Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders
2008; Elsevier BV; Volume: 49; Issue: 8 Linguagem: Inglês
10.1194/jlr.d800019-jlr200
ISSN1539-7262
AutoresOsama Y. Al-Dirbashi, Tomofumi Santa, Mohamed S. Rashed, Zuhair N. Al‐Hassnan, Nobuyuki Shimozawa, Aziza Chedrawi, Minnie Jacob, Manhal A. Al‐Mokhadab,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoQuantification 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 trimethylaminoethyl ester derivative. Although this made possible the simultaneous determination of Pri, Phy, and VLCFAs, the method was hampered by the multi-step derivatization and the long chromatographic time of ∼30 min (19Johnson D.W. Trinh M.U. Oe T. Measurement of plasma pristanic, phytanic and very long chain fatty acids by liquid chromatography electrospray tandem mass spectrometry for the diagnosis of peroxisomal disorders.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003; 798: 159-162Crossref PubMed Scopus (26) Google Scholar). Recently, we described the synthesis of 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE) and demonstrated its potential as a derivatization reagent for carboxylic acids (20Tsukamoto Y. Santa T. Saimaru H. Imai K. Funatsu T. Synthesis of benzofurazan derivatization reagents for carboxylic acids and its application to analysis of fatty acids in rat.Biomed. Chromatogr. 2005; 19: 802-808Crossref PubMed Scopus (40) Google Scholar, 21Santa T. Al-Dirbashi O.Y. Ichibangase T. Fukushima T. Rashed M.S. Funatsu T. Imai K. Synthesis of benzofurazan derivatization reagents for carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry.Biomed. Chromatogr. 2007; 21: 1207-1213Crossref PubMed Scopus (20) Google Scholar, 22Al-Dirbashi O.Y. Santa T. Al-Qahtani K. Al-Amoudi M. Rashed M.S. Analysis of organic acid markers relevant to inherited metabolic diseases by UPLC-MS/MS as benzofurazan derivatives.Rapid Commun. Mass Spectrom. 2007; 21: 1984-1990Crossref PubMed Scopus (25) Google Scholar). Designed specifically for ESI-MS/MS, DAABD-AE contains an ionizable moiety with high proton affinity to improve the ionization process and a hydrophobic benzofurazan structure. In this work, we describe a novel approach for the simultaneous analysis of VLCFAs, Phy, and Pri that involves derivatization with DAABD-AE and stable isotope-labeled internal standards (ISs). The method provides essential information about peroxisomal functions so that it permits identifying a multitude of PBDs and PEDs. HPLC-grade acetonitrile was purchased from Fisher Scientific (Fairlawn, NJ,). Pentadecafluorooctanoic acid was from Fluka (Buchs, Switzerland). DAABD-AE was synthesized according to the published procedure (20Tsukamoto Y. Santa T. Saimaru H. Imai K. Funatsu T. Synthesis of benzofurazan derivatization reagents for carboxylic acids and its application to analysis of fatty acids in rat.Biomed. Chromatogr. 2005; 19: 802-808Crossref PubMed Scopus (40) Google Scholar). The following chemicals were purchased from Sigma-Aldrich: C22:0, C24:0, C26:0, Phy, arachidic acid (C20:0 linear), nonadecanoic acid (C19:0 linear), 4-dimethylaminopyridine, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Pri, 2H3-Phy, and 2H3-Pri were from Dr. H. J. ten Brink (Vrije Universiteit Medical Center, Amsterdam, The Netherlands). 2H3-C22:0, 2H4-C24:0, and 2H4-C26:0 were from C/D/N Isotopes Inc. (Quebec, Canada). All other reagents were of analytical grade or better. Solutions of individual fatty acids and labeled compounds at a concentration of 1 mg/ml were prepared in a mixture of chloroform-methanol (2:1; v/v). These were stored at 4°C and were stable for a minimum of 3 months. Working solutions of the desired mixtures were prepared by dilution in the same solvent. A working IS mixture containing 2H3-Phy and 2H3-Pri (2 μg/ml), 2H3-C22:0 and 2H4-C24:0 (0.5 μg/ml), and 2H4-C26:0 (0.1 μg/ml) was prepared in acetonitrile. Linearity was established by analyzing plasma calibrators enriched with target analytes. Excess solvent was evaporated under N2 at room temperature before adding the plasma. Calibrators were enriched as follows: 1, 2, 4, 10, 20, 40, and 60 μM for Pri; 0.96, 1.92, 3.84, 9.6, 19.2, 38.4, and 57.6 μM for Phy; 4.4, 8.8, 17.6, 44, 88, 176, and 265 μM for C22:0; 4.1, 8.2, 16.4, 41, 82, 164, and 245 μM for C24:0; and 0.25, 0.5, 1, 2.5, 5, 10, and 15 μM for C26:0. Nonenriched plasma was included with each set of calibrators to correct for endogenous levels of fatty acids. Quality control (QC) samples were set at 2 and 20 μM for Pri, 1.92 and 19.2 μM for Phy, 8.8 and 88 μM for C22:0, 8.2 and 82 μM for C24:0, and 0.5 and 5 μM for C26:0 to represent low and high concentrations. Calibrators and QCs were divided into small portions and kept at −20°C. On the analysis day, one set of calibrators and QCs was allowed to thaw at room temperature just prior to use. Our institution's Internal Review Board (King Faisl Specialist Hospital and Research Centre, Riyadh, Saudi Arabia) approved this study, and all samples used were provided to the analysts after removal of all personal identification information. Reference intervals were determined by analyzing plasma specimens collected in EDTA tubes (n = 250) that were initially submitted to our lab for VLCFA analysis by GC-MS and reported as "unremarkable." Archived plasma specimens from patients with established PDs were also analyzed (n = 20). Control and patient samples were stored at −20°C for up to 1 year and 10 years, respectively, and were allowed to thaw naturally at room temperature before use. Twenty microliters of patients plasma, QCs, or calibrators were transferred into disposable screw-capped borosilicate tubes (13 × 100 mm; Fisher Scientific) and mixed with 50 μl of 5 M HCl and 0.4 ml of the working IS. The sealed tubes were then heated at 100°C for 1 h. After cooling to room temperature, 1 ml of n-hexane was added to the tubes, and they were vortex-mixed (3 min) and centrifuged (3,800 rpm, 5 min). After collecting and evaporating the upper layer under N2, the following were added successively to the residue: 25 μl of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (25 mM in water), 25 μl of 4-dimethylaminopyridine (25 mM in acetonitrile), and 50 μl of DAABD-AE (2 mM in acetonitrile). Tubes were then tightly sealed and incubated at 60°C. After 45 min, the reaction was stopped by adding 0.1 ml of mobile phase A, and 5 μl was injected onto the liquid chromatography tandem mass spectrometry (LC-MS/MS) system. Stability of DAABD-AE derivatives was assessed by analyzing samples kept at 4°C in the autosampler tray at 0, 1, 2, 4, 8, 12, and 24 h post reaction. A Waters ACQUITY Ultra Performance liquid chromatograph (Waters; Milford, MA) composed of a binary pump, an autosampler, and a thermostatted column compartment maintained at 40°C was used. All column effluent was directed into a Quattro micro atmospheric pressure ionization bench-top triple quadrupole mass spectrometer (Micromass; Manchester, UK), equipped with a Z-spray ESI source and a switching valve. MassLynx software (v 4.0; Micromass) running under Microsoft Windows XP professional environment was used to control the instruments and for data acquisition. Separation was performed on a 2.1 × 50 mm column packed with 1.7 μm particles (ACQUITY UPLC BEH C18 column; Waters). Mobile phase A was 80% acetonitrile, mobile phase B was 100% acetonitrile, and both contained 0.5 g/l pentadecafluorooctanoic acid. The gradient program was as follows: 0–1 min 100% of A, 1–2 min from 100% to 50% of A, and 2–3.5 min 50% A at a flow rate of 0.4 ml/min. The column was reequilibrated with mobile phase A for an additional 1.5 min at a flow rate of 1 ml/min. The injection-to-injection time was 5 min. The ESI source was operated in the positive-ion mode at a capillary and cone voltage of 4.0 kV and 45 V, respectively with collision energy of 30 eV. Argon was used as collision gas, and nitrogen was used as the nebulizing and desolvation gas. Ion source and desolvation temperatures were maintained at 125°C and 350°C, respectively. Scanning was in the selected-reaction monitoring (SRM), where the transitions to the common fragment at m/z 151 generated from the following protonated parent ions were recorded: 609 (Pri), 612 (2H3-Pri), 623 (Phy), 626 (2H3-Phy), 651 (C22:0), 654 (2H3-C22:0), 679 (C24:0), 683 (2H4-C24:0), 707 (C26:0), and 711 (2H4-C26:0). Intraday (n = 12) and interday (n = 10) imprecision was assessed by repeatedly analyzing high and low QC samples every working day over a period of 2 weeks. The reproducibility was evaluated by analyzing one normal and one abnormal plasma specimen repeatedly to generate intraday (n = 10) and interday variations (n = 5). Analytical recovery was calculated from the QC sample results according to the following equation: [Recovery (%) = 100 × (concentration measured−concentration in nonenriched plasma)/concentration added]. Potential interference from isobaric compounds was