Establishment of detection method for simultaneous determination of plasma concentrations of paraquat and diquat by high performance liquid chromatography

Abstract

Objective To establish a detection method for simultaneous determination of plasma concentrations of paraquat and diquat by high performance liquid chromatography (HPLC). Methods All the plasma samples were pretreated using 35% perchloric acid and microporous filtering film. The chromatographic peaks of paraquat and diquat were measured by high performance liquid chromatograph (Waters 2695). The mobile phase was acetonitrile∶ buffer (6∶ 94). The buffer contained 20 mmol/L reversed-phase ion pair sodium 1-heptane sulfonate and 1.5% phosphoric acid. The buffer′s pH was adjusted to 2.0 with triethylamine. The column temperature was 40 ℃. The flow speed was 0.8 ml/min. The detection wavelengths of paraquat and diquat were 254 nm and 309 nm. The plasma concentrations of paraquat and diquat were calculated according to the chromatographic peak area of paraquat and diquat and the standard curves were run on the same day. Results The paraquat and diquat chromatographic peaks detected by reversed phase ion-pair liquid chromatography showed good shape. The retention times of paraquat and diquat were 4.652 and 5.066 min, respectively. Peaks of paraquat and diquat were separated well and not disturbed by impurity peaks. Paraquat had a good linear relationship between the chromatographic peak area and the concentrations in the range of 0.097 7-50.000 0 μg/ml, the lowest detection concentration was 0.090 0 μg/ml. Diquat had a good linear relationship between the chromatographic peak area and the concentrations in the range of 0.097 7-50.000 0 μg/ml, the lowest detection concentration was 0.070 0 μg/ml. The absolute recovery and relative recovery of paraquat at high, medium and low concentrations were 98.75% to 103.86% and 102.29% to 107.26%, respectively. The absolute recoveries and relative recoveries of diquat at high, medium and low concentrations were 99.67% to 111.55% and 100.85% to 101.94%, respectively. Their relative standard deviations (RSD) were all <3%. The mean values of within-day precision in high, medium, and low concentrations of paraquat were 51.030 0, 6.429 6, and 0.767 0 μg/ml, respectively. The mean values of within-day precision in high, medium, and low concentrations of diquat were 50.178 6, 6.509 1, and 0.829 4 μg/ml, respectively. The RSD of within-day precisions of paraquat and diquat were 0.40%-3.22%. The mean values of day to day precision in high, medium, and low concentrations of paraquat were 52.707 5, 6.442 5 and 0.767 5 μg/ml, respectively. The mean values of day to day precision in high, medium, and low concentrations of diquat were 51.650 1, 6.519 2 and, 0.829 3 μg/ml, respectively. The day to day precisions (RSD) of paraquat and diquat were 0.20%-2.49%. The detection results of the standard plasma samples in high, medium and low concentrations which were repeatedly frozen and thawed (1 and 2 times) at 70 ℃, preserved at -20 ℃ at different time (3 and 7 days), lied at room temperature at different time (8 and 24 hours), lied at 4 ℃ at different time (1 and 2 days), and the standard plasma samples which were not treated with above-mentioned measures showed that all RSDs were <10%. Conclusions A detection method for simultaneous determination of plasma concentrations of paraquat and diquat by reversed-phase ion-pair liquid chromatography was established. The method is fast and accurate. Key words: Paraquat; Diquat; Drug monitoring; Chromatography, liquid