Lipids

, Volume 48, Issue 9, pp 949–956 | Cite as

Quantitative Analysis of Phytosterols in Edible Oils Using APCI Liquid Chromatography–Tandem Mass Spectrometry

  • Shunyan Mo
  • Linlin Dong
  • W. Jeffrey Hurst
  • Richard B. van Breemen
Methods

Abstract

Previous methods for the quantitative analysis of phytosterols have usually used GC–MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC–tandem mass spectrometry (LC–MS–MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, β-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC–MS–MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was β-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC–MS–MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays.

Keywords

Phytosterols Brassicasterol Campesterol Cycloartenol β-Sitosterol Stigmasterol Lupeol Edible oil LC–MS–MS Quantitation 

Abbreviations

APCI

Atmospheric pressure chemical ionization

HPLC

High performance liquid chromatography

GC–MS

Gas chromatography–mass spectrometry

LC–MS–MS

Liquid chromatography–tandem mass spectrometry

LLOQ

Lower limit of quantitation

LOD

Limit of detection

SRM

Selected reaction monitoring

References

  1. 1.
    Marangoni F, Poli A (2010) Phytosterols and cardiovascular health. Pharmacol Res 61:193–199PubMedCrossRefGoogle Scholar
  2. 2.
    Othman RA, Moghadasian MH (2011) Beyond cholesterol-lowering effects of plant sterols: clinical and experimental evidence of anti-inflammatory properties. Nutr Rev 69:371–382PubMedCrossRefGoogle Scholar
  3. 3.
    Siddique HR, Saleem M (2011) Beneficial health effects of lupeol triterpene: a review of preclinical studies. Life Sci 88:285–293PubMedCrossRefGoogle Scholar
  4. 4.
    Amiot MJ, Knol D, Cardinault N, Nowicki M, Bott R, Antona C, Borel P, Bernard J-P, Duchateau G, Lairon D (2011) Phytosterol ester processing in the small intestine: impact on cholesterol availability for absorption and chylomicron cholesterol incorporation in healthy humans. J Lipid Res 52:1256–1264PubMedCrossRefGoogle Scholar
  5. 5.
    De Smet E, Mensink RP, Plat J (2012) Effects of plant sterols and stanols on intestinal cholesterol metabolism: suggested mechanisms from past to present. Mol Nutr Food Res 56:1058–1072PubMedCrossRefGoogle Scholar
  6. 6.
    Sanclemente T, Marques-Lopes I, Fajó-Pascual M, Cofán M, Jarauta E, Ros E, Puzo J, García-Otín AL (2012) Naturally-occurring phytosterols in the usual diet influence cholesterol metabolism in healthy subjects. Nutr Metab Cardiovasc Dis 22:849–855PubMedCrossRefGoogle Scholar
  7. 7.
    Polagruto JA, Wang-Polagruto JF, Braun MM, Lee L, Kwik-Uribe C, Keen CL (2006) Cocoa flavanol-enriched snack bars containing phytosterols effectively lower total and low-density lipoprotein cholesterol levels. J Am Diet Assoc 106:1804–1813PubMedCrossRefGoogle Scholar
  8. 8.
    Genser B, Silbernagel G, De Backer G, Bruckert E, Carmena R, Chapman MJ, Deanfield J, Descamps OS, Rietzschel ER, Dias KC, März W (2012) Plant sterols and cardiovascular disease: a systematic review and meta-analysis. Eur Heart J 33:444–451PubMedCrossRefGoogle Scholar
  9. 9.
    Weingärtner O, Böhm M, Laufs U (2008) Controversial role of plant sterol esters in the management of hypercholesterolemia. Eur Heart J 30:404–409CrossRefGoogle Scholar
  10. 10.
    Lerman RH, Minich DM, Darland G, Lamb JJ, Chang J-L, Hsi A, Bland JS, Tripp ML (2010) Subjects with elevated LDL cholesterol and metabolic syndrome benefit from supplementation with soy protein, phytosterols, hops rho iso-alpha acids, and Acacia nilotica proanthocyanidins. J Clin Lipidol 4:59–68PubMedCrossRefGoogle Scholar
  11. 11.
    Redberg RF, Katz MH (2012) Healthy men should not take statins. JAMA 307:1491–1492PubMedCrossRefGoogle Scholar
  12. 12.
    Azadmard-Damirchi S (2009) Review of the use of phytosterols as a detection tool for adulteration of olive oil with hazelnut oil. Food Addit Contam Part A 27:1–10CrossRefGoogle Scholar
  13. 13.
    Cunha SS, Fernandes JO, Oliveira MB (2006) Quantification of free and esterified sterols in Portuguese olive oils by solid-phase extraction and gas chromatography-mass spectrometry. J Chromatogr A 1128:220–227PubMedCrossRefGoogle Scholar
  14. 14.
    Nang Lau H, Puah C, Choo Y, Ma A, Chuah C (2005) Simultaneous quantification of free fatty acids, free sterols, squalene, and acylglycerol molecular species in palm oil by high-temperature gas chromatography–flame ionization detection. Lipids 40:523–528CrossRefGoogle Scholar