evaluated by analyzing individual standard solutions. We compared the levels of VLCFAs obtained in 20 plasma samples by the current LC-MS/MS versus a routine reference method (23Moser H.W. Moser A.B. Measurement of saturated very long chain fatty acids in plasma.in: Hommes F.A. In Techniques in Diagnostic Human Biochemical Genetics. Wiley-Liss, New York1991: 177-191Google Scholar). Derivatization conditions used in this work were identical to those reported earlier for dicarboxylic acid (22Al-Dirbashi O.Y. Santa T. Al-Qahtani K. Al-Amoudi M. Rashed M.S. Analysis of organic acid markers relevant to inherited metabolic diseases by UPLC-MS/MS as benzofurazan derivatives.Rapid Commun. Mass Spectrom. 2007; 21: 1984-1990Crossref PubMed Scopus (25) Google Scholar). Continuous infusion of reaction mixtures into the first quadrupole and the subsequent ESI-MS scanning revealed intense ions at m/z of 609, 623, 651, 679, and 707 corresponding to [MH]+ of DAABD-AE derivatives of Pri, Phy, C22:0, C24:0, and C26:0, respectively. The transmission of these ions into the collision cell and the subsequent scanning by the second resolving quadrupole for fragments revealed a simple fragmentation pattern with an intense fragment at m/z of 151 common to all studied analytes. Fig. 1shows the structures and product ion mass spectra of Phy (Fig. 1A) and C26:0 derivatives (Fig. 1B). As anticipated, the fragmentation pattern and optimal ESI-MS/MS conditions for all analytes and their isotope-labeled ISs were identical and were used to design SRM experiments. The chromatographic system was optimized to retain DAABD-AE derivatives on the column while allowing other substances that may have an ion-suppressing effect, including excess reagents, to elute. This was achieved by a high-organic-content mobile phase with a gradient program that increases the acetonitrile concentration from 80% to 90% (v/v) over the course of the chromatographic run. Addition of pentadecafluorooctanoic acid, a volatile additive, to the mobile phase enhanced the ESI process and improved the peak shape of Pri and Phy, which eluted early in the run. Fig. 2shows extracted mass chromatograms obtained with a standard fatty acid mixture (Fig. 2A), a plasma sample from a normal subject (Fig. 2B), and a plasma sample from a patient with a PBD (Fig. 2C). As shown in Fig. 2A, arachidic and nonadecanoic acids, the linear-chain C20:0 and C19:0, respectively, were completely resolved and did not interfere with quantification of their isobaric branched antipodes. Pri, the analyte with the lowest molecular weight, eluted first at ∼1 min, whereas C26:0 appeared last at 3.25 min. After each sample, the column was reequilibrated with mobile phase A for 1.5 min; therefore, the injection-to-injection time was 5 min. To avoid overloading the ESI source with potential ion-suppressing contaminants from samples or reagents, the automatic switching valve supplied with the MS/MS detector was programmed to divert the column effluent to waste for the first 0.6 min and the last 1 min of each run. Isotopic purity of IS materials was assessed by analyzing individual solutions using MS/MS and LC-MS/MS, because contamination with nondeuterated antipodes could have led to overestimation. No contamination was observed, and this revealed that the IS materials were isotopically pure. Not unexpectedly, each analyte coeluted with the corresponding IS as a single peak (Fig. 2). Derivatives were stable for at least 24 h when kept in the autosampler tray at 4°C. Regression analyses of analyte-to-IS peak area ratios versus concentration in plasma revealed linear relationships in the following ranges (μM): Pri, 1–60; Phy, 0.96–57.6; C22:0, 4.4–265; C24:0, 4.1–245; and C26:0, 0.25–15. Calibration was reproducible, and as a representative example, Fig. 3depicts three overlaid calibration plots of C26:0 in plasma obtained on three different days. Method performance characteristics, including intraday and interday precision of QCs and clinical samples, expressed as coefficient of variation (%), analytical recovery, and limits of detection, are summarized in Table 1.TABLE 1Precision, recovery and limits of detection of Pri, Phy and VLCFAs in plasmaIntradayInterdayRecoverycRecovery (%) = 100 × (concentration measured−concentration in nonenriched plasma)/concentration added.CompoundConcentration AddedMeanCVnMeanCVnMean (CV)LODdLimits of detection at signal-to-noise ratio of 3 in femtomoles per 5 μl injection.