建立了移液枪头式固相微萃取（ SPME）?高效液相色谱检测细胞培养液中4种生物碱（黄柏碱、药根碱、巴马丁和小檗碱）的分析方法。以甲基丙烯酸为反应单体、乙二醇二甲基丙烯酸酯为交联剂、偶氮二异丁腈为引发剂，在移液枪头内进行原位聚合反应，制备含有弱阳离子交换整体固定相的SPME枪头。样品经SPME净化后，用高效液相色谱?紫外检测法进行分析，流动相为乙腈?磷酸二氢钾溶液（0?05 mol／L， pH 4），紫外检测波长为270 nm，外标法定量。4种生物碱的检出限（ S／N＝3）为0?16～0?39μg／L；以空白细胞培养液进行加标回收率试验，加标回收率为92?73％～97?91％，相对标准偏差（ RSD）为0?14％～3?31％。该方法简单、灵敏、准确，能够用于细胞培养液中微量生物碱的分析研究。
A method for the determination of four alkaloids ( phellodendrine, jatrorrhizine, pal?matine, berberine) in cell culture fluids was developed by pipette tip solid phase microextrac?tion (SPME) and high performance liquid chromatography (HPLC). A weak cation exchange monolithic stationary phase was prepared by in?situ polymerization in a pipette tip. Methacrylate acid, ethylene dimethacrylate and azodiisobutyronitrile were the functional monomer, the cross?linker and the initiator, respectively. The samples were cleaned?up using the microextrac?tion tips. Phellodendrine, jatrorrhizine, palmatine and berberine were extracted from cell cul?ture fluids. The analytes were separated on a reversed?phase Diamonsil C18 column (150 mm× 4?6 mm, 5 μm) and eluted gradiently with acetonitrile and 0?05 mol/L potassium dihydrogen phosphate solution. The detection wavelength was 270 nm. The extraction performance was investigated by varying the elution solution types, the extraction and elution time and the molar ratios among monomers, cross?linkers and porogens. The analytes were quantified using exter?nal standard method. The established method was successfully applied for the pretreatment and determination of four alkaloids from Cortex Phellodendri in cell culture fluids. Under the optimal conditions, the limits of detection ( S/N=3) for the analytes were 0?16-0?39μg/L. The recov?eries were in the range of 92?73%-97?91% and the relative standard deviations ( RSDs ) were ranged from 0?14% to 3?31% ( n=6) . The method is simple, sensitive and accurate. It can be used in the determination of alkaloids in cell culture fluids.
参考文献
| [1] | Wei, Z.;Sandron, S.;Townsend, A. T.;Nesterenko, P. N.;Paull, B..Determination of trace labile copper in environmental waters by magnetic nanoparticle solid phase extraction and high-performance chelation ion chromatography[J].Talanta: The International Journal of Pure and Applied Analytical Chemistry,2015:155-162. |
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