J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D800019-JLR200 on April 25, 2008

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Journal of Lipid Research, Vol. 49, 1855-1862, August 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders

Osama Y. Al-Dirbashi1,*,{dagger}, Tomofumi Santa**, Mohamed S. Rashed*, Zuhair Al-Hassnan{dagger}, Nobuyuki Shimozawa{dagger}{dagger}, Aziza Chedrawi§, Minnie Jacob* and Manhal Al-Mokhadab*

* National Laboratory for Newborn Screening, Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh 11211 Saudi Arabia
{dagger} Department of Medical Genetics, King Faisal Specialist Hospital and Research Center, Riyadh 11211 Saudi Arabia
§ Department of Neuroscience, King Faisal Specialist Hospital and Research Center, Riyadh 11211 Saudi Arabia
** Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033 Japan
{dagger}{dagger} Division of Genomics Research, Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193 Japan

This study was funded in part by a grant from Prince Salman Center for Disability Research, Riyadh, Saudi Arabia.

Published, JLR Papers in Press, April 25, 2008.

1 To whom correspondence should be addressed. e-mail: dirbashi{at}kfshrc.edu.sa

Quantification of pristanic acid, phytanic acid, and very long chain fatty acids (i.e., hexacosanoic, tetracosanoic, and docosanoic acids) in plasma is the primary method for investigateing a multitude of peroxisomal disorders (PDs). Typically based on GC-MS, existing methods are time-consuming and laborious. In this paper, we present a rapid and specific liquid chromatography tandem mass spectrometric method based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was undertaken to improve the poor mass spectrometric properties of these fatty acids. Analytes in plasma (20 µl) were hydrolyzed, extracted, and derivatized with DAABD-AE in ~2 h. Derivatives were separated on a reverse-phase column and detected by positive-ion electrospray ionization tandem mass spectrometry with a 5 min injection-to-injection time. Calibration plots were linear over ranges that cover physiological and pathological concentrations. Intraday (n = 12) and interday (n = 10) variations at low and high concentrations were less than 9.2%. Reference intervals in normal plasma (n = 250) were established for each compound and were in agreement with the literature. Using specimens from patients with established diagnosis (n = 20), various PDs were reliably detected. In conclusion, this method allows for the detection of at least nine PDs in a 5 min analytical run. Furthermore, this derivatization approach is potentially applicable to other disease markers carrying the carboxylic group.

Supplementary key words peroxisomes • Zellweger syndrome • DAABD-AE • hexacosanoic acid • tetracosanoic acid • docosanoic acid • derivatization

Abbreviations: DAABD-AE, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole; ESI-MS/MS, electrospray ionization tandem mass spectrometry; IS, internal standard; LC-MS/MS, liquid chromatography tandem mass spectrometry; PBD, peroxisome biogenesis disorder; PD, peroxisomal disorder; PED, single enzyme/transporter deficiency; Phy, phytanic acid; Pri, pristanic acid; QC, quality control; RD, Refsum disease; SRM, selected-reaction monitoring; UPLC, ultra performance liquid chromatography; VLCFA, very long chain fatty acid; X-ALD, X-linked adrenoleukodystrophy; ZS, Zellweger syndrome


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