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

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. 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. Peroxisomal disorders (PDs) are a heterogeneous group of congenital diseases caused by defective peroxisomes. Dysfunctions of these cellular organelles disturb several metabolic pathways that mainly involve lipids and often result in a progressive multisystem disease (1Gould S.J. Raymond G.V. Valle D. The peroxisome biogenesis disorders.In The Metabolic and Molecular Bases of Inherited Disease. C. R. Scriver, A. L. Beaudet, D. Valle, and W. S. Sly, editors. McGraw-Hill, Inc., New York. 2001; : 3181-3217Google Scholar, 2Poll-The B.T. Aubourg P. Wanders R.J.A. Peroxisomal disorders.In Inborn Metabolic Diseases Diagnosis and Treatment. J. Fernandes, J. M. Saudubray, G. van den Berghe, and J. H. Walter, editors. Springer, Berlin Heidelberg New York. 2006; : 509-522Google Scholar, 3Wanders R.J.A. Barth P.G. Poll-The B.T. Peroxisomal disorders.In Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. N. Blau, M. Duran, M. E. Blaskovics, and K. M. Gibson, editors. Springer, Berlin Heidelberg New York. 2006; : 481-508Google Scholar, 4Shimozawa N Molecular and clinical aspects of peroxisomal diseases.J. Inherit. Metab. Dis. 2007; 30: 193-197Crossref PubMed Scopus (41) Google Scholar). In the peroxisome, both anabolic and catabolic functions occur. The former include synthesis of bile acids, docosahexanoic acid, and plasmalogens. Catabolic reactions encompass α- and β-oxidation of certain fatty acids, among other reactions (1Gould S.J. Raymond G.V. Valle D. The peroxisome biogenesis disorders.In The Metabolic and Molecular Bases of Inherited Disease. C. R. Scriver, A. L. Beaudet, D. Valle, and W. S. Sly, editors. McGraw-Hill, Inc., New York. 2001; : 3181-3217Google Scholar, 2Poll-The B.T. Aubourg P. Wanders R.J.A. Peroxisomal disorders.In Inborn Metabolic Diseases Diagnosis and Treatment. J. Fernandes, J. M. Saudubray, G. van den Berghe, and J. H. Walter, editors. Springer, Berlin Heidelberg New York. 2006; : 509-522Google Scholar, 3Wanders R.J.A. Barth P.G. Poll-The B.T. Peroxisomal disorders.In Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. N. Blau, M. Duran, M. E. Blaskovics, and K. M. Gibson, editors. Springer, Berlin Heidelberg New York. 2006; : 481-508Google Scholar, 4Shimozawa N Molecular and clinical aspects of peroxisomal diseases.J. Inherit. Metab. Dis. 2007; 30: 193-197Crossref PubMed Scopus (41) Google Scholar, 5Hoffmann G.F. Nyhan W.L. Zschoche J. Kahler S.G. Mayatepek E. Inherited Metabolic Diseases. Lippincott Williams and Wilkins, Philadelphia2002: 328-333Google Scholar, 6Wanders R A J. Peroxisomes, lipid metabolism, and peroxisomal disorders.Mol. Genet. Metab. 2004; 83: 16-27Crossref PubMed Scopus (161) Google Scholar, 7Oglesbee D An overview of peroxisomal biogenesis disorders.Mol. Genet. Metab. 2005; 84: 299-301Crossref PubMed Scopus (7) Google Scholar). PDs are categorized into two main groups: peroxisome biogenesis disorders (PBDs) and single enzyme/transporter deficiencies (PEDs). Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (RD), which belong to PBD group, are caused by different mutations in the same genes (7Oglesbee D An overview of peroxisomal biogenesis disorders.Mol. Genet. Metab. 2005; 84: 299-301Crossref PubMed Scopus (7) Google Scholar, 8Wanders R A J. Metabolic and molecular basis of peroxisomal disorders: a review.Am. J. Med. Genet. A. 2004; 126: 355-375Crossref Scopus (135) Google Scholar, 9Wanders R.J. Waterham H.R. Peroxisomal disorders: the single peroxisomal enzyme deficency.Biochim. Biophys. Acta. 2006; 1763: 1707-1720Crossref PubMed Scopus (202) Google Scholar, 10Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Shirayoshi Y. Mori T. Fujiki Y. A human gene responsible for Zellweger syndrome that affects peroxisome assembly.Science. 1992; 255: 1132-1134Crossref PubMed Scopus (310) Google Scholar). The PED list was recently shortened to ten disorders after it was proven that mevalonate kinase is a cytosolic enzyme (9Wanders R.J. Waterham H.R. Peroxisomal disorders: the single peroxisomal enzyme deficency.Biochim. Biophys. Acta. 2006; 1763: 1707-1720Crossref PubMed Scopus (202) Google Scholar, 11Hogenboom S. Tuyp J.J. Espeel M. Koster J. Wanders R.J. Waterham H.R. Mevalonate kinase is a cytosolic enzyme in humans.J. Cell Sci. 2004; 117: 631-639Crossref PubMed Scopus (46) Google Scholar). The wide spectrum of clinical heterogeneity of PBDs is caused by losing the diverse functions of peroxisomes. ZS, characterized by craniofacial dysmorphia, severe neurological and hepatic abnormalities, and early death, is the most severe. Other PDs share some of these symptoms, with widely varying severity, organ involvement, and survival (12Baumgartner M.R. Saudubray J.M. Peroxisomal disorders.Semin. Neonatol. 