Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.

Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples.

High-speed analysis of complex indoor VOC mixtures by vacuum-outlet GC with air carrier gas and programmable retention.

A pressure-tunable, series-coupled column ensemble was used with atmospheric pressure air as carrier gas for the vacuum-outlet GC analysis of 42 volatile and semivolatile organic compounds commonly encountered as indoor air pollutants. Separation strategies applicable to a field-portable instrument that will employ a dual-stage preconcentrator and a microsensor array as the detector were developed, where coelution of certain analytes can be tolerated. The capillary column ensemble consists of a 4.5-m segment of nonpolar dimethyl polysiloxane followed by a 7.5-m segment of polar trifluoropropylmethyl polysiloxane. Good long-term thermal stability of the column ensemble was observed for continuous operation in air at temperatures up to 210 degrees C. A computer-driven pressure controller at the column junction point is used to adjust vapor retention for specified sets of target compounds. The compounds were divided into two groups according to retention order, and high-speed analysis conditions were determined for the two groups individually as well as for the entire mixture. The earlier eluting group of 21 compounds was analyzed isothermally at 30 degrees C in about 160 s using a single, on-the-fly junction-point pressure change during the separation. The later eluting group of 21 compounds was analyzed in about 200 s with temperature programming and a constant (tuned) junction-point pressure. The entire mixture was analyzed in about 400 s using a two-step temperature program and a three-step pressure program, with minimal overlap in eluting peaks. Separations are adequate for analysis by a sensor array capable of discriminating among small groups of coeluting vapors on the basis of their response patterns.

Pressure-tunable column selectivity for high-speed vacuum-outlet GC.

A pressure-tunable ensemble of two series-coupled capillary columns operated at subambient outlet pressure is described. The ensemble consists of a 4.5-m length of nonpolar dimethyl polysiloxane column followed by a 7.5-m length of polar trifluoropropylmethyl polysiloxane column. Air at an inlet pressure of 1.0 atm is used as carrier gas, and a vacuum pump is used to pull the carrier gas and injected samples through the column ensemble. Detection is provided by a photoionization detector operated at a pressure of 0.3 psia. Ensemble selectivity is controlled by means of an electronic pressure controller located at the junction point between the columns. The minimum pressure step size is 0.1 psi, and 50 different set-point pressures can be used, each one producing a different pattern of peaks eluting from the column ensemble. Measured ensemble retention factors for a set of target compounds produce straight lines when plotted versus the ratio of the calculated holdup time of the first column in the ensemble to the total ensemble holdup time. A component band trajectory model is used to describe the effects of ensemble junction-point pressure on the elution patterns generated by the ensemble. Ensemble retention times predicted by the model are in good agreement with values obtained from chromatograms. The use of on-the-fly set-point pressure changes during a separation (selectivity programming) is demonstrated and used to improve the quality of the separation of a 19-component test mixture.

Column performance and stability for high-speed vacuum-outlet GC of volatile organic compounds using atmospheric pressure air as carrier gas.

The development of lightweight, portable GC instrumentation is handicapped by the need for compressed carrier gas to drive the separation. The use of air as carrier gas eliminates the need for compressed gas tanks. If a vacuum pump is used to pull carrier gas and injected samples through the column, atmospheric pressure air can be used as carrier gas. Vacuum outlet operation also improves performance for high-speed separations by reducing detector dead time and by shifting optimal carrier gas velocity to higher values. Under vacuum outlet conditions using atmospheric pressure air as carrier gas, a 6-m-long, 0.25-mm-i.d. capillary column can generate approximately 12,500 theoretical plates, and a 12-m-long column can generate approximately 44,000 plates but with a 3-4-fold increase in separation time. The principal issues in column selection for high-speed GC with air as a carrier gas are efficiency and stability. Several bonded and nonbonded stationary phases were evaluated for use with air as carrier gas in the analysis of volatile organic compounds of interest in airmonitoring applications. These include dimethylpolysiloxane, 50% phenyl-50% methyl polysiloxane, 50% cycanopropylphenyl-50% methyl polysiloxane, trifluoropropyl polysiloxane, poly(ethylene glycol), and dicyanoallyl polysiloxane (nonbonded). The dimethyl polysiloxane and the trifluoropropyl polysiloxane columns showed good efficiency and no significant deterioration after 5 days of continuous operation with air as carrier gas. The 50% phenyl-50% methyl polysiloxane and the 50% cycanopropylphenyl-50% methyl polysiloxane columns showed poorer efficiency, and the poly(ethylene glycol) and dicyanoallyl polysiloxane columns showed excessive deterioration in air.

Biodistribution of model 105Rh-labeled tetradentate thiamacrocycles in rats.

105Rh(III)Cl2 complexes with a limited series of [14]ane- and [16]ane- thia macrocycles were prepared and their biodistributions in Sprague-Dawley rats studied. These studies demonstrate that modifications in the structure and composition of the 105Rh-thia macrocycle complexes produce significant differences in their uptake and retention in both the liver and kidneys. The results indicate that the cis-Rh(III)Cl2-[14]ane thiamacrocycles exhibit less kidney retention than the corresponding trans-Rh(III)Cl2-[16]ane thiamacrocycles. In addition, the presence of a side chain containing a carboxylate group will produce decreased retention of activity in the kidneys. HPLC analysis of urine from these animals indicates no observable in vivo metabolism or dissociation of these chelates in the blood stream.