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

PHR1551

Supelco

Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material

270997

Sigma-Aldrich

Dichloromethane, anhydrous, ≥99.8%, contains 40-150 ppm amylene as stabilizer

PHR1237

Supelco

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

1570428

USP

2-Propanol, United States Pharmacopeia (USP) Reference Standard

PHR1238

Supelco

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

1601340

USP

Residual Solvent Class 2 - Acetonitrile, United States Pharmacopeia (USP) Reference Standard

1424109

USP

Methyl alcohol, United States Pharmacopeia (USP) Reference Standard

650188

Sigma-Aldrich

Methanol solution, (Methanol:Dimethyl sulfoxide 1:1 (v/v))

733458

Sigma-Aldrich

2-Propanol, electronic grade, 99.999% trace metals basis

733466

Sigma-Aldrich

Acetonitrile, electronic grade, 99.999% trace metals basis

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^®

740292

Sigma-Aldrich

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

740284

Sigma-Aldrich

Vitamin D₃(6,19,19-d₃) solution, 100 μg/mL in ethanol, 97 atom % D, 97% (CP)

E0900000

Ergocalciferol, European Pharmacopoeia (EP) Reference Standard

PHR1311

Supelco

Toluene, Pharmaceutical Secondary Standard; Certified Reference Material

H-083

Supelco

25-Hydroxyvitamin D3 solution, 100 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant^®

L010300

Sigma-Aldrich

Ultrapure Acetonitrile

PHR1317

Supelco

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

PHR1557

Supelco

Methylene Chloride, Pharmaceutical Secondary Standard; Certified Reference Material

676756

Sigma-Aldrich

Toluene, ACS reagent, ≥99.5%

I9030

Sigma-Aldrich

Isopropyl alcohol, meets USP testing specifications

650579

Sigma-Aldrich

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

109827

Sigma-Aldrich

2-Propanol, Laboratory Reagent, ≥99.5%

439134

Sigma-Aldrich

Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%

34998

Sigma-Aldrich

Acetonitrile, HPLC Plus, ≥99.9%

34863

Sigma-Aldrich

2-Propanol, suitable for HPLC, 99.9%

34856

Sigma-Aldrich

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

650447

Sigma-Aldrich

2-Propanol, HPLC Plus, for HPLC, GC, and residue analysis, 99.9%

34851

Sigma-Aldrich

Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%