2002; 7: 85-94Abstract Full Text PDF PubMed Scopus (31) Google Scholar). Together with clinical presentation and imaging studies, biochemical assays are essential for PD diagnosis and subclassification. However, no single assay is capable of detecting all PDs. Quantification of plasma hexacosanoic (C26:0), tetracosanoic (C24:0), and docosanoic (C22:0) acids, collectively known as very long chain fatty acids (VLCFAs), is the primary screening test for PBDs and a multitude of PEDs, including X-linked adrenoleukodystrophy (X-ALD), the most prevalent PD (1Gould S.J. Raymond G.V. Valle D. The peroxisome biogenesis disorders.In The Metabolic and Molecular Bases of Inherited Disease. C. R. Scriver, A. L. Beaudet, D. Valle, and W. S. Sly, editors. McGraw-Hill, Inc., New York. 2001; : 3181-3217Google Scholar, 2Poll-The B.T. Aubourg P. Wanders R.J.A. Peroxisomal disorders.In Inborn Metabolic Diseases Diagnosis and Treatment. J. Fernandes, J. M. Saudubray, G. van den Berghe, and J. H. Walter, editors. Springer, Berlin Heidelberg New York. 2006; : 509-522Google Scholar, 13Berger J. Gärtner J. X-linked adrenoleukodystrophy: clinical, biochemical and pathogenic aspects.Biochim. Biophys. Acta. 2006; 1763: 1721-1732Crossref PubMed Scopus (147) Google Scholar). Although additional biochemical assays in patients with normal levels of VLCFAs are seldom warranted (5Hoffmann G.F. Nyhan W.L. Zschoche J. Kahler S.G. Mayatepek E. Inherited Metabolic Diseases. Lippincott Williams and Wilkins, Philadelphia2002: 328-333Google Scholar), analysis of phytanic acid (Phy, C20:0 branched) and plasmalogens is valuable when RD and all types of rhizomelic chondrodysplasia punctata are clinically suspected. Determination of VLCFAs, with or without Phy and pristanic acid (Pri, C19:0 branched), is traditionally carried out by GC-MS (14Vallanve H. Applegarth D. An improved method for quantification of very long chain fatty acids in plasma.Clin. Biochem. 1994; 27: 183-186Crossref PubMed Scopus (21) Google Scholar, 15Vreken P. E. M. van Lint, A. H. Bootsma, H. Overmars, R. J. A. Wanders, and A. H. van Gennip A Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1998; 713: 281-287Crossref PubMed Scopus (79) Google Scholar, 16Jakobs C. M. M. van den Heuvel, F. Stellaard, C. Largilliere, F. Skovby, and E. Christensen C. Diagnosis of Zellweger syndrome by analysis of very long-chain fatty acids in stored blood spots collected at neonatal screening.J. Inherit. Metab. Dis. 1993; 16: 63-66Crossref PubMed Scopus (14) Google Scholar). These well-documented and highly specific assays suffer the eternal GC draw back of tedious sample preparation and lengthy chromatographic times. Methods based on positive-ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) (17Johnson D.W. A rapid screening procedure for the diagnosis of peroxisomal disorders: quantification of very long chain fatty acids as dimethylaminoethyl esters in plasma and blood spots by electrospray tandem mass spectrometry.J. Inherit. Metab. Dis. 2000; 23: 475-486Crossref PubMed Scopus (37) Google Scholar) and negative-ion ESI-MS (18Valianpour F. Selhorst J.J.M. E. M. van Lint, A. H. van Gennip, R. J. A. Wanders, and S. Kemp L. Analysis of very long chain fatty acids using electrospray ionization mass spectrometry.Mol. Genet. Metab. 2003; 79: 189-196Crossref PubMed Scopus (111) Google Scholar) are limited to VLCFAs and are incapable of differentiating Phy and Pri from their straight-chain isomers. Recently, Johnson (17Johnson D.W. A rapid screening procedure for the diagnosis of peroxisomal disorders: quantification of very long chain fatty acids as dimethylaminoethyl esters in plasma and blood spots by electrospray tandem mass spectrometry.J. Inherit. Metab. Dis. 2000; 23: 475-486Crossref PubMed Scopus (37) Google Scholar) improved his original method by including a liquid chromatography step and a second derivatization to form the Although this the of and VLCFAs, the method was by the derivatization and the long chromatographic of min D.W. T. of plasma phytanic and very long chain fatty acids by liquid chromatography electrospray tandem mass spectrometry for the diagnosis of peroxisomal Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003; PubMed Scopus Google Scholar). Recently, we the synthesis of 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole and as a derivatization for carboxylic acids Y. T. H. K. T. of derivatization for carboxylic acids and to analysis of fatty acids in Chromatogr. 