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

H4014

Sigma-Aldrich

25-Hydroxycholecalciferol, ≥98% (HPLC)

E5750

Sigma-Aldrich

Ergocalciferol, 40,000,000 USP units/g

650188

Sigma-Aldrich

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

721956

Sigma-Aldrich

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

733466

Sigma-Aldrich

Acetonitrile, electronic grade, 99.999% trace metals basis

95220

Sigma-Aldrich

Ergocalciferol, ≥98.0% (sum of enantiomers, HPLC)

30800

Sigma-Aldrich

7-Dehydrocholesterol, ≥95.0% (HPLC)

439223

Sigma-Aldrich

Dichloromethane, suitable for HPLC, ≥99.9%, contains 40-150 ppm amylene as stabilizer

606723

Sigma-Aldrich

Methanol-¹²C, 99.95 atom % ¹²C

675431

Sigma-Aldrich

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

PHR1072

Supelco

2-Propanol, Pharmaceutical Secondary Standard; Certified Reference Material

110086

Sigma-Aldrich

Acetonitrile, ReagentPlus^®, 99%

320269

Sigma-Aldrich

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

34442

Sigma-Aldrich

Acetonitrile, suitable for DNA synthesis, ≥99.9% (GC)

L010300

Sigma-Aldrich

Ultrapure Acetonitrile

676853

Sigma-Aldrich

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

1601340

USP

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

1570428

USP

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

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

E0900000

Ergocalciferol, European Pharmacopoeia (EP) Reference Standard

439207

Sigma-Aldrich

2-Propanol, suitable for HPLC, 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%

1239005

USP

Ergocalciferol, United States Pharmacopeia (USP) Reference Standard

650463

Sigma-Aldrich

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

PHR1557

Supelco

Methylene Chloride, Pharmaceutical Secondary Standard; Certified Reference Material

34851

Sigma-Aldrich

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

34998

Sigma-Aldrich

Acetonitrile, HPLC Plus, ≥99.9%