μMμM%μM%%fmol/5μlPristanic22.95.5122.99.21098.9 (6.5)122019.35.31219.74.41095.1 (5.1)NormalaA plasma sample from a healthy individual.0.993.1101.16.45AbnormalbA plasma sample from a patient with a peroxisome biogenesis disorder (PBD).1.034.1101.212.45Phytanic1.95.65.4125.16.91096.8 (9.2)6.41920.91.21220.56.01095.7 (4.8)Normal5.84.2105.96.85Abnormal2.57.8102.25.85C22:08.853.41.71254.36.310109.1 (10.2)0.5988148.64.812139.66.410102.3 (5.6)Normal66.33.31067.55.25Abnormal34.11.81034.56.45C24:08.246.72.41244.84.210105.1 (6.9)0.6082126.40.512119.74.610101.7 (1.2)Normal64.72.61063.33.35Abnormal64.65.91065.24.25C26:00.51.31.1121.17.71091.3 (8.7)0.4556.31.2126.14.010103.9 (2.4)Normal1.05.2101.088.35Abnormal3.30.7103.45.45CV, coefficient of variation; Pri, pristanic acid; Phy, phytanic acid; LOD, limits of detection; VLCFA, very long chain fatty acid.a A plasma sample from a healthy individual.b A plasma sample from a patient with a peroxisome biogenesis disorder (PBD).c Recovery (%) = 100 × (concentration measured−concentration in nonenriched plasma)/concentration added.d Limits of detection at signal-to-noise ratio of 3 in femtomoles per 5 μl injection. Open table in a new tab CV, coefficient of variation; Pri, pristanic acid; Phy, phytanic acid; LOD, limits of detection; VLCFA, very long chain fatty acid. Fig. 4illustrates Bland-Altman plots that show the distribution of average concentrations of VLCFAs in 20 plasma samples measured by the current method and in parallel by our standard GC-MS method versus the difference in the paired values. Pri and Phy are not measured by our GC-MS; hence, they were not included in this comparison. Reference intervals of absolute fatty acid concentrations and ratios (C24:0/C22:0 and C26:0/C22:0) obtained in plasma from controls (n = 250) and patients with confirmed PDs (n = 20) are summarized in Table 2. For comparison, shown also are reference ranges obtained by GC-MS in other populations.TABLE 2Concentrations of Pri, Phy, and VLCFAs in plasma of controls and patients with various PDsPriPhyC22:0C24:0C26:0C24:0/C22:0C26:0/C22:0RemarksμMμMμMμMμMX-ALD/Median1.250.8440.667.03.31.610.080This studyAMNaThe adult form of X-ALD. (n = 4)Range0.34–1.790.32–1.7729.7–64.347.6–103.42.0–5.61.57–1.750.068–0.089RD (n = 1)0.20269.951.738.030.790.740.015This studyPBD (n = 15)Median1.421.4526.738.28.61.650.32This studyRange0.27–41.20.14–137.99.7–34.022.3–64.12.7–14.30.99–2.30.08–0.62Controls (n = 250)Median0.571.5851.0837.70.640.710.012This studyRange0.0–3.4bThe upper limits of Pri for age groups <1 year and 1–2 years are 1.54 and 2.0 μM, respectively.0.04–11.5cThe upper limits of Phy for age groups <1 year and 1–2 years are 6.8 and 5.3 μM, respectively.9.6–100.53.4–91.70.04–1.460.15–1.150.001–0.02895th %1.676.1786.766.31.170.950.022ControlsRange0.01–2.980.04–9.8817–96N/A0.22–1.31N/A0.003–0.021Ref 28Verhoeven N.M. Kulik W. M. M. van den Heuvel, and C. Jakobs C. Pre- and postnatal diagnosis of peroxisomal disorders using stable-isotope dilution gas chromatography-mass spectrometry.J. Inherit. Metab. Dis. 1995; 18: 45-60Crossref PubMed Scopus (28) Google ScholarControlsRanged5%–95% interval.0.0–1.50.3–11.541.1–90.337.4–74.90.6–1.20.69–1.00.011–0.022Ref 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 ScholarPD, peroxisomal disorder; X-ALD, X-linked adrenoleukodystrophy; AMN, adrenomyeloneuropathy; RD, Refsum disease; N/A, not available.a The adult form of X-ALD.b The upper limits of Pri for age groups <1 year and 1–2 years are 1.54 and 2.0 μM, respectively.c The upper limits of Phy for age groups <1 year and 1–2 years are 6.8 and 5.3 μM, respectively.d 5%–95% interval. Open table in a new tab PD, peroxisomal disorder; X-ALD, X-linked adrenoleukodystrophy; AMN, adrenomyeloneuropathy; RD, Refsum disease; N/A, not available. The importance of measuring VLCFAs for assessing peroxisomal functions has been recognized since the 1980s (24Moser A.E. Singh I. Brown F.R. Solish G.I. Kelly R.I. Benke P.J. Moser H.W. The cerebrohepatorenal (Zellweger) syndrome. Increased levels and impaired degradation of very-long chain fatty acids and their use in prenatal diagnosis.N. Engl. J. Med. 1984; 310: 1141-1146Crossref PubMed Scopus (193) Google Scholar). Quantifying these analytes in plasma is indicated when a PD is clinically evident or as part of investigating nonspecific presentations in newborns (i.e., profound hypotonia, seizures, and neuronal migration defect) or in olde
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