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Monolithic chromatography on conjoint liquid chromatography columns for speciation of platinum-based chemotherapeutics in serum of cancer patients.
Journal of Trace Elements in Medicine and Biology ( IF 3.6 ) Pub Date : 2019-09-18 , DOI: 10.1016/j.jtemb.2019.09.011 Katarina Marković 1 , Radmila Milačič 1 , Janja Vidmar 2 , Stefan Marković 1 , Katja Uršič 3 , Martina Nikšić Žakelj 3 , Maja Cemazar 3 , Gregor Sersa 3 , Mojca Unk 4 , Janez Ščančar 1
Journal of Trace Elements in Medicine and Biology ( IF 3.6 ) Pub Date : 2019-09-18 , DOI: 10.1016/j.jtemb.2019.09.011 Katarina Marković 1 , Radmila Milačič 1 , Janja Vidmar 2 , Stefan Marković 1 , Katja Uršič 3 , Martina Nikšić Žakelj 3 , Maja Cemazar 3 , Gregor Sersa 3 , Mojca Unk 4 , Janez Ščančar 1
Affiliation
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BACKGROUND
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.
METHODS
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.
RESULTS
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%.
CONCLUSIONS
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.
中文翻译：
联合液相色谱柱上的整体色谱，用于癌症患者血清中铂基化学治疗剂的形态分析。
背景技术使用对流相互作用介质（CIM）盘或柱的整体色谱法可用于形态分析的分离步骤。当将不同的整体式磁盘放置在一个外壳中，形成联合液相色谱（CLC）整体式色谱柱时，在一次色谱操作中即可实现二维分离。方法在这里，我们组装了低压（最大50 bar）的CLC整体色谱柱，该色谱柱由两个0.34 mL浅CIM整体板和0.1 mL高性能分析型短床CIMac整体板的高压CLC色谱柱（最大150 bar）组成。由亲和蛋白G和弱阴离子交换二乙胺（DEAE）盘构建的CLC色谱柱均用于加标标准血清蛋白中的顺铂，奥沙利铂和卡铂的形态分析，加标的人血清和癌症患者的血清。通过比较它们的耐用性，选择性，可重复性和可重复性，对所用CLC色谱柱的分析性能进行了评估。分离的血清蛋白通过紫外线（UV）在线检测，并通过电感耦合等离子体质谱（ICP-MS）洗脱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）相关的物种的未结合的基于Pt的化学疗法），使用了柱后同位素稀释（ID）-ICP-MS 。结果分析数据表明，两种测试的CLC整体柱均提供了统计上可比的结果，而低压CLC柱表现出更好的分离能力和耐用性。与高压CLC色谱柱相比，它还可以更有效地清洗整体式磁盘并分析更大系列的血清样品。对接受顺铂或卡铂治疗的癌症患者的血清样本的分析表明，Pt化疗药物优先结合HSA（约80％）。未结合的Pt的比例一般不超过2％，最高5％的Pt与Tf相关，约20％与IgG相关。色谱柱回收率接近100％，该回收率以洗脱的Pt物种总浓度与总Pt浓度之比计算。结论低压CLC色谱柱比高压CLC色谱柱具有更大的潜力，因此可推荐用于金属基生物分子的形态分析。
更新日期：2019-09-18
中文翻译：
联合液相色谱柱上的整体色谱，用于癌症患者血清中铂基化学治疗剂的形态分析。
背景技术使用对流相互作用介质（CIM）盘或柱的整体色谱法可用于形态分析的分离步骤。当将不同的整体式磁盘放置在一个外壳中，形成联合液相色谱（CLC）整体式色谱柱时，在一次色谱操作中即可实现二维分离。方法在这里，我们组装了低压（最大50 bar）的CLC整体色谱柱，该色谱柱由两个0.34 mL浅CIM整体板和0.1 mL高性能分析型短床CIMac整体板的高压CLC色谱柱（最大150 bar）组成。由亲和蛋白G和弱阴离子交换二乙胺（DEAE）盘构建的CLC色谱柱均用于加标标准血清蛋白中的顺铂，奥沙利铂和卡铂的形态分析，加标的人血清和癌症患者的血清。通过比较它们的耐用性，选择性，可重复性和可重复性，对所用CLC色谱柱的分析性能进行了评估。分离的血清蛋白通过紫外线（UV）在线检测，并通过电感耦合等离子体质谱（ICP-MS）洗脱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）相关的物种的未结合的基于Pt的化学疗法），使用了柱后同位素稀释（ID）-ICP-MS 。结果分析数据表明，两种测试的CLC整体柱均提供了统计上可比的结果，而低压CLC柱表现出更好的分离能力和耐用性。与高压CLC色谱柱相比，它还可以更有效地清洗整体式磁盘并分析更大系列的血清样品。对接受顺铂或卡铂治疗的癌症患者的血清样本的分析表明，Pt化疗药物优先结合HSA（约80％）。未结合的Pt的比例一般不超过2％，最高5％的Pt与Tf相关，约20％与IgG相关。色谱柱回收率接近100％，该回收率以洗脱的Pt物种总浓度与总Pt浓度之比计算。结论低压CLC色谱柱比高压CLC色谱柱具有更大的潜力，因此可推荐用于金属基生物分子的形态分析。
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