Indicine-N-oxide was analyzed by gas chromatography mass spectrometry with nanogram sensitivity after trimethylsilylation. Two different products were produced by altering the conditions of this reaction. Mass spectral evidence is presented to show that one of these was the expected trisubstituted pyrrolizidine product while the other was a trisubstituted pyrrole. The latter derivative is useful for distinguishing between indicine-N-oxide and indicine which dies not form this novel product under the same conditions. Analogous pyrrole and pyrrolizidine products were formed from heliotrine-N-oxide, a compound that can serve as an internal standard from measuring indicine-N-oxide and its metabolites in biological samples. A method for purifying such samples by strong cation exchange chromatography prior to derivatization is also discussed.

Indicine-N-oxide was analyzed quantitatively in biological samples using a direct partial purification method involving acetonitrite precipitation or methanol precipitation followed by ion exchange chromatography. Trimethylsilyl derivatization of the resultant provided either of two derivatives, depending on the reaction conditions used, both of which had good gas chromatographic qualities. Heliotrine-N-oxide was used as the internal standard for this work. Data are presented to show that this is a reliable and useful internal standard based on its behavior in the partial purification method and on the gas chromatographic characteristics of its two derivatives. In addition, both low and high resolution mass spectral data indicate that heliotrine-N-oxide produces two trimethylsilyl derivatives analogous to those produced by indicine-N-oxide under the same conditions. Application of this procedure to urine and blood samples from cancer patients in clinical trials indicates that over 95% of the drug is removed from the circulation and excreted in the urine over the course of 48 h.

Methyl-G was analyzed by gas chromatography and mass spectrometry after trimethylsilylation. A means of quantitatively measuring methyl-G by gas chromatography of the TMS derivative is presented using the analogous derivative of methylethyl-G as an internal standard. The value of this compound as an internal standard for measurements of methyl-G is discussed based on comparisons of the mass spectral and gas chromatographic properties of their derivatives as well as their similar behavior in the ion exchange method used for partial purification. The latter procedure is discussed in some detail, and the results of applying it to biological samples of human and cell culture origin are presented.