Jianqing (Ben) Lu (Presenter)
Prince of Wales Hospital
Bio: I have completed a diploma of laboratory technology (major: clinical chemistry pathology testing) with a distinction average from Ultimo college, Sydney. I currently work at Prince of Wales Hospital, Sydney in the clinical chemistry and endocrinology department. My pathology career started in the endocrinology department, where I became well versed in immunoassays for endocrinology tests. Following a merger between endocrinology and clinical chemistry, I began working on clinical chemistry methods and now have extensive knowledge in both chemistry and endocrinology assays. As part of my training I have gained experience in a range of mass spectrometry assays and as a result of this, I am now part of the method development team.
Authorship: Ben Lu (1), Michael Wright (1), Lawrence Ward (1)
(1) Prince of Wales Hospital, SEALS, Sydney, Australia
| Short Abstract Traditionally HPLC methods have been used to analyse serum vitamins A, E and K, which involves extensive sample clean-up and long chromatographic run times. The aim of this study was to develop a single LC-MS/MS method for the determination of these fat soluble vitamins. Relatively low serum concentrations, the lack of availability of some authentic standards and occurrence of interfering lipids provided significant hurdles to method development. Here we present a simple and rapid method for the extraction and analysis of fat soluble vitamins in human serum and plasma, and the investigation of possibilities offered by MRM3 transitions for improved selectivity and sensitivity either as a front-line test or as a confirmation method. |
Long Abstract
Introduction
Vitamin A (retinol), vitamin E (á-tocopherol) and vitamin K (phylloquinone) are a group of fat soluble vitamins required for healthy growth. These compounds are essential nutrients for normal physiological functioning but can be toxic at high levels.
Vitamin A (retinol) directs normal differentiation of epithelia and affects the plasma membrane. Deficiency may lead to blindness and epithelial metaplasia. á-Tocopherol, the most biologically active form of vitamin E, is associated with poly-unsaturated fatty acids and is present in most conventional diets. Vitamin E deficiency is caused by cystic fibrosis, malabsorption and neurologic abnormality. Vitamin K is required for the post-translational biosynthesis of gamma-carboxyglutamic acid in certain proteins, which are required for blood clotting and are involved in metabolic pathways of bone and other tissue. The most naturally occurring form is vitamin K1 (phylloquinone).
Traditionally HPLC methods have been used to analyse serum vitamin A & E, with UV detection, and vitamin K with post-column reduction and fluorescence detection. Both methods involve extensive sample clean-up and long chromatographic run times. The aim of this study was to develop a single LC-MS/MS method for the determination of these fat soluble vitamins. Relatively low serum concentrations, the lack of availability of some authentic standards and occurrence of interfering lipids provided significant hurdles to method development. Here we demonstrate a simple and rapid method for the extraction and analysis of fat soluble vitamins in human serum and plasma.
Methods
A SCIEX QTRAP 5500 system in positive APCI mode coupled to a Shimadzu Prominence liquid chromatography system were utilized for analysis. Plasma and serum samples were mixed with internal standards followed by supported liquid extraction utilising Biotage ISOLUTE SLE+ 96-well plates. Sample extracts were separated on an Ascentis Express 100x3.0mm, 2.7µm, F5 column at flow rate of 0.8 mL/min over an 8.5 minute gradient from 85% methanol in water to 100% methanol. The column temperature was maintained at 40°C and an injection volume of 5µL (Vitamin A&E) or 50µL (Vitamin K) was used. Quantitative analysis was performed by multiple reaction monitoring (MRM) for vitamin A, E and K. Multistage fragmentation (MRM3), using the linear ion trap, was also investigated for vitamin K.
Results
Preliminary data demonstrated linearity for all three vitamins across the measurable biological range. Pure standards for Vitamin A and E & internal standard for Vitamin A were difficult to obtain so initial work focussed on Vitamin K. Deming regression analysis of patient serum samples demonstrated a positive bias by LC-MS/MS compared to HPLC-FD (y=1.11x-0.02). Within-batch and between-batch imprecision at multiple levels had %CV of between 3.0-7.3% for Vitamin K. Initial investigations using MRM3 transitions for the quantitation of Vitamin K demonstrated improved selectivity and good linearity. Total dwell times for Vitamin K, when using MRM3 transitions, becomes an issue when combined with the MRM transitions for all three vitamins.
Conclusions
Supported liquid extraction allow the automation of sample handling to reduce preparation time and human error.
The preliminary data demonstrates linearity for all analytes measured by LC-MS/MS in positive APCI mode.
Whilst MRM3 transitions did improve selectivity for Vitamin K, the longer dwell times and additional instrument maintenance were initially deemed excessive for the minor gains achieved.
Future Work:
• The positive bias seen by LC-MS/MS compared to HPLC-FD for Vitamin K will be investigated by acquiring KEQAS plasma standard reference material for Vitamin K to assess calibrator accuracy and by comparison to the MRM3 method to investigate selectivity of the MRM method.
• Validation of the described LC-MS/MS method to replace current HPLC methods for Vitamin K.
• Validation of the Vitamin A&E LC-MS/MS assay once appropriate standards are obtained.
References & Acknowledgements:
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