Department of Environmental Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia.
Department of Environmental Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
J Trace Elem Med Biol. 2020 Jan;57:28-39. doi: 10.1016/j.jtemb.2019.09.011. Epub 2019 Sep 18.
Monolithic chromatography using convective interaction media (CIM) disks or columns can be used in the separation step of speciation analysis. When different monolithic disks are placed in one housing, forming conjoint liquid chromatography (CLC) monolithic column, two-dimensional separation is achieved in a single chromatographic run.
Here, we assembled low-pressure (maximum 50 bar) CLC monolithic column, which consists of two 0.34 mL shallow CIM monolithic disks and high-pressure CLC column (maximum 150 bar) from 0.1 mL analytical high performance short bed CIMac monolithic disks. Both the CLC columns constructed from affinity Protein G and weak anion exchange diethylamine (DEAE) disks, were applied for the speciation of cisplatin, oxaliplatin and carboplatin in spiked standard serum proteins, spiked human serum and serum of cancer patients. The analytical performances of the CLC columns used were evaluated by comparing their robustness, selectivity, repeatability and reproducibility. The separated serum proteins were detected on-line by ultraviolet (UV) and eluted Pt species by inductively coupled plasma mass spectrometry (ICP-MS). For accurate quantification of the separated Pt species (unbound Pt-based chemotherapeutic from species associated to transferrin (Tf), human serum albumin (HSA) and Immunoglobulin G (IgG)), post column isotope dilution (ID)-ICP-MS was used.
The data from analyses showed that both tested CLC monolithic columns gave statistically comparable results, with the low-pressure CLC column exhibiting better resolving power and robustness. It also enables more effective cleaning of monolithic disks and to analyse larger series of serum samples than the high-pressure CLC column. Analyses of serum samples of cancer patients treated with cisplatin or carboplatin showed that Pt-chemotherapeutics were bound preferentially to HSA (around 80%). The portion of unbound Pt in general did not exceed 2%, up to 5% of Pt was associated with Tf and approximately 20% with IgG. Column recoveries, calculated as a ratio between the sum of concentrations of Pt species eluted and concentration of total Pt in serum samples, were close to 100%.
Low-pressure CLC column exhibited greater potential than high-pressure CLC column, and can be thus recommended for its intended use in speciation analysis of metal-based biomolecules.
使用对流相互作用介质（CIM）磁盘或柱的整体式色谱可用于形态分析的分离步骤。当不同的整体式磁盘放置在一个外壳中，形成组合液相色谱（CLC）整体式柱时，可在单个色谱运行中实现二维分离。
在这里，我们组装了低压（最高 50 巴）CLC 整体式柱，它由两个 0.34 mL 浅 CIM 整体式磁盘和高压 CLC 柱（最高 150 巴）组成，由 0.1 mL 分析性高性能短床 CIMac 整体式磁盘组成。由亲和蛋白 G 和弱阴离子交换二乙胺（DEAE）磁盘构建的两个 CLC 柱均用于缀合标准血清蛋白、缀合人血清和癌症患者血清中顺铂、奥沙利铂和卡铂的形态分析。通过比较其稳健性、选择性、重复性和重现性来评估 CLC 柱的分析性能。通过紫外（UV）在线检测分离的血清蛋白，并通过电感耦合等离子体质谱（ICP-MS）洗脱洗脱的 Pt 物种。为了准确定量分离的 Pt 物种（与转铁蛋白（Tf）、人血清白蛋白（HSA）和免疫球蛋白 G（IgG）结合的游离基于 Pt 的化疗药物），使用柱后同位素稀释（ID）-ICP-MS。
分析数据表明，两种测试的 CLC 整体式柱给出了统计学上可比的结果，低压 CLC 柱表现出更好的分辨率和稳健性。它还能够更有效地清洁整体式磁盘，并比高压 CLC 柱分析更大系列的血清样本。对接受顺铂或卡铂治疗的癌症患者的血清样本进行分析表明，Pt 化疗药物优先与 HSA 结合（约 80%）。未结合的 Pt 部分通常不超过 2%，高达 5%的 Pt 与 Tf 结合，约 20%与 IgG 结合。柱回收率，计算为洗脱的 Pt 物种浓度之和与血清样品中总 Pt 浓度的比值，接近 100%。
低压 CLC 柱比高压 CLC 柱表现出更大的潜力，因此可推荐其用于金属基生物分子形态分析。
