8_PO-CON1692E
PO-CON1692E Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with full-automated sample preparation MSACL 2016 EU Daisuke Kawakami 1 , Davide Vecchietti 2 , Maura Brambilla 3 1 Shimadzu Corporation, Kyoto, Japan, 2 S…
PO-CON1692E
Analysis of 25-OH Vitamin D2/D3
in Serum by LC-MS/MS with
full-automated sample preparation
MSACL 2016 EU
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
2
Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with
full-automated sample preparation
Introduction
Vitamin D measurement has become an important
component in clinical assays largely because deciency 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 specicity; 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 efciency, 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, “For research use only. Not for use in
clinical diagnostics” Shimadzu) to process large sample
sets (Fig. 1). The CLAM-2000 has the ability to perform a
variety of steps appropriate for automated sample
preparation by LC-MS/MS including precipitation,
ltration, heating, shaking, and pipetting. This system is
seamlessly integrated with the LC-MS/MS 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.
Fig. 1 CLAM-2000 and LCMS-8050 system
Place blood collection tubes
in CLAM-2000
Blood serum
3
Analysis of 25-OH Vitamin D2/D3 in Serum by LC-MS/MS with
full-automated sample preparation
Method
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 precipitant solution followed by ltration
and sample collection. Sample preparation involved
taking 30 μL of sample, adding to it 90 μL of precipitant
solution (containing internal standard). Following
ltration, the ltrated 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 (Fig. 2).
The LC-MS/MS instrument was equipped with an
atmospheric pressure chemical ionization source (APCI).
Sample Preparation
5min
LC/MS/MS analysis
3.2min
Sample Preparation
5min
LC/MS/MS analysis
3.2min
4.2min 4.2min
Sample
injection
Sample
injection
Sample
injection
Sample
Preparation
5min
Fig. 2 Analytical Flow with Parallel Processing
Reagent
Dispensing
• 20 µL of MeOH
Sample
Dispensing
• 30 µL of Serum
Reagent
Dispensing
• 90 µL IS
Shaking
• 30sec
Filtration
• 90sec