High pressure liquid chromatography purification of human urinary samples for thyrotropin-releasing hormone radioimmunoassay.

Urine samples from healthy adult subjects and patients who had received TRH orally were prepurified in SP-Sephadex-C-25 cation exchange chromatography, subjected to reverse phase high pressure liquid chromatography (HPLC) starting with 0.01 M ammonium acetate, pH 4, followed by 1%/min gradient of acetonitrile or isopropanol and assayed in a TRH RIA. Two TRH immunoreactive peaks (A and B) were detected by HPLC with an RP-8 column, peak A eluting at 4-10 min and B at 12-14 min. Serial dilutions of peak B produced a line parallel with synthetic TRH by RIA. Synthetic TRH added to urinary samples and urinary TRH immunoreactivity from TRH-treated patients were eluted at 12-14 min. These results suggest that peak B represents endogenous urinary TRH. Urinary TRH levels of eight normal human males and four females were 5.0 +/- 3.4 pg/ml and 5.2 +/- 1.2 pg/ml (mean +/- SD). These values are many times lower than those presented in previous studies.