2005; PubMed Scopus Google Scholar, T. T. T. T. K. of derivatization for carboxylic acids in liquid ionization tandem mass Chromatogr. 2007; PubMed Scopus Google Scholar, T. K. M. Analysis of acid markers to metabolic diseases by as 2007; PubMed Scopus Google Scholar). for DAABD-AE with high to improve the ionization and a In this we a approach for the analysis of VLCFAs, and Pri that derivatization with DAABD-AE and stable The method essential peroxisomal functions that it a multitude of PBDs and was from acid was from DAABD-AE was to the procedure Y. T. H. K. T. of derivatization for carboxylic acids and to analysis of fatty acids in Chromatogr. 2005; PubMed Scopus Google Scholar). The were from acid acid and and were from H. J. ten The and were from other were of analytical or of fatty acids and at a of were in a of These were stored at and were stable for a of of the were by dilution in the same A and and and was in was established by plasma with was at the were as and for and for and for and for and and for plasma was with each of to for levels of fatty acids. were at and for and for and for and for and and 5 for to low and high concentrations. and were into and at the analysis of and was to at to and this and all were to the after of all Reference intervals were by plasma specimens collected in (n = 250) that were to for analysis by GC-MS and as plasma specimens from patients with established PDs were (n = and were stored at for to and and were to at of patients or were into and with of 5 and of the The were at for h. to of was to the and were and 5 and the the were to the of in of in and of DAABD-AE in were and at the was by of and 5 was the liquid chromatography tandem mass spectrometry of DAABD-AE was by at in the at and A liquid of a and a column at was column was into a ionization mass with a and a was to the and for was on a column with A was B was and both The was as min of min from to of and min A at a of The column was with A for additional min at a of The injection-to-injection was 5 The was in the positive-ion at a and of and with of was as and was as the and and were at and was in the the to the at from the were (C24:0), (C26:0), and Intraday (n = 12) and interday (n = 10) was by high and low over a of The was by normal and plasma to (n = 10) and interday variations (n = was from the sample to the = in from was by the levels of VLCFAs in plasma by the a method of very long chain fatty acids in In in New Scholar). Derivatization in this were to for acid T. K. M. Analysis of acid markers to metabolic diseases by as 2007; PubMed Scopus Google Scholar). of into the and the ESI-MS at of and to of DAABD-AE of and The of these into the and the by the second for a with at of to all the and mass of Phy and the and for all and their were and were to The chromatographic was to DAABD-AE on the column other that including to was by a with a that the from to over the of the chromatographic run. of acid, a to the the and improved the of Pri and which early in the run. mass with a fatty acid a plasma sample from a normal and a plasma sample from a with a PBD in and acids, the C20:0 and were and with quantification of their the with the molecular at at each the column was with A for the injection-to-injection was 5 the with from or the with the was to the column to for the min and the min of each run. of was by using and with to was and this that the were each with the as a single Derivatives were stable for at least when in the at of in plasma linear in the ranges and Calibration was and as a plots of in plasma on different including and interday of and clinical as of analytical and of are in and of detection of Phy and VLCFAs in = in of detection at of in 5 plasma sample from a plasma sample from a with a peroxisome biogenesis of pristanic phytanic of very long chain fatty A plasma sample from a A plasma sample from a with a peroxisome biogenesis = in of detection at of in 5 in a of pristanic phytanic of very long chain fatty plots that the of concentrations of