Jessica Gifford (Presenter)
Calgary Laboratory Services
Bio: I have a PhD in structural biochemistry from the University of Calgary as well as postdoctoral training from the University of Alberta. My background is in the use of nuclear-magnetic resonance spectroscopy to study protein-ligand interactions and I have published 11 manuscripts and 13 abstracts. I am currently a first year fellow in the Clinical Chemistry Fellowship Program at the University of Calgary and Calgary Laboratory Services (CLS). Thus far in my Clinical Chemistry training, I have developed a keen interest in both quality improvement and analytical method development. I am particularly drawn to the use of mass spectrometry to improve both the throughput of the diagnostic lab as well as the analytical utility of the tests we perform.
Authorship: Jessica L. Gifford, Joshua M. Buse, Jessica M. Boyd, and S.M. Hossein Sadrzadeh
Calgary Laboratory Services and University of Calgary, Calgary, AB, Canada
| Short Abstract Beta carotene is a fat-soluble compound routinely measured in serum because of its physiological importance as a vitamin A precursor. Most analytical techniques measure beta-carotene by liquid chromatography coupled to a diode array detector. These analytical techniques, however, suffer from poor analyte specificity and accuracy, and an inadequate variety of commercial kits that are expensive. To address this, we have developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method that measures beta-carotene in serum; a matrix that contains other carotenoids including alpha-carotene and lycopene (isobars of beta-carotene) and a variety of lipophilic molecules. |
Long Abstract
Introduction: Beta-carotene is an important member of the carotenoid family that is routinely measured in serum. Unable to be synthesized de novo in humans and only available ingested in the diet, beta-carotene is a highly fat-soluble compound that is carried in the blood bound to lipoproteins. Physiologically, beta-carotene is the precursor of vitamin A and possesses additional free radical scavenging properties. In the clinical laboratory, beta-carotene is measured to assess malabsorption conditions associated with small bowel disease, cystic fibrosis, and intestinal parasitic infection. In addition, it can be measured to diagnose carotenodema, a benign condition associated with the excess dietary intake of carotene rich foods or vitamin supplements, as well as to assess liver disease. To date, most techniques measure beta-carotene by chromatography coupled to a diode array detector. However, these techniques suffer from poor analyte specificity and accuracy. Also, the limited variety of commercial kits that are currently available are expensive. Furthermore, due to its instability and hydrophobicity, beta-carotene requires additional measures during specimen preparation, analyte extraction, and final analysis to prevent artificial degradation. To address these issues, we have developed an LC-ESI-MS/MS method for measuring beta-carotene in serum, a matrix that contains other carotenoids including alpha-carotene and lycopene (isobars of beta-carotene) and a variety of lipophilic molecules.
Methods: The evaluated sample preparation procedures utilized ethyl acetate, methyl-tert-butyl-ether (MTBE), and tetrahydrofuran (THF) as liquid:liquid extraction solvents to isolate β-carotene from 100 µL serum samples; some samples had butylated hydroxytoluene (BHT) added as a stabilizing agent. To each sample, 250 µL of one solvent or combination of different solvents was added to each sample followed by vigorous vortexing for 1 minute. Samples were subsequently centrifuged at 12000 x g for 10 minutes at 20 °C, with 2.5 µL of supernatant injected into the LC-MS/MS system. The LC-MS/MS system consisted of an Agilent series 1290 binary pump with an online degasser, auto sampler and column oven (Agilent Technologies, Mississauga, ON, Canada) coupled to a 6460 triple quadrupole MS/MS via a JetStream(R) electrospray interface. Chromatographic evaluation of β-carotene was achieved using an Agilent Zorbax Eclipse Plus C18 column (50 x 2.1 mm, 2.7 µ) and separation of β-carotene from other carotenoids was achieved using a YMC Carotenoid C30 column (250 x 4.6 mm, 5 µ); both columns were protected by a Phenomenex HPLC KrudKatcher Ultra Column In-line filter. Separation of β-carotene using the C18 column utilized acetonitrile (A) and ethanol (B) (with 0.1 % formic acid) mobile phases, with a gradient from 70:30 to 20:80 (%A:%B) over 9 minutes at a flow rate of 0.3 mL/min. Conversely, the C30 column attained isocratic separation of β-carotene using a mobile phase of 75% ethanol, 20% methanol and 5% THF over 15 minutes at a flow rate of 0.5 mL/min. Sample carryover in the autosampler was assessed by the injection of a double blank following injection of the highest standard curve concentration. Columns and samples were maintained at a temperature of 8 °C during analysis. The Agilent 6460 triple quadrupole MS/MS utilized a nebulizer flow of 35 psi, sheath gas flow of 11 psi and sheath gas temperature of 250 °C. The capillary voltage was set at 3000 V and the V charging was set at 500 V. The mass spectrometer utilized multiple reaction monitoring (MRM) run in positive mode to monitor β-carotene [536.4 to 444.5, 143.1, 105.1]; each MRM transition monitored relied upon a dwell time of 20 ms, fragmentation voltage of 177 V, and cell acceleration voltage of 2 V. Collision energy (CE) increased inversely to the monitored m/z values, with 444.5 utilize a CE of 14 V, 143.1 a CE of 62 V and 105.1 a CE of 74 V.
Results: Our preliminary results show that beta-carotene can be extracted from serum using a simple liquid:liquid extraction method that does not require concentration or evaporation to dryness and subsequent reconstruction of the sample. Optimal liquid: liquid extraction of β-carotene was attained using 100% ethyl acetate as the organic solvent system and was amenable to direct injection into the LC-MS/MS. Specific consideration of evaporation of the solvent highlighted the need for an internal standard for all quantitative measurements. The inability to separate β-carotene from other cartenoids using the C18 column highlighted the need to utilize the C30 column for adequate separation of the different carotenoids found in serum samples. Achieving a LLQ of 200 nmol/L relied upon using the MRM transition 536.4 to 444.5 as the quantifier ion and 536.4 to 143.1, and to 105.1 as the qualifier ions. Additionally, the addition of 0.1 % BHT to preparations of beta-carotene standards significantly increased the stability of the calibrator at room temperature (22 °C) and autosampler conditions (4 °C), but was not found to significantly improve stability at -70 °C. No carryover was detected. Studies to further assess LLQ, linearity, precision, accuracy, and correlation with a commercially available assay are ongoing.
Conclusion: We developed a simple liquid: liquid extraction procedure using ethyl acetate and a LC-ESI-MS/MS method using a C30 LC column to extract and measure beta-carotene in patient serum, a matrix that contains other types of carotenoids.
References & Acknowledgements:
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