1. Direct determination of free phenylacetic acid in human plasma and urine by column-switching high performance liquid chromatography with fluorescence detection
T Iwata, T Ishimaru, M Nakamura, M Yamaguchi Biomed Chromatogr. 1994 Nov-Dec;8(6):283-7. doi: 10.1002/bmc.1130080606.
A simple and highly sensitive column-switching high performance liquid chromatographic method with fluorescence detection for the determination of free phenylacetic acid (PAA) in human plasma and urine is described. The method is based on the direct derivatization of plasma and urine PAA with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide (DMEQ-hydrazide). The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide at 37 degrees C. The resulting DMEQ derivative of PAA is separated from endogenous interfering substances by a column-switching chromatographic system consisting of a precolumn (YMC-Pack C4) for sample clean-up and an analytical column (L-Column ODS) for the complete separation of the derivative. The derivative is detected fluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal to noise ratio = 3) for PAA is 10 fmol in a 10 microL injection volume. The recoveries from plasma and urine are 75 and 96%, respectively. The present method is highly sensitive and simple compared to conventional liquid-liquid extraction procedures. The sensitivity allows the direct determination of free PAA in an extremely small amount (5 microL) of plasma and urine.
2. Fluorimetric high-performance liquid chromatography of prostaglandins and its application to their determination in human seminal fluid
M Yamaguchi, K Fukuda, S Hara, M Nakamura, Y Ohkura J Chromatogr. 1986 Aug 2;380(2):257-65. doi: 10.1016/s0378-4347(00)83654-3.
A highly sensitive and simple high-performance liquid chromatographic method with fluorescence detection for the determination of prostaglandins is described. Prostaglandins are converted into the corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated simultaneously within 34 min (the total run time per injection, 56 min) on a reversed-phase column (YMC Pack C8) by a stepwise elution with mixtures of acetonitrile, methanol and water and detected fluorimetrically. The detection limits are 10-15 fmol at a signal-to-noise ratio of 5 in a 10-microliter injection volume. Prostaglandins E1, E2, F1 alpha and F2 alpha in human seminal fluids are measured by this method.
3. Highly sensitive determination of free fatty acids in human serum by high-performance liquid chromatography with fluorescence detection
M Yamaguchi, R Matsunaga, S Hara, M Nakamura, Y Ohkura J Chromatogr. 1986 Feb 14;375(1):27-35. doi: 10.1016/s0378-4347(00)83688-9.
A highly sensitive and rapid high-performance liquid chromatographic method for the determination of free fatty acids in human serum is described. The fatty acids are converted into the corresponding fluorescent derivatives by the reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium carbonate and 18-crown-6 in acetonitrile. The derivatives are separated simultaneously within 44 min on a reversed-phase column (YMC-Pack C8) with a gradient elution of aqueous methanol and detected fluorimetrically. The detection limits are 0.5-2 fmol in a 10-microliters injection volume. This sensitivity permits precise determination of free fatty acids including lauric, myristoleic and linolenic acids, which occur in serum at very low concentrations, in 5 microliters of sera from healthy subjects and patients with diabetes.
