Investigations of modified Edman reagents and modified anabolic steroids for the electrospray mass spectrometric detection

Electrospray mass spectrometry (ESI/MS) has become a widely used technique owing to its excellent sensitivity and capacity to mass analyze high-molecular weight compounds due to multiply charging. This technique has gained wide acceptance in biological mass spectrometry due to the ease of coupling with liquid chromatographic systems. In this study, an electrospray source was designed and fabricated for the HP 5985 single quadrupole mass spectrometer. An interface between the mass spectrometer and a personal computer has been developed.; The molecules containing pre-formed charged groups or basic functionalities greatly improve the ESI/MS response. The phenylthiohydantoin amino acids in the Edman degradation methodology are identified solely on the retention time on the chromatogram after the separation by high performance liquid chromatography followed by UV detection. New Edman reagents containing a basic pyridyl group were introduced to improve the detectability and the reliability of thiohydantoin derivatives using ESI/MS.; The low level detection of anabolic steroids is difficult due to the extensive fragmentations under the conventional gas chromatography-mass spectrometric techniques. Anabolic ketosteroids were modified to form hydrazone derivatives or oxime derivatives with Girard's T reagent or hydroxylamine hydrochloride, respectively. The modifications improve the detectability into the femtogram level using electrospray mass spectrometry. Tandem mass spectral studies showed that a loss of 59 Daltons was common to all derivatives. A selective method to detect the hydrazone derivatives was developed using the neutral loss scanning mode of a triple quadrupole mass spectrometer. Feasibility studies based on analysis of stanozolol using electrospray mass spectrometry combined with the neutral loss scanning mode or liquid chromatography showed that this technology is suitable for detecting stanozolol in urine samples.