Elsevier

Clinical Biochemistry

Volume 48, Issues 16–17, November 2015, Pages 1105-1112
Clinical Biochemistry

Vitamins D and A can be successfully measured by LC–MS/MS in cord blood diluted plasma

https://doi.org/10.1016/j.clinbiochem.2015.04.014Get rights and content

Highlights

  • •

    Diluted UCB plasma can be used for the quantification of retinol and 25(OH)D3 by LC-MS/MS.

  • •

    Two laboratories using two methods have demonstrated close agreement for 25(OH)D3 in UCB serum versus diluted UCB plasma.

  • •

    Measurement of 25(OH)D3 epimer and vitamin E in diluted UCB plasma is not supported by this study due to limitations in assay sensitivity.

Abstract

Objectives

In widely used protocols for the collection and isolation of cord blood mononuclear cells, investigators are left with substantial volumes of diluted plasma which could be used for other measurements. The aim of this study was to ascertain the validity of umbilical cord blood (UCB) diluted plasma samples for vitamin D, A and E analysis compared to UCB serum samples.

Design & methods

Twenty UCB matched samples of diluted plasma and serum were collected. The samples were analysed by two liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods on two separate occasions.

Results

The results of 25(OH)D3 obtained by the two laboratories demonstrated close agreement with a mean difference of 0.14 nmol/L [95% confidence interval (95% CI), − 6.8 to 7.1]. Both methods demonstrate close agreement for 25(OH)D3 in UCB serum versus diluted UCB plasma; mean difference 2.2 nmol/L [95% CI, − 9.5 to 13.9] and 4.1 nmol/L [95% CI, − 14.5 to 6.1] for the results from Lab A and Lab B, respectively. Vitamin A was quantified by Lab A in UCB serum and diluted UCB plasma; mean difference 0.07 μmol/L [95% CI, − 0.41 to 0.28]. Results of 25(OH)D3 epimer and vitamin E in the diluted UCB plasma were below the limit of quantification, and could not be compared with UCB serum.

Conclusions

Diluted UCB plasma can be used for the quantification of retinol and 25(OH)D3 by LC–MS/MS. By contrast, quantification of 25(OH)D3 epimer and vitamin E in diluted UCB plasma is not supported by this study due to limitations in analytical sensitivity.

Introduction

Fat soluble vitamin deficiency is classically associated with complications of diseases presenting in neonates [1]. Of the four vitamins in this group, vitamins A, D and also K have pleiotropic actions whilst vitamin E has important anti-oxidant activity. Of these, vitamin D has received a lot of attention recently as a result of the meteoric rise in the number of publications showing that this secosteroid plays a crucial role in a plethora of physiological functions and is associated with many acute and chronic illnesses. In particular, there is mounting interest in the potential importance of vitamin D status, and to a lesser extent vitamin A, during early life for a wide range of health outcomes [2].

Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) quantification of each of these fat soluble vitamins, including separation of epi-25(OH)D3, is now established [3], [4], [5]. Serum, and also undiluted plasma, are the validated matrixes for analysis of vitamins A (retinol), D (25(OH)D3) and E (α-tocopherol). However the diluted plasma matrix, which is widely used in protocols for the collection and isolation of viable mononuclear cells, has not been validated for use in the LC–MS/MS analysis of small molecules. Given the limited volumes of blood available in birth cohort studies, and the implicit value of these in the context of a research intensive large-scale epidemiological projects, it is of interest to determine whether vitamins D, A and E may be adequately measured in diluted plasma from umbilical cord blood (UCB).

The aim of this study was to validate the measurement of vitamins D plus vitamins A and E using LC–MS/MS in diluted UCB plasma versus UCB serum.

Section snippets

Subjects

Twenty participants, recruited as part of the Barwon Infant Study (BIS), were randomly selected for comparison of matched serum and diluted plasma of UCB samples. BIS is a population derived birth cohort study conducted in south-eastern Australia that has been designed to investigate the early life origins of immune dysregulation. UCB was collected and stored as part of the BIS protocol. The project was approved by the Barwon Health Human Research Ethics Committee (10/24) and written informed

Results

Twenty matched samples of UCB serum and diluted UCB plasma were analysed by two LC–MS/MS laboratories (Lab A measured vitamin D and its epimer, plus vitamins A and E; Lab B measured vitamin D and its epimer) on two consecutive occasions; Fig. 2. As part of the continued monitoring of ion suppression by Lab A, two transition ions for phospholipids (104 → 104 and 184 → 184) were monitored for each sample that was analysed. These phospholipids could be a source of ion suppression due to their effects

Discussion

This study examined the suitability of diluted UCB plasma, compared with undiluted UCB serum, and provides the first report on its utility for the quantification of vitamins A and D by LC–MS/MS. This study also demonstrates the agreement between results obtained across two LC–MS/MS laboratories for vitamin D, plus the continued challenges faced in the quantitation of epi-25(OH)D3.

The appropriate selection of sample matrix is an important issue in the assessment of a number of blood analytes in

Conclusion

Diluted UCB plasma can be used for the quantification of 25(OH)D3 and vitamin A by LC–MS/MS. By contrast, measurement of the 25(OH)D3 epimer and vitamin E in diluted UCB plasma is not supported by this study due to the current limitation of analytical sensitivity for quantification. Potentially this limitation could be addressed in the future through the use of increased sample volume.

Funding source

This work was supported in part by NHMRC Grant ID 1029927

Financial disclosure

The authors have no financial relationships relevant to this article to disclose.

Conflicts of interest

There is no conflict of interest that could be perceived as prejudicing the impartiality of this manuscript.

Authorship statement

All authors listed contributed to this work. Professor Allen, Professor Ponsonby and Dr Vuillermin developed the initial study concept. Dr Collier organised and distributed the de-identified samples. Mr Albarhani and Dr Clarke analysed the samples in the respective laboratories and performed the statistical analysis in conjunction with Dr Collier. Dr Greaves and Dr Roche supervised and guided Mr Albarhani's work, which formed part of his PhD candidature. Mr Albarhani wrote the first draft of

Data sharing statement

Additional information including analytical protocol and raw data may be obtained by contacting the corresponding author.

Acknowledgements

The Barwon Infant Study (BIS) Steering Committee consists of Katie Allen, David Burgner, John Carlin, Terry Dwyer, Anne-Louise Ponsonby, Sarath Ranganathan, Richard Saffery, Mimi Tang and Peter Vuillermin. A-L Ponsonby held an NHMRC Senior Research Fellowship.

The work performed at RMIT University was conducted in the RMIT-Agilent Clinical Biochemistry Mass Spectrometry Collaboration Laboratory.

References (20)

There are more references available in the full text version of this article.

Cited by (0)

View full text
Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.