Developing a trace level GC-MS method for detecting methylhydrazine in an experimental drug substance.

Methylhydrazine (NH(2)NHCH(3), CAS 60-34-4) is a highly reactive reducing agent used as an intermediate for synthesizing an experimental drug substance. Methylhydrazine is a known mutagen, an animal carcinogen, and a suspected human carcinogen. A gas chromatography-mass spectrometry method was developed as a limit test method for analyzing trace levels of methylhydrazine in the experimental drug substance. The method utilizes acetone as a dissolving solvent for the drug substance and a derivatizing agent for methylhydrazine in the meantime, thus eliminating the need for post-derivatization sample clean-up prior to analysis. The gas chromatographic (direct injection) conditions provide good separation for the acetone-methylhydrazine derivative (acetone methylhydrazone) from matrix interference, and mass spectrometric detection (selected ion monitoring mode, m/z 86) allows sufficient sensitivity for detecting 1 part per million methylhydrazine relative to the drug substance.

Influence of the hofmeister series on the retention of amines in reversed-phase liquid chromatography.

The chromatographic behavior of protonated amines in reversed-phase liquid chromatography (RPLC) is influenced markedly by the identity of the mobile-phase anion. For example, retention factor values, k, obtained from protonated nordoxepin, nortriptyline, and amitriptyline increase almost 1 order of magnitude across the following series of anions employed as mobile-phase modifiers: H2PO4- < HCOO- < CH3SO3- < Cl- < NO3- < CF3COO- < BF4- < ClO4- < PF6-. Early eluting primary, secondary, and tertiary benzylamines are retained and resolved using BF4-, ClO4-, and PF6- but elute in or very near the void using all other mobile-phase anions tested. In contrast, a neutral hydrophobic marker, acenaphthene, shows no significant changes in retention with mobile-phase anion identity. Such large differences in amine retention with anion identity can be rationalized via both an ion-pairing model and the Hofmeister effect. Two key findings are reported. First, the dependence of amine retention on mobile-phase anion identity is attributed unambiguously to the Hofmeister effect and is quantified using a simple equation based solely on differences in the solvation of anions. Accurate prediction of k values from the excess chemical potential of anions in water suggests that anion-solvent interactions dominate the retention of amines in RPLC. Thus, controlling amine retention depends critically on judicious selection of mobile-phase anion (in addition to the usual experimental parameters such as organic modifier, temperature, pH, and stationary phase). Second, more lipophilic molecular anions can provide retention and tailing properties comparable to those obtained from traditional amphiphilic ion-pairing reagents such as octanesulfonate, but with the benefit of a superior gradient background and solubility at high concentrations of organic modifier.