Rapid quantification of free and esterified phytosterols in human serum using APPI-LC-MS/MS

• DOI: 10.1194/jlr.c400004-jlr200
• Publication Date: 2004-10-17T00:12:57Z
• Authors: Jan Lembcke; Uta Ceglarek; Georg Martin Fiedler; Sven Baumann; Alexander Leichtle; Joachim Thiery
• Keywords: Stigmasterol

A novel analytical platform based on liquid chromatography and tandem mass spectrometry using atmospheric pressure photoionization was applied for the simultaneous quantification of free esterified β-sitosterol, campesterol, brassicasterol, stigmasterol. The total time sample pretreatment analysis could be reduced from ∼3 h [gas chromatography-mass (GC-MS)] to 15 min. detection limits different phytosterols ranged between 0.25 0.68 μg/l. Linear ranges were 1 1,000 within-run between-run variabilities 1.4% 9.9%. sensitivity at least 150-fold higher compared with GC-MS.Our new method allows a rapid determination in serum. GC-MS. Our Phytosterols are common components plant foods, especially vegetable oils, seeds, nuts, cereals (1Weihrauch J.L. Gardner J.M. Sterol content foods origin.J. Am. Diet. Assoc. 1978; 73: 39-47Abstract Full Text PDF PubMed Google Scholar). They structurally similar cholesterol, differing only number carbons or double bonds side chain. An average Western diet contains ∼200–400 mg per day (e.g., stigmasterol, brassicasterol). All serum tissue derived exclusively by intestinal absorption. Therefore, levels reflect dietary sterol intake absorption (2Miettinen T.A. Tilvis R.S. Kesaniemi Y.A. Serum sterols cholesterol precursors synthesis volunteers randomly selected male population.Am. J. Epidemiol. 1990; 131: 20-31Crossref Scopus (558) individual differences plasma phytosterol concentrations highly heritable (3Boomsma D.I. Princen H.M. Frants R.R. Gevers Leuven J.A. Kempen H.J. Genetic indicators absorption: lathosterol Dutch twins their parents.Twin Res. 2003; 6: 307-314Crossref (16) Compared rate is markedly lower, because bulk absorbed immediately secreted into intestine ATP-binding cassette half-transporters ABCG5 ABCG8 enterocytes (4Lee M.H. Lu K. Patel S.B. basis sitosterolemia.Curr. Opin. Lipidol. 2001; 12: 141-149Crossref (167) Mutations either transporter genes have been identified as cause sitosterolemia, rare autosomal recessive lipid disorder that characterized increased β-sitosterol > 50 mg/l) consequence hyperabsorption impaired biliary secretion neutral (5Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Accumulation sitosterolemia caused mutations adjacent ABC transporters.Science. 2000; 290: 1771-1775Crossref (1354) Scholar, 6Heimer S. Langmann T. Moehle C. Mauerer Dean M. Beil F.U. Bergmann von Schmitz G. human transporters sitosterolemia.Hum. Mutat. 2002; 20: 151Crossref (58) 7Salen Lutjohann D. Kane Musliner Stein Musser Ezetimibe effectively reduces patients sitosterolemia.Circulation. 2004; 109: 966-971Crossref (251) These develop premature coronary heart disease (8Salen Horak I. Rothkopf Cohen Speck Tint G.S. Shore V. Dayal Chen Shefer Lethal atherosclerosis associated abnormal composition xanthomatosis.J. Lipid 1985; 26: 1126-1133Abstract 9Katayama Satoh Yagi Hirose N. Kurita Y. Anzai Asakura Yoshikawa Mitamura Ogawa 19-year-old man myocardial infarction sitosterolemia.Intern. Med. 42: 591-594Crossref (25) recent study admitted elective artery bypass graft surgery supports hypothesis even slightly campesterol (3.8 ± 1.6 (3.1 1.1 may contribute risk (10Sudhop Gottwald B.M. Von potential factor disease.Metabolism. 