A method for detection and quantification of hydroxyethyl starch in plasma

The use of hydroxyethylstarch (HES) is a controversial issue due to increasing evidence that HES accumulates in plasma and various tissues and therefore leads to unfavourable outcome in critically ill patients [1, 2]. No simple methods are available for monitoring HES plasma levels; present technologies to measure HES are based on gas chromatography-mass spectrometry, which are time-consuming and need advanced equipment [3]. Here, we applied Lugol's iodine solution (LUGOL; Sigma-Aldrich, Steinheim, Germany) to determinate HES concentrations in plasma and compare the results with those obtained by high-performance liquid chromatography (HPLC).

The study was approved by the institutional ethical committee (Friedrich-Schiller-University, Jena) as well as by the animal welfare committee (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz, Germany). Blood samples from healthy volunteers were diluted with balanced 6% HES 130/0.4 (Fresenius Kabi, Bad Homburg, Germany) to get concentrations up to 30 mg/ml. Samples were centrifuged (10 minutes, 4,700 × g, 4°C), and plasma aliquots were mixed with 10% trichloroacetic acid and re-centrifuged (5 minutes, 4,700 × g, 4°C). The supernatant was mixed (4:1) with LUGOL and optical density was measured at 530 nm. For in vivo experiments, 5 ml or 10 ml of 6% HES 130/0.4 was infused into wistar rats over 1 hour via a central venous catheter. Blood samples were obtained prior and up to 24 hours after infusion. Plasma samples were prepared as described. For HPLC analysis, plasma samples were diluted 1:10 with water, then mixed with 60% perchloric acid, heated at 90°C for 60 minutes, diluted again (1:50) and subjected to HPLC [4]. For correlation analysis the Pearson correlation coefficient was calculated.

Using the LUGOL method, we found a linear correlation (r² > 0.99) between calculated and measured plasma HES concentrations after serial dilutions of HES 130/0.4 in human whole blood (Figure 1a). At a concentration of 6 mg/ml the half life of HES 130/0.4 was estimated to be 1.8 hours (Figure 1b). In rats, plasma levels of 2.9 mg/ml and 4.6 mg/ml were measured immediately after infusion of 5 or 10 ml HES 130/0.4. Two hours after infusion the measured levels were only 10% of the initial levels (Figure 2). Comparing the plasma concentrations measured by LUGOL and HPLC revealed a correlation coefficient of r² > 0.90 (P < 0.001).

Figure 1

Quantification of HES by Lugol's iodine solution. (a) Measurement of HES concentration using LUGOL's iodine solution in samples of human whole blood incubated with different HES 130/0.4 concentrations. (b) Time course of HES concentration in human whole blood after incubating with HES 130/0.4 at a final concentration of 6 mg/ml.

Figure 2

Time course of HES plasma levels after infusion of 5 or 10 ml HES 130/0.4 solution over 1 hour. Significance was tested using ANOVA (*P < 0.05).

The new method described here allows the rapid and simple detection of HES in plasma samples. LUGOL is known to stain polysaccharides, including amylose, amylopectin and glycogen. Amylose and amylopectin, due to their herbal origin, should not be found in blood. However, glycogen might interfere with the measurements, but it is present in only negligible amounts in plasma [5].

Measurement of HES plasma levels in patients could help to gain new insights into HES degradation and plasma accumulation after infusion.