Quantitative determination of glufosinate in biological samples by liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization.
We have established a new HPLC method for derivatizing and quantifying glufosinate (GLUF) in human serum and urine using p-nitrobenzoyl chloride (PNBC). The p-nitrobenzoyl derivative of GLUF (PNB-GLUF) was produced quantitatively over 10 min at room temperature. PNB-GLUF possesses the property of ultraviolet (UV) light absorption with a lambda(max) of 272.8 nm, and was isolated from biological specimens by reversed-phase chromatography using Inertsil Ph-3. In experiments at a UV wavelength of 273 nm, GLUF has a quantitative detection limit of 0.005 microg/ml, and when it was added to both serum and urine to yield concentrations of 0.1-1000 microg/ml, its recovery rate was quite satisfactory: at least 93.8% in all cases. Further, the measured amounts of GLUF in 23 serum samples from patients intoxicated by ingestion of GLUF compared favorably with those obtained by fluorescence derivatization-HPLC using 9-fluorenylmethyl chloroformate (R=0.998). This technique of analysis is, in addition, applicable for Glyphosat, which possesses a chemical structure resembling that of GLUF, and it will be of great use in the determination of these two compounds.
Solid-phase extraction of phosphorous-containing amino acid herbicides from biological specimens with a zirconia-coated silica cartridge.
We report a rapid solid-phase extraction method for glyphosate (Glyp), glufosinate (Gluf), and bialaphos (Bial) using a zirconia-coated silica cartridge, which interacts specifically with phosphorous-containing amino acid herbicides (PAAHs). We extracted PAAHs from serum and urine samples. The PAAHs were derivatized with trimethyl orthoacetate-acetic acid and analyzed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). The intra-day and inter-day accuracy was within ±13% RE, the intra-day and inter-day precision was less than 12% RSD, and the total recovery was more than 60% for Glyp and more than 80% for Gluf and Bial. The linearity ranges of the calibration curves of the serum samples were 0.2-10,000μg/mL for Glyp, 0.1-1000μg/L for Gluf, and 0.5-1000μg/L for Bial; and those of the urine samples were 0.4-20,000μg/L for Glyp, 0.2-2000μg/L for Gluf, and 0.1-2000μg/L for Bial. This range covers almost all the reported poisoning cases involving these compounds, from very mild to fatal cases. The present paper offers a universal cleanup method for PAAHs in serum and urine samples for clinical and forensic analysis.
Equilibrium dialysis for determination of protein binding or imipramine--evaluation of a method.
A range of methodological problems associated with equilibrium dialysis for serum or plasma protein binding assay of a weakly basic drug imipramine, in man has been carried out in order to establish a reliable procedure for large scale studies. Freezing of serum samples did not change the binding significantly, but a slight tendency towards increased binding to samples stored for 30 days was found. Protein binding was significantly higher in serum than in heparinized or citrated plasma, whereas there was no significant difference between plasma obtained by use of heparin or citrate as anticoagulant. There was a linear correlation between temperature (25-42 degrees) and binding such that binding decreased 0.1% per centigrade increase in temperature. Increase in pH resulted in increase in binding (0.6% per 0.1 unit pH). Equilibrium between serum and buffer phase was obtained within 5-6 hrs. The changes following osmotic equilibrium necessitated volume corrections of about 4-5% after 5-6 hrs, and 20-25% after 16-20 hrs (overnight experiments). With this correction the free fraction estimate remained constant for 26 hrs. Application of small volumes in the dialysis chamber may make the volume correction difficult and inaccurate. The use of Tris-Ringer buffer gave free fraction values about 20% higher (absolute values of free fraction) than that with phosphate buffer. With different imipramine concentrations a constant degree of binding was found at "therapeutic" levels, whereas a significantly decreased binding was found at a concentration of 1000 ng/ml. Control of these methodological factors is a prerequisite for obtaining reproducible and valid results, and authors should give more details on methodology in this field.