Detection and quantitation of fatty acid acyl conjugates of triamcinolone acetonide via gas chromatography–electron-capture negative-ion mass spectrometry

doi:10.1016/j.ab.2003.08.006

Analytical Biochemistry

Volume 322, Issue 2, 15 November 2003, Pages 243-250

https://doi.org/10.1016/j.ab.2003.08.006 Get rights and content

Abstract

The inherent electron-capture properties of triamcinolone acetonide (TAA) fatty acid conjugates were exploited for development of a GC–MS technique for quantitation of C21 long-chain fatty esters of TAA synthesized in BEAS-2B cells, an immortalized airway epithelium cell line. TAA esters extracted from BEAS-2B cells were purified and detected via selected ion monitoring of the molecular anions generated from the TAA esters under electron-capture negative-ion mass spectrometric conditions. Standard curves were linear over a range of 0.0 to >4.5 ng/mg protein with r² values = 1. Levels of TAA conjugates extracted from BEAS-2B treated with 10⁻⁵ M TAA for 24 h ranged from 0.024 to 0.301 ng/mg protein. Further evidence for confirmation of the identity of TAA fatty esters formed in BEAS-2B cells was obtained via selected reaction monitoring. The transition monitored was formation of the carboxy anion generated from each of the respective molecular anions of the TAA esters during collision-induced decomposition. These findings indicate that the GC–MS analysis is suitable for studies of the kinetics of the TAA fatty acid conjugates formation in vitro and may be directly applicable to determination of the kinetics of TAA fatty acid conjugation in vivo.

Section snippets

Standards and reagents

Five C16 and C18 fatty acid conjugates of TAA were obtained via custom synthesis (Biomol Research Laboratories, Plymouth Meeting, PA). The fatty acid acyl conjugates were the palmitate ester (TAA-21-16:0), palmitoleate ester (TAA-21-16:1), stearate ester (TAA-21-18:0), oleate ester (TAA-21-18:1), and linoleate ester (TAA-21-18:2). Multideuterated analogs of TAA-21-18:0 and TAA-21-16:0 were also obtained via custom synthesis. The multideuterated analogs obtained were 18,18,18-²H₃-stearate ester (

GC properties and electron capture negative-ion fragmentation of TAA conjugates

The inherent electron-capture properties of a TAA-21 ester, the C21 acetate, were illustrated in studies described in an earlier report [8]. The TAA C21 long-chain fatty acid conjugates of the steroid should also possess strong inherent electron properties similar to those observed for the TAA-21 acetate. A distinct advantage of strong inherent electron-capture properties of the TAA fatty acid esters should be a low background that would facilitate measurement of low nanogram to subnanogram

Conclusions

The inherent electron-capture properties of C21 fatty acid esters of TAA were exploited for development of GC–MS assay methods for determination of the identities and quantities of C16 and C18 fatty acid esters of the antiasthma steroid synthesized in BEAS-2B cells in vitro. The assay displays linearity over a range of 0.0 to >4.5 ng/mg protein. Two C16 fatty acid esters of TAA, TAA-21-16:1 and TAA-21-16:0, and two C18 fatty acid conjugates of TAA, TAA-21-18:1 and TAA-21-18:0, were isolated from

Acknowledgements

The authors thank Rhone–Poulenc–Rorer (now Aventis) for providing the multideuterated analogs of TAA-21-16:0 and TAA-21-18:0 and protium forms of the C16 and C18 TAA-21 fatty acid standards employed in these studies.

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