After the bile was treated with 8-glucuronidase, additional peaks were observed on the gas chromatogram (Fig. 1). The major peak has the same retention time after enzymatic cleavage as does the parent probenecid (Fig. 1).
Further evidence was obtained that this peak was probenecid by the combined GC-mass spectrometry described previously. To establish firmly the masses of the prominent ions in the mass spectrum of the probenecid methyl ester, a high resolution mass spectrum was obtained (Table 111) on the standard sample introduced into the mass spectrometer in the direct probe. The mass spectra of probenecid and that obtained by enzymatic hydrolysis of the glucuronide conjugate were determined by combined GC-mass spectrometry, and both spectra were identical (Fig. 3). The computer printout of probenecid spectrum was identical to that reported by Guarino et af. (6). Thus, no attempt will be made to identify frao tions or fragmentation pattern.
Therefore, from this study, it would appear that the major metabolic product of probenecid in the rat is the simple acyl glucuronide. The authors do not feel that this is the only metabolite of probenecid, but it is the major metabolite because it is the only easily detectable peak observed by GLC when the probenecid peak remains on scale. However, when a larger quantity of metabolites is placed on the column so that the probenecid peak goes off scale, additional peaks are observed. By examining the data reported by Guarino er a[. (6), it would appear that their results are similar but that they inadvertently disregarded their major metabolite, the metabolite that was in such great quantity that it went off scale and whose retention time was identical to the simple acyl glucuronide of probenecid. Therefore, it would appear that the major metabolic product of probenecid found in rat bile is the simple acyl glucuronide of probenecid.
CONCLUSION
The GC method described is accurate, sensitive, and specific for the determination of probenecid. The method is applicable to bio-
D R U G S T A N D A R D S
logical fluids such as blood, bile, and urine, using only very small amounts of these fluids.
The major metabolic product of probenecid was identified to be the simple acyl glucuronide conjugate. Conclusive evidence was obtained from the retention time and mass spectrometry following enzymatic hydrolysis. The mass spectra are simple and can be described mainly in terms of bond fission.