LC/MS/MS determination of omapatrilat, a sulfhydryl-containing vasopeptidase inhibitor, and its sulfhydryl- and thioether-containing metabolites in human plasma.

Omapatrilat, the most clinically advanced member of a new class of cardiovascular agents, vasopeptidase inhibitors, is under development at Bristol-Myers Squibb Pharmaceutical Research Institute for the treatment of hypertension and heart failure. An electrospray LC/MS/MS method has been developed and validated for the simultaneous determination of omapatrilat and its four metabolites in human plasma. Since omapatrilat and two of the metabolites are sulfhydryl-containing compounds, methyl acrylate was used to stabilize these compounds in human blood and plasma samples. Methyl acrylate reacted instantly with the sulfhydryl group to form a derivative that was stable in blood and plasma. Extraction of the analytes from plasma samples was achieved by semiautomated liquid-liquid extraction, where a robotic liquid handler performed the liquid-transferring steps. The mass spectrometer was operated in the negative ion selected-reaction-monitoring mode. The calibration curve ranges were 0.5-250 ng/mL for omapatrilat and one metabolite and 2.0-250 ng/mL for the other three metabolites.

An automated method of sample preparation of biofluids using pierceable caps to eliminate the uncapping of the sample tubes during sample transfer.

Biological samples are normally collected and stored frozen in capped tubes until analysis. To obtain aliquots of biological samples for analysis, the sample tubes have to be thawed, uncapped, samples removed and then recapped for further storage. In this paper, we report an automated method of sample transfer devised to eliminate the uncapping and recapping process. This sampling method was incorporated into an automated liquid-liquid extraction procedure of plasma samples. Using a robotic system, the plasma samples were transferred directly from pierceable capped tubes into microtubes contained in a 96-position block. The aliquoted samples were extracted with methyl-tert-butyl ether in the same microtubes. The supernatant organic layers were transferred to a 96-well collection plate and evaporated to dryness. The dried extracts were reconstituted and injected from the same plate for analysis by liquid chromatography with tandem mass spectrometry.

High performance liquid chromatography mobile phase composition optimization for the quantitative determination of a carboxylic acid compound in human plasma by negative ion electrospray high performance liquid chromatography tandem mass spectrometry.

A systematic investigation was undertaken to study the effects of varying concentrations of additives in the acetonitrile/water high performance liquid chromatography mobile phase, especially formic acid and ammonium formate, on the negative ion electrospray response of a carboxylic acid compound. The study showed that the response progressively decreased with increase in the formic acid concentration. While such a decrease in the response could be qualitatively explained by the decrease in the concentration of the ionized form of the carboxylic acid compound due to the lower pH of the mobile phase, the change in response was not as large as expected from the change of the concentration of the ionized form. The response also progressively decreased with increase in the ammonium formate concentration but the decrease cannot be explained by the change in the pH of the mobile phase. Although the best negative ion electrospray response was obtained with a water/acetonitrile mobile phase that contained no additives at all, the retention time of the analyte was not found to be adequately reproducible on repeated injections. Thus, this mobile phase was deemed unacceptable for practical, routine use. Comparing formic acid against ammonium formate, the former was preferable since it caused a smaller attenuation of the negative ion response. Equally important was the fact that addition of formic acid had the desirable effect of maintaining a reasonably high capacity factor (k') for the analyte even at a relatively high acetonitrile concentration. A concentration of 1 mM formic acid in the mobile phase was large enough to achieve the reproducible elongated retention time for the analyte, with a loss in the analyte response of about 60% only. It should be noted that the sensitivity achieved with the 1 mM formic acid mobile phase, in which the carboxylic acid is expected to be about 10% in the ionized form, is about 9 times better than the sensitivity achieved in the 1 mM ammonium formate mobile phase, in which the carboxylic acid is expected to be about 99% in the ionized form.

Quantitative bioanalysis utilizing high-performance liquid chromatography/electrospray mass spectrometry via selected-ion monitoring of the sodium ion adduct [M+Na]+.

A high-performance liquid chromatography (HPLC)/electrospray mass spectrometric method for quantitative determination of a compound in dog plasma was developed and validated via the selected-ion monitoring of the electrospray-generated [M+Na]+ adduct of the compound. The plasma samples were acidified with HCl and then extracted with methyl tert-butyl ether. The reconstituted extracts were injected into an HPLC/positive-ion electrospray ionization mass spectrometry system. The HPLC mobile phase consisted of acetonitrile, water, formic acid (3 mM) and sodium acetate (0.3 mM). This composition of mobile phase provided the optimum electrospray condition for the formation of the [M+Na](+)-ion. This work demonstrates that the addition of sodium acetate into the HPLC mobile phase and the subsequent selected-ion monitoring of the sodium ion adduct of the analyte is a viable approach in quantitative bioanalysis. The facile formation of the sodium ion adduct of the analyte, which does not contain functional groups that are known to be strong proton acceptors, appears to be a function of the particular electrospray instrument used.

Direct injection for high sample throughput capillary gas chromatographic-mass spectrometric bioanalysis.

Because of the drawback of the relatively long analysis times inherent to temperature-programmed splitless injection capillary GC-MS, isothermal direct injection capillary GC-MS was investigated for quantitative bioanalysis. Using extracts from spiked plasma samples, we showed that high quality chromatography with a run time much shorter than that achievable with splitless injection can be achieved with direct injection. Sensitivity and other performance parameters were as good as or better than those of the splitless method. Since sample throughput is of great importance in laboratories that analyze thousands of biological samples, it is recommended that, when possible, splitless injection, which has traditionally been used in trace level GC-MS bioanalytical methods, be replaced by direct injection.