A sensitive and accurate stable
isotope dilution assay was developed for the measurement of
pipecolic acid in body fluids using electron capture negative ion mass fragmentography. The method utilizes [2H11]
pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous
solution (pH 11.5) of the
amine moiety with
methyl chloroformate to the
N-methylcarbamate, followed by acidic
ethyl acetate extraction (pH 2) and further derivatization of the carboxyl moiety to the pentafluorobenzyl
ester. Normal values have been determined in cerebrospinal fluid (mean means = 0.041 mumol/l, range 0.010-0.120 mumol/l), in plasma of at term infants (age less than 1 wk, means = 5.73 mumol/l, range 3.75-10.8 mumol/l; age greater than 1 wk, means = 1.46 mumol/l, range 0.70-2.46 mumol/l), in urine of at term infants (age less than 6 mth, means = 32.5 mumol/g. creat., range 9.81-84.5 mumol/g. creat; age greater than 6 mth, means = 6.35 mumol/g. creat., range 0.15-13.6 mumol/g. creat.) and in amniotic fluid (means = 4.65 mumol/l, range 2.24-8.40 mumol/l). The utility of the method was demonstrated for the
pipecolic acid quantification in these biofluids of patients with
peroxisomal disorders. As affected fetuses with
infantile Refsum's disease and
Zellweger syndrome showed no significant elevation of
pipecolic acid in their surrounding amniotic fluids, the measurement of
pipecolic acid in amniotic fluid seemed not to be useful for prenatal diagnosis in these disorders.