Chocholouš Petr, Vinklárek Jan, Semerádová Eva, Miekh Yuliia, Marshall Graham D, Solich Petr
Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic.
Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic.
Talanta. 2023 Jun 1;258:124420. doi: 10.1016/j.talanta.2023.124420. Epub 2023 Mar 7.
Reaction-based assays are commonly automated and miniaturized via flow analysis. However, aggressive reagents can affect or destroy even the chemically resistant manifold during long-term use. Using on-line solid-phase extraction (SPE) can eliminate this drawback and allow for high reproducibility and further advanced automation, as presented in this work. Determination of creatinine in human urine, an important clinical marker, by sequential injection analysis was achieved using bead injection on-line SPE with specific UV spectrophotometric detection, providing the necessary sensitivity and selectivity of the method for bioanalysis. The automated SPE column packing and disposal, calibration, and fast measurement highlighted the improvements in our approach. Variable sample volumes and a single working standard solution eliminated matrix effects, broadened the calibration range, and accelerated the quantification. Our method comprised an injection of 20 μL of 100 × times diluted urine with aqueous acetic acid solution pH 2.4, sorption of creatinine in a strong cation exchanger SPE column, washing out urine matrix with 50% aqueous acetonitrile, and elution of creatinine with 1% ammonium hydroxide. The SPE step was accelerated by a single flush of the column when the eluent/matrix wash/sample/standard zones sequence was created in the pump holding coil, and then the sequence of the zones was flushed into the column at once. The whole process was continually spectrophotometrically detected at 235 nm, subtracted from the signal at 270 nm. A single run duration was less than 3.5 min. Method relative standard deviation was <5.0% (n = 6). A calibration range was linear within the range of 0.02-0.30 μg creatinine (R > 0.999), covering 1.0-15.0 mmol/L creatinine in urine. The standard addition method used two different volumes of a single working standard solution for quantification. Results proved the effectiveness of our improvements in the flow manifold, bead injection, and automated quantification. The accuracy of our method was comparable to the routine enzymatic assay of real urine samples in a clinical laboratory.
基于反应的分析方法通常通过流动分析实现自动化和小型化。然而，腐蚀性试剂在长期使用过程中甚至会影响或破坏化学抗性流路。如本研究所示，使用在线固相萃取（SPE）可以消除这一缺点，并实现高重现性以及进一步的高级自动化。通过采用带特定紫外分光光度检测的珠进样在线SPE，利用顺序注射分析法测定了人体尿液中的肌酐（一种重要的临床标志物），为生物分析提供了该方法所需的灵敏度和选择性。自动化的SPE柱填充和处理、校准以及快速测量突出了我们方法的改进之处。可变的样品体积和单一工作标准溶液消除了基质效应，拓宽了校准范围，并加快了定量分析。我们的方法包括注入20 μL用pH 2.4的乙酸水溶液稀释100倍的尿液，在强阳离子交换剂SPE柱上吸附肌酐，用50%的乙腈水溶液冲洗掉尿液基质，并用1%的氢氧化铵洗脱肌酐。当在泵的保持盘管中创建洗脱液/基质冲洗液/样品/标准品区域序列时，通过单次冲洗柱子加速SPE步骤，然后将区域序列一次性冲洗到柱子中。整个过程在235 nm处持续进行分光光度检测，并从270 nm处的信号中扣除。单次运行时间少于3.5分钟。方法的相对标准偏差<5.0%（n = 6）。校准范围在0.02 - 0.30 μg肌酐范围内呈线性（R > 0.999），涵盖尿液中1.0 - 15.0 mmol/L的肌酐。标准加入法使用单一工作标准溶液的两种不同体积进行定量。结果证明了我们在流路、珠进样和自动定量方面改进的有效性。我们方法的准确性与临床实验室中实际尿液样品的常规酶法测定相当。
