ABSTRACT
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.
