ADV06-15_1-412

www.restek.com

By Shun-Hsin Liang, Sharon Lupo, Frances Carroll, Ty Kahler, and Paul Connolly

• Separate target analytes in just minutes for faster sample throughput.

• Report accurate results with confidence based on validated method performance.

• ARC-18 column endures low-pH mobile phases without sacrificing retention or peak quality.

Vitamin D deficiency has been linked to an increased risk for many

chronic diseases including diabetes, heart disease, autoimmune

diseases, and some cancers. Vitamin D exists in two forms: vitamin

D2 and vitamin D3. While vitamin D3 is an endogenous nutrient

that the human body can synthesize, vitamin D2 must be obtained

from dietary sources, such as dairy products and fish. These parent

compounds undergo metabolism to form 25‐hydroxyvitamin D2 and

25‐hydroxyvitamin D3. For accurate determination of vitamin D levels

in the blood, it is important to distinguish between these metabolites

and to separate them from major matrix interferences.

Separating fat‐soluble vitamins by LC can be quite time-consuming,

taking up to 20 minutes or longer by some methods. However, the

new Raptor™ ARC‐18 LC column can analyze these difficult com-

pounds using reversed-phase chromatography (RPC) in less time than

traditional columns, which helps increase sample throughput and

overall lab productivity. In the method developed here, the Raptor™

ARC‐18 column combines the speed of superficially porous particles

(SPP) with the resolution of highly selective USLC® technology to pro-

duce a simple and accurate method for the determination of vitamin

D metabolites in plasma.

Fast Analysis Times Improve Productivity

The Raptor™ ARC-18 column was selected for this method because

its resolving power allows accurate determination of both forms of

vitamin D as well as the metabolites. It was also chosen because it

performs well with the low pH mobile phases used to promote ion-

ization in MS detection. Prior to evaluating the method with fortified

samples, the suitability of the Raptor™ ARC-18 column for the analysis

of vitamin D metabolites was established using a neat standard solu-

tion. As demonstrated in Figure 1, all compounds were separated with

an analysis time of less than 4 minutes, while the metabolites specifi-

cally targeted here eluted in less than 2 minutes. This allows reliable

quantitative data to be generated quickly, so sample throughput can

be increased.

Improve Sample Throughput for LC‐MS/MS

Analysis of Vitamin D Metabolites in Plasma

With a New Raptor™ ARC-18 Column

Figure 1:

The Raptor™ ARC-18 column makes quick work

of analyzing vitamin D and metabolites by LC-MS/MS.

Time (min)

0.0 0.5 1.0

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Conc.

Peaks

t R (min) (ng/mL)

1. 1,25-Dihydroxyvitamin D3 0.88

200 399.4 381.5

2. 25-Hydroxyvitamin D3

1.33

200 401.5 383.5

3. 25-Hydroxyvitamin D2

1.41

200 413.5 395.5

4. Vitamin D2

3.47

200 397.5 379.6

5. Vitamin D3

3.53

200 385.5 367.5

LC_CF0586

Column:

Raptor™ ARC-18 (cat.# 9314A12); Dimensions: 100 mm x 2.1 mm ID; Particle Size: 2.7 µm;

Temp.: 40 °C;

Sample:

Diluent: Methanol; Conc.: 200 ng/mL; Inj. Vol.: 5 µL;

Mobile Phase:

A: 0.1%

Formic acid + 5 mM ammonium formate in water B: 0.1% Formic acid + 5 mM ammonium formate in

methanol;

Gradient (%B):

0.00 min (90%), 4.00 min (100%), 4.01 min (90%), 6.00 (90%);

Flow:

0.5 mL/min;

Detector:

ABSCIEX API 4000™; Ion Source: TurboIonSpray®; Ion Mode: ESI+;

Instrument:

Shimadzu UFLC

Separate target

analytes in just

minutes!