A strategy for quantitative bioanalysis of non‐polar neutral compounds by liquid chromatography/electrospray ionization tandem mass spectrometry: determination of TS‐962, a novel acyl‐CoA:cholesterol acyltransferase inhibitor, in rabbit aorta and liver tissues

Abstract

A strategy for the sensitive and reliable quantitative determination of non-polar neutral compounds in biological matrices by liquid chromatography/electrospray ionization tandem mass spectrometry is described in the context of assay development for TS-962, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, in rabbit aorta and liver tissues. The electrospray ionization (ESI) mass spectrum of this compound with a mobile phase of water/acetonitrile did not give abundant [M + H]+ ions, but did give alkali metal cation adducts such as [M + Na]+, [M + CH3CN + Na]+ and [M + K]+ ions. The cationized species are acknowledged as unsuitable precursor ions for selected reaction monitoring (SRM) for various reasons, such as difficulty in obtaining characteristic product ions in low-energy collision-induced dissociation, and irreproducibility of the adduct-ion intensities. To overcome this problem, a solution of 3.4 mM trifluoroacetic acid in 2-propanol was added to the mobile phase as a postcolumn additive, resulting in a decrease of the undesirable adduct formation and significant enhancement of [M + H]+ ion intensity. An attempt was then made to prevent the matrix effect by employing a column-switching system, which allowed direct injection of a large volume of 2-propanolic tissue homogenate (950 microL) followed by sufficient clean-up, separation, and ESI-SRM on-line. This enabled development of a sensitive and reliable assay method for TS-962 in rabbit aorta and liver tissues in the concentration range of 5-500 ng/g wet tissue using a 25-mg aliquot of tissue sample. Application of this method to the determination of aortic TS-962 levels at 24 h after repeated oral administration of this compound (3 mg/kg) once a day for 12 weeks to 1% cholesterol-fed rabbits is also presented. Results showed that TS-962 is well distributed to both the thoracic and abdominal aorta tissues, at levels higher than the 50% inhibitory concentration value of this compound for microsomal ACAT activity from rabbit aorta.

Restricted access materials and large particle supports for on-line sample preparation: an attractive approach for biological fluids analysis

“…In the case of RAM sorbents, TAT depends not only on the separation time but also on the extraction time. Indeed, conventional flow rates are used for sample extraction on RAM supports and total analysis time are usually longer to 5 min [42,43,67,78,88,95,108,123,140]. Nevertheless, alkyl-diol-silica material have, recently, been found suitable for a fast and efficient sample extraction at a high flow rate [16,167].…”

“…Several ml of biological fluids can be directly injected on this extraction material without loss of performances [115,116]. Capcell Pak MF [115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130][131][132][133] and Capcell Pak SCX [134] materials were suitable tools for the direct determination of drug-containing biofluids.…”

Increase of the LC–MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection

“…In addition to posing challenges to quantification, the matrix effect also results in the suppression of the analyte signal and lower sensitivity. Several approaches have been investigated to address matrix effects and improve sensitivity [6][7][8][9][10][11][12][13][14]. Some researchers have used post-column additions of methanoic acid [6] and trifluoroacetic acid in 2-propanol [7] into the LC flow to reduce or prevent analyte ionization suppression.…”

“…Several approaches have been investigated to address matrix effects and improve sensitivity [6][7][8][9][10][11][12][13][14]. Some researchers have used post-column additions of methanoic acid [6] and trifluoroacetic acid in 2-propanol [7] into the LC flow to reduce or prevent analyte ionization suppression. Others investigated different off-line clean-up procedures [8] and on-line LC-LC [9][10][11] to clean up the samples and to address matrix effect and reduce signal suppression.…”