High-pressure liquid chromatographic assay for the determination of thyrotropin-releasing hormone and its common metabolites in a physiological salt solution circulated through the isolated perfused rat lung.

A specific and sensitive assay for TRH (L-pyroglutamyl-L-histidyl-L-proline amide), 3H-TRH (L-proline 3,4-3H(N), histidyl-3-3H(N)), and four possible metabolites of TRH, present in the recirculated perfusate of an isolated perfused rat lung preparation, was developed. Unlike previous methods, the method developed does not require extraction of the analytes from the biological matrix. The crude sample was adjusted to a pH of 3.2 with concentrated trifluoroacetic acid and injected on to a PRP-1 (polystyrene divinylbenzene) column (10 microns, 25 cm x 4.6 mm i.d.). The mobile phase was 10% v/v acetonitrile and 90% v/v 0.75 g l-1 1-hepantanesulfonic acid in 0.004 M trifluoroacetic acid, adjusted to a pH of 2.4 with concentrated NaOH. The flow rate was 0.5 ml min-1 and the analytes were detected by UV absorption at a wavelength of 26 nm and by radiochemical detection utilizing a liquid scintillation counter. The nominal retention times for L-PRO, L-PRO-NH2, TRH, cyclo(HIS-PRO) and TRH-OH were 4.0 +/- 0.9, 10.0 +/- 0.2, 15.5 +/- 0.4, 19.2 +/- 0.5 and 25.3 +/- 0.5 min respectively. The assay performs well in terms of precision and accuracy as indicated by linear regression and intra-assay variability analysis.