴哈尔古丽·黄尔汗,等.柱后衍生阳离子交换色谱法测定桦菌芝中氨基酸的含量[J].药物分析杂志, 2012, 32(11):1972 AZIZ MHMT, SHA LN, BAHARGULI HEh, et al. Determination of amino acids in Pyropolyporus fomentarius by post-column derivatization cation-exchange chromatography[J]. Chin J Pharm Anal, 2012, 32(11):1972