51: 1519-1521Abstract (185) Sensitive methods necessary detect physiological Currently available measurement However, this time-consuming requires laborious procedure (hydrolysis, liquid-liquid extraction, derivatization) large volume (11Phillips K.M. Ruggio D.M. Bailey Precise quantitative phytosterols, stanols, metabolites capillary gas-liquid chromatography.J. Chromatogr. B Biomed. Sci. Appl. 1999; 732: 17-29Crossref (65) 12Kuksis A. Plasma non-cholesterol sterols.J. 935: 203-236Crossref (53) Recently described (LC-MS) oxidized also include extraction hydrolysis steps (13Burkard Rentsch Eckardstein Determination 24S- 27-hydroxycholesterol high-performance spectrometry.J. 45: 776-781Abstract (74) 14Plat Brzezinka Mensink R.P. Oxidized infusions measured combined 2030-2038Abstract aim our spectrometric simple one-step small volumes. For purpose, we used (APPI) technique chromatography-tandem (LC-MS/MS) (15Robb D.B. Covey T.R. Bruins A.P. Atmospheric photoionization: an ionization spectrometry.Anal. Chem. 72: 3653-3659Crossref (725) Stigmasterol, brassicasterol purchased Steraloids (Newport, RI). Labeled standards not development lack commercial supplier. Thus, [25,26,26,26,27,27,27-2H7]-cholesterol (Euriso-top, Saarbrücken, Germany) internal standard. [3,4-13C2]cholesterol obtained Cambridge Isotope Laboratory (Andover, MA). Cholesterol, cholesteryl stearate, toluene, all other chemicals Sigma-Aldrich (München, Germany). Lipoprotein-deficient (LPDS) prepared ultracentrifugation pooled (16Teupser Thiery Walli A.K. Seidel LDL- scavenger-receptor activity adherent non-adherent cultured cells single-step fluorometric assay.Biochim. Biophys. Acta. 1996; 1303: 193-198Crossref (68) Blood samples collected 49 healthy mean age 22.5 years (20–25 years). After coagulation centrifugation 1,400 g, stored polypropylene tubes −80°C until analysis. Stock solutions isopropanol (1 g/l). Four calibrators mixing diluting stock methanol containing 750 mg/l [13C2]cholesterol final 0.1, 0.5, 2, 10 added methanolic standard same concentration range samples. In-house-made LPDS controls each calculate accuracy. Daily precision determined in-house control preparation (final concentration, 2.14, 5.80, 0.62, 1.41 respectively). Internal solution (100 μg/l [2H7]cholesterol methanol) g/l isopropanol. 20 μl calibrators, controls, mixed 980 solution. min 11,400 supernatant transferred glass vial. butylated hydroxyl toluene g/l) cholesterol-cholesteryl stearate (51.7 μmol/l each) control. 2 ml freshly M ethanolic sodium hydroxide, performed 68°C. Thereafter, deionized water added. extracted twice 3 cyclohexane. extract dried 65°C under stream nitrogen reconstituted 200 chromatographic system consisted series autosampler, column oven, binary micro pump Perkin-Elmer (Rotgau-Jügesheim, Chromatographic separation Chromolith SpeedRod RP-18e (50 × 4.6 mm) monolithic (Merck KGaA, Darmstadt, 40°C. Supernatants (25 μl) injected onto column, which equilibrated methanol-water (75:25, v/v) flow 600 μl/min. these isocratic conditions, linear gradient step 100% kept 3.5 separation, reequilibrated starting solvent mixture 4.5 MDS SCIEX API 3000 triple quadrupole spectrometer Photospray™ Applied Biosystems (Darmstadt, used. APPI measurements, eluent introduced ion source without splitting μl/min 400°C. Ions formed positive mode lamp voltage 1,700 V orifice 45 Toluene dopant 90 delivered additional pump. investigation interfering effects matrix recovery done spiked [2H7]cholesterol. HPLC response analytes signal Because ester standards, (1.65 evaluate LC-MS/MS measurements. calculated adding result after hydrolysis. analyzed before alkaline saponification. Standard curves plotting peak-area ratio external against calibrator concentration. limit signal-to-noise 3. investigated up Variability assessed in-house-made Within-run variability measuring times one run. Between-run consecutive working days. Accuracy Carry-over routinely controlled injecting blanks series. [M+H-H2O]+ ions during process. fragmentation, characteristic product detected (multiple reaction monitoring). respective transitions follows: m/z 397.4/257.3 383.4/161.3 381.4/297.3 395.4/297.3 376.4/161.3 corresponding esters, bond cleavage process, observed stearate. influence solvents (methanol, isopropanol, acetonitrile, water) additives