  15. 15.
    Kpoviéssi DSS, Gbaguidi F, Gbénou J, Accrombessi G, Moudachirou M, Rozet E, Hubert P, Quetin-Leclercq J (2008) Validation of a method for the determination of sterols and triterpenes in the aerial part of Justicia anselliana (Nees) T. Anders by capillary gas chromatography. J Pharm Biomed Anal 48:1127–1135PubMedCrossRefGoogle Scholar
  16. 16.
    Careri M, Elviri L, Mangia A (2001) Liquid chromatography–UV determination and liquid chromatography–atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soybean oil. J Chromatogr A 935:249–257PubMedCrossRefGoogle Scholar
  17. 17.
    Nair VDP, Kanfer I, Hoogmartens J (2006) Determination of stigmasterol, β-sitosterol and stigmastanol in oral dosage forms using high performance liquid chromatography with evaporative light scattering detection. J Pharm Biomed Anal 41:731–737PubMedCrossRefGoogle Scholar
  18. 18.
    Lerma-García MAJS, Concha-Herrera V, Herrero-Martínez JM, Simó-Alfonso EF (2009) Classification of extra virgin olive oils produced at La Comunitat Valenciana according to their genetic variety using sterol profiles established by high-performance liquid chromatography with mass spectrometry detection. J Agric Food Chem 57:10512–10517PubMedCrossRefGoogle Scholar
  19. 19.
    Zarrouk W, Carrasco-Pancorbo AA, Segura-Carretero A, Fernández-Gutiérrez A, Zarrouk M (2010) Exploratory characterization of the unsaponifiable fraction of Tunisian virgin olive oils by a global approach with HPLC–APCI–IT MS/MS analysis. J Agric Food Chem 58:6418–6426PubMedCrossRefGoogle Scholar
  20. 20.
    Bedner M, Schantz MM, Sander LC, Sharpless KE (2008) Development of liquid chromatographic methods for the determination of phytosterols in Standard Reference Materials containing saw palmetto. J Chromatogr A 1192:74–80PubMedCrossRefGoogle Scholar
  21. 21.
    Sánchez-Machado DI, López-Hernández J, Paseiro-Losada P, López-Cervantes J (2004) An HPLC method for the quantification of sterols in edible seaweeds. Biomed Chromatogr 18:183–190PubMedCrossRefGoogle Scholar
  22. 22.
    Lembcke J, Ceglarek U, Fiedler GM, Baumann S, Leichtle A, Thiery J (2005) Rapid quantification of free and esterified phytosterols in human serum using APPI–LC–MS/MS. J Lipid Res 46:21–26PubMedCrossRefGoogle Scholar
  23. 23.
    Cañabate-Díaz B, Segura Carretero A, Fernández-Gutiérrez A, Belmonte Vega A, Garrido Frenich A, Martínez Vidal JL, Duran Martos J (2007) Separation and determination of sterols in olive oil by HPLC–MS. Food Chem 102:593–598CrossRefGoogle Scholar
  24. 24.
    Noppe H, Le Bizec B, Verheyden K, De Brabander HF (2008) Novel analytical methods for the determination of steroid hormones in edible matrices. Anal Chim Acta 611:1–16PubMedCrossRefGoogle Scholar
  25. 25.
    Saraiva D, Semedo R, Castilho MD, Silva JM, Ramos F (2011) Selection of the derivatization reagent—the case of human blood cholesterol, its precursors and phytosterols GC-MS analyses. J Chromatogr B Analyt Technol Biomed Life Sci 879:3806–3811PubMedCrossRefGoogle Scholar
  26. 26.
    Wewer V, Dombrink I, vom Dorp K, Dörmann P (2011) Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry. J Lipid Res 52:1039–1054PubMedCrossRefGoogle Scholar
  27. 27.
    Honda A, Yamashita K, Miyazaki H, Shirai M, Ikegami T, Xu G, Numazawa M, Hara T, Matsuzaki Y (2008) Highly sensitive analysis of sterol profiles in human serum by LC–ESI–MS/MS. J Lipid Res 49:2063–2073PubMedCrossRefGoogle Scholar
  28. 28.
    Lu B, Zhang Y, Wu X, Shi J (2007) Separation and determination of diversiform phytosterols in food materials using supercritical carbon dioxide extraction and ultraperformance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. Anal Chim Acta 588:50–63PubMedCrossRefGoogle Scholar
  29. 29.
    Mendiara I, Domeño C, Nerin C (2012) Development a fast sample treatment for the analysis of free and bonded sterols in human serum by LC–MS. J Sep Sci. doi:10.1002/jssc.201200519

Copyright information

© AOCS 2013

Authors and Affiliations

  • Shunyan Mo
    • 1
  • Linlin Dong
    • 1
  • W. Jeffrey Hurst
    • 2
  • Richard B. van Breemen
    • 1
  1. 1.Department of Medicinal Chemistry and Pharmacognosy, UIC/NIH Center for Botanical Dietary Supplements ResearchUniversity of Illinois College of PharmacyChicagoUSA
  2. 2.The Hershey Center for Health and NutritionThe Hershey CompanyHersheyUSA

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