Protocol for liquid chromatography/mass spectrometry of glutathione conjugates using postcolumn solvent modification.

A novel protocol for thermospray liquid chromatography/mass spectrometry (LC/MS) analysis of mixtures of glutathione conjugates is reported. Solvent conditions for optimal high-performance liquid chromatography are not always the same as for optimal thermospray ionization mass spectrometry. Labile glutathione conjugates that give poor spectra in aqueous ammonium acetate yield more intense molecular ion signals with increased percentages of acetonitrile. Direct injection thermospray ionization using 30-60% acetonitrile in aqueous ammonium acetate produced protonated molecular ions for glutathione conjugates of menadione, styrene oxide, pentachlorophenyl methyl sulfone, chlorodinitrobenzene, and chlorambucil. Since, the high percentages of organic modifier needed for good molecular ion intensity preclude chromatographic separation of these polar compounds, successful graphic separation of these polar compounds, successful LC/MS was facilitated by postcolumn addition of organic modifiers to the mobile phase. This new methodology allowed excellent chromatographic separations and thermospray ionization mass spectra to be obtained for a mixture of haloalkane glutathione conjugates. Moreover, cleavage of the gamma-glutamyl-cysteine amide bond of glutathione results in class-characteristic fragment ions. Changes in the fragmentation pathways in spectra acquired with and without organic modifiers shed light on the importance of the desolvation process in obtaining good molecular ion sensitivity in thermospray.

Solvent-sample interactions in thermospray mass spectrometry of antineoplastic nitrogen mustards.

Conditions are reported for the optimization of thermospray mass spectrometric analysis of antineoplastic nitrogen mustard alkylating agents. In aqueous ammonium acetate mobile phase, multiple sequential solvolytic reactions occur with these highly labile compounds, and protonated molecular ions of the reaction products are observed. However, when high proportions of acetonitrile or other organic modifier are added to the mobile phase, solvolytic reactions are much reduced and abundant protonated molecular ions are detected. One exception to these observations is phosphoramide mustard, which solvolyses under all conditions attempted. A lower limit for detection of melphalan using direct injection and summing the ion current between m/z 120 and 870 is about 150 ng. Successful thermospray liquid chromatography/mass spectrometry of these compounds should be possible using high percentages of methanol in the mobile phase or increasing the organic content by post-column solvent modification.

Identification of phytoestrogens in the urine of male dogs.

It is becoming increasingly apparent that dietary factors may play a role in the etiology of hormone dependent neoplasias. It has been hypothesized that estrogens play some role in the etiology of benign prostatic hyperplasia (BPH) in the canine. The presence of estrogen receptor binding activity in a fraction of canine urine purified by high performance liquid chromatography (HPLC) that did not correspond to estriol, estradiol, estrone or any of their primary metabolites was observed in the present study. We used thermospray-mass spectrometry and GC-MS to identify the phytoestrogens daidzein, equol, formononetin and genistein in HPLC purified fractions of urine obtained from male beagles. Using the same techniques we also confirmed the presence of daidzein and genistein in the commercial diet fed to these same dogs. Using the immature rat uterine cytosol estrogen receptor assay, relative binding affinities of 0.08, 1.1, less than 0.01 and 3.9% were obtained for daidzein, equol, formononetin and genistein, respectively when compared to estradiol (100%). In conclusion, phytoestrogens are present in urine of male beagles. Moreover, the commercial diet fed to these dogs contains isoflavones which can be converted to equol by intestinal microflora. These results suggest the need for investigations of phytoestrogens (e.g. equol) excreted into the urine daily and its relationship to the incidence and severity of BPH in the dog.