Neus Fabregat-Cabello (Presenter)
University of Liège, CHU de Liège
Bio: Neus Fabregat Cabello is Post-Doc at the Department of Clinical Chemistry, University Hospital of Liège (Belgium) under the supervision of Prof. Etienne Cavalier since November 2015. Her educational background includes a degree in Chemistry (2010), a Master in Applied Chromatographic Techniques (2011), and a Ph.D. in analytical chemistry (2015) from the University Jaume I (Castellón, Spain). Last years she has focused her expertise on different hyphenated techniques, involving chromatography and mass spectrometry, including LC-MS/MS, LC-ICP-MS and GC-MS. At the moment she is responsible for the development of new mass spectrometry applications based on LC-MS for endocrinology research dealing with small molecules, most of them related to vitamin D metabolites.
Authorship: Neus Fabregat-Cabello(1), Jordi Farre-Segura(1), Loreen Huyghebaert (1), Stéphanie Peeters(1), Caroline Le Goff(1) and Étienne Cavalier(1).
(1)Department of Clinical Chemistry, University of Liège, CHU Sart-Tilman, Liège, Belgium
We present here a rapid, easy, reliable and cost-effective method for the quantification of 25-hydryxoyvitamin D2 and D3, epi-25-hydroxyyvitamin D3 and 24,25-dihydroxyvitamin D3 by LC-MS/MS in serum samples. The proposed methodology has been strongly validated with both NIST and Labquality materials, obtaining mean intra-assay and inter-assay imprecision lower than 6 and 6.4% and mean recoveries within 95-104%. Besides we have compared satisfactorily samples from Vitamin D Standardization Program (n=80) with reference values and patient samples (n=281) with our reference LC-MS/MS method. The proposed methodology is prepared to be used in routine testing and permits the calculation of the Vitamin D Metabolite Ratio (VMR).
Vitamin D (VTD) is an important prohormone required for correct bone and muscle development and maintenance. Its deficiency is a risk factor for metabolic bone diseases and, as recently discovered, for numerous non-skeletal conditions such as autoimmune diseases, cardiovascular diseases and cancer, among others. The important function that this compound plays in human health has aroused, during the last decades, a great interest in accurate assessment of Vitamin D status in humans.
Nowadays there is a general agreement that measuring 25-hydroxyvitamin D (25(OH)D) levels in blood is the best way to estimate VTD status. In routine, its quantification is mainly conducted by immunoassays due to the high throughput and the reduced sample preparation. However, these techniques lack specificity since they are not able to resolve some other metabolites, such as 24,25-dihydroxyvitamin D (24,25(OH)2D) or epimers, that provide a better insight in a patient’s status when they are analyzed simultaneously.
Indeed, very recently, a new candidate for vitamin D status has been proposed, namely the ratio of serum 24,25(OH)2D to 25(OH)D, also known as Vitamin D Metabolite Ratio (VMR). In contrast to other biomarkers, the VMR does not differ significantly between races and better describes the status of patients suffering from vitamin D deficiency. Since 24,25(OH)2D is the major product of 25(OH)D catabolism, the levels of both 25(OH)D and 24,25(OH)2D are strongly related in healthy persons.
Contrary to 25(OH)D quantifications, the measurement of 24,25(OH)2D can only be achieved by LC-MS/MS approaches. Besides, its determination is still complicated due to its low ionization efficiency and its low concentration levels. Most current LC-MS/MS methodologies which are able to quantify simultaneously 25(OH)D and 24,25(OH)2D rely on time-consuming sample preparation, based on protein precipitation followed by liquid-liquid extraction (LLE), solid phase extraction (SPE) or supported liquid extraction (SLE). Then the enhancement ionization efficiency of 24,25(OH)2D usually relies on a derivatization step with a Cookson-type reagent, thus increasing even more the sample preparation time and complexity.
Liquid chromatography-mass spectrometry. Identification and quantification of the selected compounds was carried out with an UHPLC system using a Nexera X2 UPLC interfaced to a quadrupole-linear ion trap QTRAP 6500 system from Sciex. Chromatographic separation was achieved using a Kinetex PFP 100A column fitted with a UPLC SecurityGuard ULTRA cartridge from Phenomenex. The mobile phase was a time programmed gradient using A (water) and B (methanol), both modified with 0.1% formic acid.
