Sterols (ST) determination - Mezine et al.

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Contents 1 Abstract 2 Analysed Matrices 3 Analytes 4 Sample Preparation 5 Analytical Method 5.1 HPLC-APCI-MS 5.1.1 HPLC 1100 system (Agilent Technologies, Palo Alto, CA) 5.1.2 APCI-MS 6 Method Validation 6.1 Accuracy 6.2 Precision 6.3 LOD 6.4 LOQ 6.5 Linearity 7 Internal Standard 8 Reference

Abstract

Plant sterol and stanol esters were separated on a Luna hexyl-phenyl column using a gradient of acetonitrile (90-100%) in water. The eluted compounds were detected by atmospheric pressure chemical ionization (APCI)-mass spectroscopy (MS) in the positive mode. Sterol and stanol esters produced [M + H - HOOCR]+ ions. Application of the hyphenated techniques - LC-MS - allowed differentiation between a number of esters of sitosterol, campesterol, stigmasterol, and (tentatively) avenasterol, as well as sitostanol and campestanol esters. With cholesteryl decanoate used as the internal standard, the method showed good linearity, precision, and reproducibility. The method required minimal sample pretreatment and can be applied to samples with high water content (juices) as well as samples with high oil content (margarine spreads). The method could be useful for theanalysis of sterol and stanol esters in fortified food products.

Analysed Matrices

spreads, beverages

Analytes

• 6 phytosterols (+ 1 IS)

esters of ß-sitosterol; campesterol; stigmasterol; avenasterol; sitostanol; campestanol

Sample Preparation

• Orange Juice Sample Preparation

The samples of orange juice were accurately weighed in glass vials (~5 g), followed by the addition of ethyl acetate (20.0 mL) containing 0.05 mg/mL of cholesterol decanoate as IS. The samples were vigorously agitated and sonicated for 20 min and then left for 30 min to allow phase separation. Portions of the upper phase (~3 mL) were taken out and centrifuged at 1000g for 5 min. Clear supernatants were filtrated (0.45 µm PTFE filters) and analyzed by HPLC-APCI-MS. The injection volume was 5 µL. The blank orange juice samples were analyzed in a similar way, using ethyl acetate for extraction.

• Margarine Spread Preparation

Samples of spreads in triplicate(200 mg) were accurately weighed in a volumetric flask (25 mL), and the volume was adjusted by ethyl acetate containing 0.05 mg/mL of cholesteryl decanoate as IS. Samples were sonicated (5 min), and hydrophilic material was filtered (0.45 µm). The prepared samples were analyzed by HPLC-APCI-MS.

Analytical Method

HPLC-APCI-MS

HPLC 1100 system (Agilent Technologies, Palo Alto, CA)

• column: Luna hexyl-phenyl HPLC column (100 mm x 2.0 mm i.d., 3 µm, Phenomenex)
• flow rate, 0.6 mL/min
• linear gradient of CH3CN in water from 90 to 100% in 15 min
• postrun equilibration time: 5 min
• T (column compartment): 35 °C

APCI-MS

• quadrupole mass selective detector (MSD)
• positive ion mode
• optimized settings of the MSD:

fragmentator voltage: 80 V

T (vaporizer): 400 °C

T (drying gas): 300 °C

nebulizer pressure: 40 psi

corona current: 8 µA

capillary voltage: 3000 V

• scan mode over the m/z range of 350-410.

Method Validation

Accuracy

Precision

LOD

25 ng

LOQ

100 ng

Linearity

250 - 5000 ng

Internal Standard

cholesteryl decanoate

Reference

• I. Mezine, H. Zhang, C.Macku, R. Luana J. Agric. Food Chem. 2003, 51, 5639-5646.

A. M. Todd Group; 150 Domorah Drive; Montgomeryville; Pennsylvania 18936; U.S.A.

Fax: (215) 628-8651; e-mail: imezine@amtbotanicals.com