Direct high-performance liquid chromatography method for determination of glycine betaine and its metabolite, N,N-dimethylglycine, in pharmacokinetic studies

A simple and direct, without derivatization, method for the routine analysis of glycine betaine and its metabolite N,N-dimethylglycine in body fluids, serum or urine, is described. Low wavelength ultraviolet detection is achieved at 198 nm using 20 mmol/L KH2PO4 at a flow rate of 1.2 mL/min as a mobile phase and a Nucleosil 100-10 SA (10 mum, 250 x 4.6 mm) as an analytical column. The limit of detection is 7.5 ng for both compounds and the limit of quantitation is 25 ng when 50 muL is injected onto the column. A rectilinear relationship is observed up to 120 ng/muL. Method validation performed by day-to-day (n = 8) and within-day (n = 8) precision assays gave relative standard deviation values in the range of 5.1-10.5% for glycine betaine and 3.6-9.0% for N,N-dimethylglycine. 7-Methylxanthine is used as the internal standard at a concentration of 0.2 ng/muL. Analysis time is approximately 6 min. The method developed is applied to the analysis of glycine betaine in a commercial pharmaceutical preparation with high accuracy and precision results, as well as to the monitoring of glycine betaine and its metabolite N,N-dimethylglycine levels in body fluids, serum and urine, as their levels are indicative of renal disorder. Mean recovery of glycine betaine in the pharmaceutical formulation, assayed at different concentration levels, is 102.4% Percentage recovery values of analytes in spiked serum samples range from 90.9 to 106.6% for N,N-dimethylglycine and from 95.6 to 100.3% for glycine betaine, while the respective values in spiked urine samples are in the range of 93.6-106.2% and 100.9-103.9%. After the appropriate dilution of biological fluid samples, any matrix interference is eliminated. The method appears to be suitable for monitoring glycine betaine and its metabolite in body fluid, especially in urine of patients undergoing therapy with betaine.