Liquid-chromatographic measurement of biopterin and neopterin in serum and urine

Abstract We report an improved "high-performance" liquid-chromatographic (HPLC) method for measuring biopterin and neopterin in serum and urine. Specimens are acidified, treated with iodine in 0.2 mol/L trichloroacetic acid, party purified on Bio-Rad MP-50 cation-exchange columns, and analyzed by reversed-phase HPLC with fluorometric detection. The minimal concentration of biopterin detectable is 0.3 micrograms/L in a 50-microL injection. The total CV is less than or equal to 10%. Improvements over other reported methods include the use of a single, simplified sample-preparation step with a Baker-10 SPE System, and a guard column to increase analytical column stability and analyte recovery. The assay is semiautomated to reduce technician time and improve precision. Mean observed values for biopterin and neopterin in sera of normal human adults were 1.64 and 5.52 micrograms/L, respectively. The mean ratio of neopterin to biopterin in acidified adult urine samples was lower than that found in matched nonacidified samples (n = 10). Serum specimens from diagnosed phenylketonuric (PKU) and hyperphenylalaninemic patients were also analyzed for biopterin and neopterin; the findings agreed with reported values for similar patients. One patient, previously identified as an atypical PKU patient, showed serum values of neopterin and biopterin suggestive of a defect in biopterin synthesis.