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A reversed-phase isocratic high-performance liquid chromatographic method is described for the simultaneous determination of EO9, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta- en-alpha-ol (I), and its ring-opened aziridine analogue EO5A (II), employing ultraviolet detection. Solid-phase sample extraction was used without addition of an internal standard. Plots of peak heights and areas of I and II were linear in the range 5-10,000 ng/ml. The lower limit of detection of both I and II in plasma was 2 ng/ml. The between-day variation of I was 13.9% at 5 ng/ml and lower than 6.2% for concentrations > or = 10 ng/ml. The between-day variation of II at 5 ng/ml was 13.8% and lower than 4.5% for concentrations > or = 10 ng/ml. The assay was developed to enable pharmacological guiding of a phase I study of I in solid tumour cancer patients.

Two isomeric urinary melanogens, 5-hydroxy-6-methoxyindole-2-carboxylic acid and 5-methoxy-6-hydroxyindole-2-carboxylic acid, have been separated by gas chromatography with selected-ion monitoring mass spectrometry. After chemical synthesis of one of these two isomers, 5-methoxy-6-hydroxyindole-2-carboxylic acid, and the establishment of the mass spectrum of its trimethylsilylated derivative, a 30-ml sample of a melanotic 24-h urine was adjusted to pH 1 and extracted twice with 10 ml of ethyl acetate. The extract was evaporated to dryness and the residue derivatized with methyl-8, followed by Tri-Sil/TBT. Silylated derivatives were analysed by gas chromatography with selected-ion monitoring mass spectrometry. The mass spectrum of the 5-methoxy-6-hydroxyindole-2-carboxylic acid allowed the determination of the retention times of both isomers.

A high-performance liquid chromatographic method for the determination of the novel indoloquinone antitumour agent E09, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta-e n-alpha - ol, in mouse plasma and urine is described. Following protein precipitation by means of methanol (2 volumes), separation and quantification of parent drug, metabolites and internal standard E012 (5-morpholine substituted analogue) were achieved on a 5-microns Resolve C18 Rad-Pak with a 15-min linear gradient of 10-30% acetonitrile in a 0.02 M pH 7.4 sodium phosphate buffer with UV detection at 280 and 310 nm. The utility of the assay is also demonstrated for the aziridine ring-opened analogue E05A. 3-hydroxymethyl-5-beta-hydroxyethylamino-2-(1H-indole-4,7-dione)pr op-beta-en- alpha-ol. Plots of area ratios of analytes versus internal standard were linear in the range 50-15,000 ng/ml. The detection limit for indoloquinones in plasma was ca. 30 ng/ml. The within-assay and day-to-day variation were consistently lower than 12.5%. The assay was applied in preliminary pharmacokinetic investigations. One minor metabolite of E09 could be identified; further metabolites were characterized by ultraviolet-visible spectra.

Latest strategies are discussed for the routine chromatographic analysis of clinically important indole derivatives in urine. Analysis of 5-hydroxyindoleacetic acid and, perhaps more importantly, serotonin and 5-hydroxytryptophan remains attractive in the screening for carcinoid tumours and their differentiation. Analyses of two precursors of the skin pigment eumelanin seem to be promising in the monitoring of treatment of malignant melanoma and screening for pigmentation disorders and gallstone formation. Studies on the clinical relevance of the determination of tetrahydro-beta-carbolines and melatonin-related indoles await routine application of chromatographic methods designed to take into consideration the relative instability of these compounds. Application of GC-MS, although confined to larger and/or governmental laboratories remains attractive as a way of improving the specificity of analyses and in establishing reference methods. As for HPLC, the recent development of chromatographic and detection methods for the concurrent determination of different clinically important and metabolically related compounds from the same sample, preferably by direct injection techniques, seems to be fruitful and should be continued.

A combination of thermospray liquid chromatography-mass spectrometry (LC-MS) and LC MS MS has allowed the structural elucidation of a number of metabolites of 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one (SK & F 101468) in monkey urine. By using LC-MS-MS with the third quadrupole (Q3) set up in multiple ion detection (MID) mode, a number of metabolites were subsequently detected in the human urine and plasma samples despite very low dosing regimes. This was achieved with minimal sample preparation, e.g. for the urine sample centrifugation was the only preparative step, in order to remove particulate matter, prior to analysis. The good signal-to-noise ratio obtained for the human samples, using LC MS MS with Q3 set up for MID, raised the possibility of a LC-MS-MS quantitative assay. As a result, the detection limit of this method for SK&F 101468 when dissolved in methanol was determined to be in the region of 20 pg on column.

Optimized operating conditions previously developed for the determination of neuroactive indoleamines and metabolites were adapted to meet the requirements of thermospray liquid chromatography-mass spectrometry (LC-MS) in terms of the ammonium acetate buffer system needed in this technique. Mass spectra were obtained for nineteen indolic compounds in both the positive and negative ion modes. The positive thermospray mass spectra of indoles with a free primary amino group are characterized by the base peak at [M + H]+, whereas the alcohol and acid metabolites show the base peak at [M + NH4]+. In the negative mode only amino acids and acids give good mass spectra with base peaks at [M - H + ACOOH]-. Detection limits by selected ion monitoring were of the order of 50-100 pg SIM on-column, allowing the direct determination of endogenous serotonin in an extract from rat hypothalamus. Quantitation was performed by isotope dilution MS. In the same way 5-hydroxyindoleacetic, indoleacetic, indolepropionic and indolelactic acids in urine were directly determined in an ethyl acetate extract from acidified urine samples. Likewise, gamma-aminobutyric acid and tricyclic antidepressants gave detection limits of 10 pg whereas only nanogram sensitivity could be achieved with catecholamines.

The isolation of two Thormählen-positive compounds from the urine of a patient with malignant melanoma and the elucidation of their structure by gas chromatography-mass spectometry is described. The compounds were isolated using a poly-N-vinylpyrrolidone column and separated by preparative thin-layer chromatography. After elution they were analyzed by gas chromatography and gas chromatography-mass spectrometry as their trimethylsilyl derivatives and after hydrolysis also as their tert.-butyldimethylsilyl derivatives. The results showed the main Thormählen-positive compound A to be the glucuronide of 5-hydroxy-6-methoxyindole, whereas the minor compound AX appeared to be the glucuronide of its isomer 6-hydroxy-5-methoxyindole.

Tryptophan and some of its indole metabolites were separated by thin-layer chromatography, stained with the Van Urk--Salkowski reagent, and quantitated by scanning densitometry. The application of this technique for the detection of the indoles in urine samples, employing Sep-Pak C18 cartridges for extraction, was demonstrated. The proposed method is simple and accurate. The detection limits were 2 micrograms/ml 5-hydroxytryptophan, 1.75 micrograms/ml 5-hydroxyindolyl-3-acetic acid, 1.5 micrograms/ml tryptophan, 0.8 micrograms/ml indolyl-3-acetic acid, 0.9 micrograms/ml indolyl-3-butyric acid, 1.75 micrograms/ml serotonin, and 1.25 micrograms/ml tryptamine.