ABSTRACT
Non-esterified fatty acids (NEFA) from C12 to C24 are assayed in human serum or plasma in a four-step procedure: extraction, volume reduction, methylation and gas chromatography. NEFA are extracted with chloroform--heptane--methanol from 50--100 microliter of serum or plasma buffered with phosphate. After adding ethyl acetate the volume of the extract is reduced under partial reflux to 5--7 microliter. Potassium carbonate, methyl iodide and a crown ether are added to the dry concentrate and the NEFA are selectively methylated with a yield of 100% by heating in a microrefluxer for 10 min. Gas chromatography is carried out with 1 microliter of the reaction mixture on a packed column by temperature-programmed operation. Thirteen individual fatty acids are determined in sera of normal adults. The coefficients of variation for 24 determinations of a pooled serum were 2.7% for the total NEFA content and 3--10% for most of the individual NEFA.
Subject(s)
Chromatography, Gas/methods , Fatty Acids, Nonesterified/blood , Butylated Hydroxytoluene , Evaluation Studies as Topic , Humans , Methylation , MicrochemistryABSTRACT
A method for the determination of therapeutic levels of barbituric acids in 25 microliter of whole blood is described. After extraction and controlled concentration of the extract to a volume of 5 microliter, the barbituric acids are N,N'-dimethylated using a microrefluxer. Of the total extract 20-100% is injected into the gas chromatograph. Low blanks, recoveries of 70--80% and peak ratios that are comparable to those in calibration experiments are obtained provided the detailed working instructions are followed strictly. In addition, barbiturates were determined (1 ng in 25 microliter blood) using column-switching devices and nitrogen-sensitive detection.