VLCFAs in plasma by the method and in by GC-MS method the in the Pri and Phy are by were in this Reference intervals of fatty acid concentrations and and in plasma from (n = 250) and patients with PDs (n = are in are ranges by GC-MS in other of and VLCFAs in plasma of and patients with various form of (n = (n = (n = (n = of Pri for and are and of Phy for and are and W. M. M. van den Heuvel, and C. C. and diagnosis of peroxisomal disorders using dilution gas spectrometry.J. Inherit. Metab. Dis. PubMed Scopus Google 15Vreken P. E. M. van Lint, A. H. Bootsma, H. Overmars, R. J. A. Wanders, and A. H. van Gennip A Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1998; 713: 281-287Crossref PubMed Scopus (79) Google peroxisomal X-linked Refsum The form of The of Pri for and are and The of Phy for and are and in a peroxisomal X-linked Refsum The of VLCFAs for peroxisomal functions the The levels and of chain fatty acids and their in J. Med. PubMed Scopus Google Scholar). these in plasma is when a PD is clinically or as of in (i.e., and or in (i.e., progressive neurological and In methods for clinical analysis of and are often based on GC-MS (14Vallanve H. Applegarth D. An improved method for quantification of very long chain fatty acids in plasma.Clin. Biochem. 1994; 27: 183-186Crossref PubMed Scopus (21) Google Scholar, 15Vreken P. E. M. van Lint, A. H. Bootsma, H. Overmars, R. J. A. Wanders, and A. H. van Gennip A Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1998; 713: 281-287Crossref PubMed Scopus (79) Google Scholar, 16Jakobs C. M. M. van den Heuvel, F. Stellaard, C. Largilliere, F. Skovby, and E. Christensen C. Diagnosis of Zellweger syndrome by analysis of very long-chain fatty acids in stored blood spots collected at neonatal screening.J. Inherit. Metab. Dis. 1993; 16: 63-66Crossref PubMed Scopus (14) Google Scholar, of very long chain fatty acids in In in New Scholar). These methods are and and time-consuming (18Valianpour F. Selhorst J.J.M. E. M. van Lint, A. H. van Gennip, R. J. A. Wanders, and S. Kemp L. Analysis of very long chain fatty acids using electrospray ionization mass spectrometry.Mol. Genet. Metab. 2003; 79: 189-196Crossref PubMed Scopus (111) Google Scholar, D.W. clinical of analysis of carboxylic Biochem. 2005; PubMed Scopus Google Scholar). Analysis by limited to a a or for very long chain fatty acids in plasma of with peroxisomal disorders as by and by gas chromatography mass PubMed Scopus (21) Google Scholar). is a with in clinical limited to it for and VLCFAs to the that of fatty acids to a with a D.W. clinical of analysis of carboxylic Biochem. 2005; PubMed Scopus Google Scholar). and D.W. T. of plasma phytanic and very long chain fatty acids by liquid chromatography electrospray tandem mass spectrometry for the diagnosis of peroxisomal Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003; PubMed Scopus Google Scholar) a of fatty acids. Although the poor the of a the of the the chromatographic and a lengthy chromatographic of to in the often with was we at a with high ionization this we recently known to form stable with a group Y. T. H. K. T. of derivatization for carboxylic acids and to analysis of fatty acids in Chromatogr. 2005; PubMed Scopus Google Scholar, T. T. T. T. K. of derivatization for carboxylic acids in liquid ionization tandem mass Chromatogr. 2007; PubMed Scopus Google Scholar, T. K. M. Analysis of acid markers to metabolic diseases by as 2007; PubMed Scopus Google Scholar). was with the in and was to a and a The of the the of the and improved the from in the reverse-phase Furthermore, it is known that at the and the gas than in the and this the electrospray ionization to of 2000; Scholar). of the with a at that was to the from DAABD-AE is that the of fatty acids was low with their straight-chain and this to In a chromatography is for the method with very the liquid chromatography column in this and for a high of of a with the in the and to the these the to at The injection-to-injection was 5 that a of linear and C19:0 and C20:0 was with GC-MS and methods (14Vallanve H. Applegarth D. An improved method for quantification of very long chain fatty acids in plasma.Clin. Biochem. 