Spectrophotometric and liquid-chromatographic studies of thymolphthalein monophosphate. Specifications for high-quality substrate for the measurement of prostatic acid phosphatase activity.
Abstract Fourteen lots of thymolphthalein monophosphate (TMP), disodium salt, obtained from 10 commercial suppliers were compared spectrophotometrically at 445 and 595 nm, liquid-chromatographically with monitoring at 254 nm, and enzymically by measurements of activity of prostatic acid phosphatase in human serum. Eight lots were classified as "unacceptable," six as "acceptable." Spectrophotometric testing revealed four lots with excessive thymolphthalein and three lots with grossly deficient amounts of TMP. In general, the chromatographic results paralleled those obtained by spectrophotometry, and both results correlated well with enzymic activity. Changing water content in this hygroscopic salt was a major problem, which resulted in great uncertainty as to the formula weight and therefore as to the moles of TMP actually taken. From these studies, specifications for high-quality TMP were determined. The critical importance of simultaneous enzymic activity measurements in comparisons with other "acceptable" lots in defining an adequate TMP substrate is stressed. Use of these specifications for selecting TMP for acid phosphatase activity measurements should improve intra- and inter-laboratory analytical performance.