Analysis of oxysterols and vitamin D metabolites in mouse brain and cell line samples by ultra-high-performance liquid chromatography-atmospheric pressure photoionization-mass spectrometry.

Analysis of oxysterols and vitamin D metabolites in mouse brain and cell line samples by ultra-high-performance liquid chromatography-atmospheric pressure photoionization-mass spectrometry.

Journal of chromatography. A (2014-09-11)

Linda Ahonen, Florian B R Maire, Mari Savolainen, Jaakko Kopra, Rob J Vreeken, Thomas Hankemeier, Timo Myöhänen, Petri Kylli, Risto Kostiainen

PMID25204266

ABSTRACT

We have developed an ultra-high-performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometric (UHPLC-APPI-MS/MS) method for the simultaneous quantitative analyses of several oxysterols and vitamin D metabolites in mouse brain and cell line samples. An UHPLC-APPI-high resolution mass spectrometric (UHPLC-APPI-HRMS) method that uses a quadrupole-time of flight mass spectrometer was also developed for confirmatory analysis and for the identification of non-targeted oxysterols. Both methods showed good quantitative performance. Furthermore, APPI provides high ionization efficiency for determining oxysterols and vitamin D related compounds without the time consuming derivatization step needed in the conventionally used electrospray ionization method to achieve acceptable sensitivity. Several oxysterols were quantified in mouse brain and cell line samples. Additionally, 25-hydroxyvitamin D3 was detected in mouse brain samples for the first time.

MATERIALS

Product Number

Brand

Product Description

E5750

Sigma-Aldrich

Ergocalciferol, 40,000,000 USP units/g

H4014

Sigma-Aldrich

25-Hydroxycholecalciferol, ≥98% (HPLC)

66742

Supelco

Dichloromethane, Selectophore^™, ≥99.5%

89680

Supelco

Toluene, analytical standard

650579

Sigma-Aldrich

Toluene, HPLC Plus, for HPLC, GC, and residue analysis, ≥99.9%

676756

Sigma-Aldrich

Toluene, ACS reagent, ≥99.5%

179418

Sigma-Aldrich

Toluene, ACS reagent, ≥99.5%

D65100

Sigma-Aldrich

Dichloromethane, contains 40-150 ppm amylene as stabilizer, ACS reagent, ≥99.5%

320269

Sigma-Aldrich

Dichloromethane, ACS reagent, ≥99.5%, contains 40-150 ppm amylene as stabilizer

676853

Sigma-Aldrich

Dichloromethane, ACS reagent, ≥99.5%, contains 40-150 ppm amylene as stabilizer

PHR1317

Supelco

Residual Solvent - Toluene, Pharmaceutical Secondary Standard; Certified Reference Material

650463

Sigma-Aldrich

Dichloromethane, HPLC Plus, for HPLC, GC, and residue analysis, ≥99.9%, contains 50-150 ppm amylene as stabilizer

494453

Sigma-Aldrich

Dichloromethane, biotech. grade, 99.9%, contains 40-150 ppm amylene as stabilizer

V-025

Supelco

Vitamin D3 solution, 1 mg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant^®

V-024

Supelco

Vitamin D2 solution, 1 mg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant^®

E0900000

Ergocalciferol, European Pharmacopoeia (EP) Reference Standard

PHR1311

Supelco

Toluene, Pharmaceutical Secondary Standard; Certified Reference Material

PHR1237

Supelco

Cholecalciferol (Vitamin D3), Pharmaceutical Secondary Standard; Certified Reference Material

PHR1238

Supelco

Ergocalciferol (Vitamin D2), Pharmaceutical Secondary Standard; Certified Reference Material

1424109

USP

Methyl alcohol, United States Pharmacopeia (USP) Reference Standard

721956

Sigma-Aldrich

Methanol solution, NMR reference standard, 4% in methanol-d₄ (99.8 atom % D), NMR tube size 3 mm × 8 in.

740292

Sigma-Aldrich

Vitamin D₃ solution, 100 μg/mL in ethanol, 97% (CP)

45983

Supelco

Acetonitrile, analytical standard

91237

Supelco

2-Propanol, analytical standard

I9516

Sigma-Aldrich

2-Propanol, for molecular biology, BioReagent, ≥99.5%

02575

Supelco

Dichloromethane, analytical standard

34856

Sigma-Aldrich

Dichloromethane, suitable for HPLC, ≥99.8%, contains amylene as stabilizer

PHR1557

Supelco

Methylene Chloride, Pharmaceutical Secondary Standard; Certified Reference Material