1994; 27: 183-186Crossref PubMed Scopus (21) Google Scholar, 15Vreken P. E. M. van Lint, A. H. Bootsma, H. Overmars, R. J. A. Wanders, and A. H. van Gennip A Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1998; 713: 281-287Crossref PubMed Scopus (79) Google Scholar, 16Jakobs C. M. M. van den Heuvel, F. Stellaard, C. Largilliere, F. Skovby, and E. Christensen C. Diagnosis of Zellweger syndrome by analysis of very long-chain fatty acids in stored blood spots collected at neonatal screening.J. Inherit. Metab. Dis. 1993; 16: 63-66Crossref PubMed Scopus (14) Google Scholar, D.W. T. of plasma phytanic and very long chain fatty acids by liquid chromatography electrospray tandem mass spectrometry for the diagnosis of peroxisomal Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003; PubMed Scopus Google Scholar, of very long chain fatty acids in In in New Scholar), method is to and at least of the time. we chromatographic on a column 5 with a of min using the same and this to with no to The fatty acids were from as and to (17Johnson D.W. A rapid screening procedure for the diagnosis of peroxisomal disorders: quantification of very long chain fatty acids as dimethylaminoethyl esters in plasma and blood spots by electrospray tandem mass spectrometry.J. Inherit. Metab. Dis. 2000; 23: 475-486Crossref PubMed Scopus (37) Google Scholar, D.W. T. of plasma phytanic and very long chain fatty acids by liquid chromatography electrospray tandem mass spectrometry for the diagnosis of peroxisomal Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003; PubMed Scopus Google Scholar). was in a single step with was and from to Derivatization with DAABD-AE for and the was with no this method as the with certain (18Valianpour F. Selhorst J.J.M. E. M. van Lint, A. H. van Gennip, R. J. A. Wanders, and S. Kemp L. Analysis of very long chain fatty acids using electrospray ionization mass spectrometry.Mol. Genet. Metab. 2003; 79: 189-196Crossref PubMed Scopus (111) Google Scholar), and a of patients with the and ∼2 for sample preparation and of time. The of this which from the of tandem mass in low and high we were to reliably fatty acids at low physiological as Pri and using a very of plasma (20 is in D.W. T. of plasma phytanic and very long chain fatty acids by liquid chromatography electrospray tandem mass spectrometry for the diagnosis of peroxisomal Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003; PubMed Scopus Google Scholar) when with the often for The of stable as to for sample preparation and analysis was very and the assay was and with established GC-MS plots that of the two methods are and the clinically the include intervals and pathological concentrations. the most for PD was reliably in plasma of all known to from at levels that among was detected in all was in of the intervals by this in which are in agreement with established by GC-MS in P. E. M. van Lint, A. H. Bootsma, H. Overmars, R. J. A. Wanders, and A. H. van Gennip A Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1998; 713: 281-287Crossref PubMed Scopus (79) Google Scholar, W. M. M. van den Heuvel, and C. C. and diagnosis of peroxisomal disorders using dilution gas spectrometry.J. Inherit. Metab. Dis. PubMed Scopus Google Scholar). is that plasma levels of Pri and Phy were was to by ten and F. van den Wanders R.J.A. C. acid and phytanic acid in plasma from patients with peroxisomal disorders: stable isotope dilution analysis with mass 1992; Full Text PDF PubMed Google Scholar), levels of these acids in The for PDs involve concentrations of and their In patients with ZS, neonatal adrenoleukodystrophy, infantile and a multitude of PEDs, levels of and are and are These are over and which are in and peroxisomal W. M. M. van den Heuvel, and C. C. and diagnosis of peroxisomal disorders using dilution gas spectrometry.J. Inherit. Metab. Dis. PubMed Scopus Google Scholar). in of Phy was in the with the of that the of Pri to the of from patients with this method was to the analysis of than clinical and was in the for and of Diagnosis and of Inherited of established the and of this which the GC-MS in In we a approach to the of and VLCFAs, the of PDs. allows for of these in a plasma (20 μl) a analytical of 5 The method is with a single and derivatization Reference intervals in were in agreement with the and the method was to of PDs with The method is capable of detecting the nine ZS, neonatal adrenoleukodystrophy, infantile rhizomelic chondrodysplasia punctata peroxisomal and this and the existing in and of this derivatization approach to other disease markers carrying a carboxylic is it limited of DAABD-AE to Y. The their to the of the of for their to the Laboratory for to the and the of of and for and this