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The chiral separation and simultaneous determination of D- and L-pantothenic acids and D- and L-hopantenic acids in rat plasma using gas chromatography-mass fragmentography are described. The method is based on deproteinization by ion-exchange resin, extraction with ethyl acetate under acidic conditions, and derivatization to form several interesting compounds. After methyl esterification for carboxylic acid of D- and L-pantothenic acids, D- and L-hopantenic acids could be derivatized to trifluoroacetate, cyclic sulphite, and cyclic n-butylboronate for hydroxy groups. D- and L-forms of these derivatives were completely separated by mass fragmentographic technique with quasi-molecular ions. Calcium salt of D-5-[(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)amino]pentanoic acid was used as an internal standard for the determination of DL-pantothenic acids and DL-hopantenic acids. The detection limits of DL-pantothenic acids and DL-hopantenic acids in this method were 5 and 12 ng, respectively. This method could be applied to the study of plasma levels of D-, L-pantothenic acids and D-, L-hopantenic acids in rat.