such ammonium acetate formic acid intensity investigated. highest intensities pure use dopants anisole acetone showed no better characteristics than toluene. To investigate ion-suppressing excess serum, (each presence 0.75 cholesterol. ratios 0.72, 0.60, 0.71, 0.54, 0.49 [2H7]-cholesterol, respectively. LPDS, simulating conditions adapting behavior shown Fig. 1. phytosterol/[2H7]-cholesterol influenced g/l. Characteristic chromatograms (total current) presented 2A, high specificity method, coeluting quantified any within 6 Mass esters 2C, E, G, I, In contrast, 2D (β-sitosterol), 2F (campesterol), 2H (brassicasterol), 2J (stigmasterol). 2K, L, ([2H7]-cholesterol) presented. 33.8 (equivalent cholesterol) 97.5% 97.7% originated 90.1% efficiency comparing phytosterol-spiked standards. 100.3% 102.1% 98.9% 98.3% 97.9% Detection 0.24, 0.28, 0.42, throughout calibration Pearson correlation coefficients r ≥ 0.999. data free, esterified, Table variation 2.4% 9.1%, 3.9% 9.9%.TABLE 1Variation photoionization-LC-MS/MSWithin RunBetween RunAnalyteMeanCoefficient VariationMeanCoefficient Variationmg/l%mg/l%β-SitosterolFree0.696.70.709.2Esterified1.439.11.449.9Total2.126.02.148.6CampesterolFree1.573.81.507.0Esterified3.532.93.395.8Total5.102.45.806.7BrassicasterolFree0.247.30.256.7Esterified0.415.50.385.1Total0.644.10.623.9StigmasterolFree0.384.60.366.7Esterified1.085.71.027.8Total1.464.31.417.4LC-MS/MS, spectrometry. Open table tab LC-MS/MS, accuracy (0.5, phytosterol). (96.0–107.0%) 99.7% (95.0–107.0%) 97.8% (96.4–104.0) 100.0% (96.4–108.0%) Phytosterol young (<25 age) normal levels. Results summarized 2. means 2.43 4.53 0.54 1.37 percentages fractions 65, 66, 58, 71% 2.13 g/l, percentage 74%.TABLE 2Free LC-MS/MSAnalyteMeanSDRangemg/lmg/lβ-SitosterolFree0.850.360.32–2.29Esterified1.570.620.68–3.78Total2.430.961.01–6.07CampesterolFree1.570.630.55–4.73Esterified2.960.961.41–7.54Total4.531.561.96–12.27BrassicasterolFree0.230.100.10–0.73Esterified0.310.110.14–0.76Total0.540.200.27–1.49StigmasterolFree0.400.190.16–1.23Esterified0.980.210.47–1.48Total1.370.310.64–2.28 We describe APPI-LC-MS/MS MS/MS detection, elaborating GC-MS can omitted. workup single specific multiple monitoring transitions. Free fragmentation very complicated polycyclic structure. identification direct fragments difficult mechanisms (i.e., retro-Diels-Alder reaction, molecule elimination) (17Pelillo Galletti Lercker spectral fragmentations oxidation products.Rapid Commun. Spectrom. 14: 1275-1279Crossref (14) optimized separate At present, cannot exclude possibility sufficiently separated. Grandgirard et al. (18Grandgirard Martine Demaison Cordelet Joffre Berdeaux O. Semon E. Oxyphytosterols present subjects.Br. Nutr. 91: 101-106Crossref Scholar), subjects 4.8 57.2 phytosterolemia, ∼1.4% (19Plat limited measurements isomers properties. there need known natural isomers. labeled important requirement Unfortunately, commercially low purity. started [2H7]-labeled quantification. structure polarity, it adequate standard, (Fig. 1). setup sensitivity, linearity over wide (from μg/l). might simultaneously. Validation good within-day between-day both sterols. 100.3%. experiments available. volunteers, platform, those reported authors Campesterol 3.2 (20Teunissen C.E. Vente De Bosma van Boxtel M.P. Bruijn Jolles Steinbusch H.W. cognitive performance aging population.Neurobiol. Aging. 24: 147-155Crossref (73) Scholar) 5.2 (1.5–9.7 (21Stalenhoef A.F. Hectors Demacker P.N. Effect sterol-enriched margarine lipids heterozygous phytosterolaemia.J. Intern. 249: 163-166Crossref (27) reported. 2.7 3.6 (0.8–6.6 found. comparison, found (1.96–12.27 (1.01–6.07 mg/l). No stigmasterol present. conclusion, volumes extensive pretreatment. suited large-scale clinical animal studies role factor. This work supported grant Deutsche Forschungsgemeinschaft (Th 374/2-3). thank Bruno Casetta excellent technical advice.