Ultra-high performance liquid chromatographic and mass spectrometric analysis of active vitamin B12 in cells of Propionibacterium and fermented cereal matrices.

Ultra-high performance liquid chromatographic and mass spectrometric analysis of active vitamin B12 in cells of Propionibacterium and fermented cereal matrices.

Food chemistry (2014-07-24)

Bhawani Chamlagain, Minnamari Edelmann, Susanna Kariluoto, Velimatti Ollilainen, Vieno Piironen

ABSTRACT

A sensitive and selective method is needed to analyse in situ produced vitamin B12 in plant-based materials, potential new dietary sources of vitamin B12. A UHPLC/UV method was developed and validated for the determination of human active vitamin B12 in cell extracts of Propionibacterium freudenreichii subsp. shermanii and after immunoaffinity purification in extracts of cereal matrices fermented by P. freudenreichii. An Acquity HSS T3 C18 column resulted in a baseline separation, a calibration curve of excellent linearity and a low limit of detection (0.075 ng/5 μL injection). As confirmed by UHPLC-MS, the active vitamin B12 could be separated from pseudovitamin B12. The recovery of vitamin B12 from purified spiked cereal matrices was good (>90%; RSD<5%). A nutritionally relevant amount of active vitamin B12 was produced by P. freudenreichii in cereal malt matrices (up to 1.9 μg/100 g) in 24h at 28 °C.

MATERIALS

Product Number

Brand

Product Description

43617

Supelco

Sodium hydroxide concentrate, 0.1 M NaOH in water (0.1N), Eluent concentrate for IC

1012772

USP

Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard

679186

Sigma-Aldrich

Acetonitrile solution, contains 0.05 % (w/v) ammonium formate, 5 % (v/v) water, 0.1 % (v/v) formic acid, suitable for HPLC

71251

Supelco

Acetic acid, analytical standard

685461

Sigma-Aldrich

Acetonitrile solution, contains 10.0% acetone, 0.05% formic acid, 40.0% 2-propanol

71687

Sigma-Aldrich

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72068

Sigma-Aldrich

Sodium hydroxide solution, BioUltra, for molecular biology, 10 M in H₂O

45726

Sigma-Aldrich

Acetic acid, for luminescence, BioUltra, ≥99.5% (GC)

72064

Supelco

Sodium hydroxide solution, 49-51% in water, eluent for IC

409332

Sigma-Aldrich

Cobalt(II) chloride, AnhydroBeads^™, −10 mesh, 99.995% trace metals basis

W200611

Sigma-Aldrich

Acetic acid, ≥99.5%, FCC, FG

W200603

Sigma-Aldrich

Acetic acid, natural, ≥99.5%, FG

449776

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Cobalt(II) chloride, AnhydroBeads^™, −10 mesh, 99.9% trace metals basis

606766

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Acetic acid-¹²C₂, 99.9 atom % ¹²C

574732

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Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC

574724

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Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid

48075

Supelco

Ethanol solution, certified reference material, 2000 μg/mL in methanol

576956

Sigma-Aldrich

Acetonitrile solution, contains 0.1 % (v/v) formic acid, suitable for HPLC

V2876

Sigma-Aldrich

Vitamin B₁₂, ≥98%

V6629

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Vitamin B₁₂, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥98%

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757527

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S8045

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282979

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3-Ethyl-2,4-pentanedione, mixture of tautomers, 98%

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Acetic acid solution, suitable for HPLC

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Supelco

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Sigma-Aldrich

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Sigma-Aldrich

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