Analytes were ionized by atmospheric pressure chemical ionization (APCI) in positive mode (+) and detected by MS/MS in Selected Reaction Monitoring (SRM).
Sample preparation. Our proposed sample preparation was based on a protein precipitation of 100 uL of serum sample by adding ZnSO4•7H2O in water and 100µL of the labelled compound mix in acetonitrile. Then samples were vortexed and centrifugated and 150 uL of the supernatant was transferred to a 96-well plate.
For comparison purposes with our LC-MS/MS VDSP (Vitamin D Standardization Certification Program) traceable method, we obtained serum samples of apparently Caucasian healthy subjects supplemented with vitamin D (n=50), stable hemodialyzed patients (n=50), 3rd trimester pregnant women (n=50), women referred to specialized osteoporosis clinic (n=50), CKD patients with glomerular filtration rate <30 (n=50) and African healthy subjects from Abidjan, Côte d’Ivoire (n=31).The study was conducted in accordance with the Ethics Committee of the CHU de Liège. All samples were stored at -80ºC a maximum of 6 months before processing.
Method validation. Precision and accuracy were evaluated by analyzing the reference material 972a from the NIST together with six levels form the reference Serum Panel for 25(OH)D, which was purchased from Labquality Oy. Accuracy was also assessed from measurement of samples from proficiency testing programs from the Center for Disease Control and Prevention (CDC), included in the VDSP standardization program (n=80) and Vitamin D External Quality Assessment Scheme (DEQAS).
Method development. Mass spectrometry parameters where optimized for the an in-source fragment with a water loss [M-H2O+H]+ for the selected compounds. However, for 24,25(OH)2D3 we found the precursor ion [M+H-2H2O]+, which provides an increased sensitivity as well as selectivity that has not been described previously.
For sample preparation we tested three different procedures: LLE with 1 mL of hexane, SLE also with hexane and protein precipitation (data not shown). In order to enhance analysis throughput while reducing cost, we focused on protein precipitation for further optimization.
Method Validation. For validation purposes ten reference materials were analyzed in triplicate during three different days. Matrix effects were obtained with triplicate experiments achieving a matrix enhancement of 4% for 24,25(OH)2D3 while a matrix suppression of 20%, 26% and 22% was observed for 25(OH)D3, 25(OH)D2 and epi-25(OH)D3, respectively.
Mean recoveries for all analytes were satisfactory, being within 95-104%. Coefficients of variation or CVs(%) for intra-assay(repeatability) and inter-assay (intermediate precision) were lower than 6.4 % in all vitamin D metabolites. The estimated lower LOQ’s were 0.5 µg/l for 24,25(OH)2D3, 1.1 µg/l for 25(OH)D3 and epi-25(OH)D3 and 1.7µg/l for 25(OH)D2.
For the clinical validation, we tested 80 reference samples provided by the CDC for the VDSP program and compared them to the established total vitamin D value by the reference method. The Passing-Bablok regression equation that we obtained for 25(OH)D3 was APCI-PFP method=-0.44+0.97CDC. A slope of 0.97 accounts for a 3% bias which is in accordance with the VDSP recommendations that accepts a ±5% bias. With a 95% CI, slope was between 0.95 and 1.00 and the intercept between −1.2 and 0.03.
Method comparison. For comparison purposes, we determined all the analytes in different population groups, achieving a total number of 281 samples. The “true” value for 25(OH)D in these samples was determined with our VDSP-traceable LC-MS/MS method from Chromsystems® (MassChrom® 25-OH-Vitamin D3/D2 (LC-MS/MS), Chromsystems, http://www.chromsystems.com).
Conclusions & Discussion
In this work we have focused on the minimization of sample manipulation and solvent consumption in order to pursue an easy implementation in routine laboratories. As a consequence we have developed a UHPLC-MS/MS method for the simultaneous quantification of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and epi-25(OH)D3 in human serum samples. The proposed assay utilizes a simple, fast, easy and cost-effective sample preparation. Besides we have obtained a low limit of quantification of 0.5 µg/l for 24,25(OH)2D3 without derivatization with a sample volume of only 100uL.
The present methodology has been strongly validated using all the certified reference materials available which makes it reliable for its application. Besides, a comparison of 80 samples with the values from VDSP demonstrates its suitability, as well as y an extensive comparison of 281 samples with our VDSP traceable LC-MS/MS.
References & Acknowledgements:
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