MSACL 2016 EU Abstract

Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with Full-automated Sample Preparation

Daisuke Kawakami (Presenter)
Shimadzu Corporation

Authorship: Daisuke Kawakami (1), Davide Vecchietti (2), Maura Brambilla (3)
(1) Shimadzu Corporation, Kyoto, Japan, (2) Shimadzu Italia, Milano, Italy, (3) Mass spectrometry Toxycology laboratory, Hospital Desio, Italy

Short Abstract

Vitamin D measurement has become an important component in clinical assays largely because deficiency is associated with a number of disorders. LC-MS/MS has become essential tool for monitoring the concentration of vitamin D2/D3 in biological samples due to its high level of sensitivity and specificity; however, manual sample preparation often involves several complicated manual steps which can introduce error into the results. In this study, we investigated the ability to analyze for 25-OH Vitamin D2 /D3 by LC-MS/MS using automated sample preparation to process large sample sets.

Long Abstract

Introduction

Vitamin D measurement has become an important component in clinical assays largely because deficiency is associated with a number of disorders, such as rickets, osteomalacia and osteoporosis.

LC-MS/MS has become essential tool for monitoring the concentration of Vitamin D2/D3 in biological samples due to its high level of sensitivity and specificity; however, manual sample preparation often involves several complicated steps which can introduce error into the results. Additionally, the time consuming nature of the sample preparation and the large number of samples makes LC-MS/MS a less desirable method. Automated sample preparation has been shown to eliminate human error, as well as increase laboratory efficiency, making LC-MS/MS a feasible method to incorporate in the clinic.

In this study, we investigated the ability to analyze for 25-OH Vitamin D2/D3 by LC-MS/MS (LCMS-8050, Shimadzu) using automated sample preparation (CLAM-2000, gFor research use only. Not for use in clinical diagnosticsh Shimadzu) to process large sample sets. The CLAM-2000 has the ability to perform a variety of steps appropriate for automated sample preparation by LC-MS/MS including precipitation, filtration, heating, shaking, and pipetting. This system is seamlessly integrated with the LCMS system requiring no human involvement after loading the biological samples into the sample chamber. We validated the automated method by using a kit containing standard compounds.

Methods

Compounds were measured using a commercially available test kit ClinMass® LC-MS/MS Complete Kit for 25-OH-Vitamin D2 / D3, MS7000 (RECIPE Chemicals + Instruments GmbH, Dessauerstraße 3, 80992 München, Germany). Calibrators, control samples, analytical column and mobile phase solvents were provided by the kit. These calibrators and controls were loaded directly into the CLAM-2000 for sample processing. The CLAM-2000 was programmed to perform protein precipitation using acetonitrile followed by filtration and sample collection. Sample preparation involved taking 30 ƒÊL of sample, adding to it 90 ƒÊL of precipitant solution (containing internal standard). Following filtration, the filtrated sample is then transported using an arm from the CLAM-2000 to the HPLC for LC-MS/MS analysis and no human intervention was required. The LC-MS instrument was equipped with an atmospheric pressure chemical ionization source (APCI).

Results

The calibration curves showed good linearity (R^2>0.998) over a clinical relevant range of 4.10-68.5 ƒÊg/L for 25-OH Vitamin D2 and 4.68- 77.3ƒÊg/L for 25-OH Vitamin D3. The reproducibility (N=7) at three concentrations, including LLOQ, of each compounds was excellent (CV<6.5%). Different day reproducibility (N=7) for 3 days at three concentrations as well (CV<7.2%). Comparison of 25-OH Vitamin D3 concentration between manual sample preparation and automated sample preparation using serum from human samples who have Vitamin D intake shows good agreement. The correlation coefficient of the automated operation against the manual operation was excellent (R^2>0.9).

Conclusions

We completed 25-OH Vitamin D2/D3 analysis using a LC-MS/MS coupled to an automated sample preparation system. The results shows the capability of the system for large sample set analyses with improved accuracy and precision by eliminating human error associated with manual sample handling.

Novel Aspect

Automated sample preparation which interfaces directly to a fast scanning LCMS triple quadrupole improved reproducibility while achieving increased sample prep efficiency.


References & Acknowledgements:


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