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Identification of 1α,25(OH)2-Vitamin D2 and D3 in Serum Samples Using the AB SCIEX Triple Quad™ 5500 System Simplified sample processing and analysis

Adam Latawiec1 and Bruno Casetta2 1 AB SCIEX, Canada; 2 AB SCIEX, Italy Introduction

Over the years Mass Spectrometry (MS) techniques have spread to an ever wider range of clinical analyses supplanting traditional analytical techniques such as immunoassays and UV-Vis

spectroscopic techniques. The challenges are to expand the high sensitivity and specificity of tandem mass spectrometry (MS/MS) to simple instrumental techniques for the preparation and

analysis of real life samples.

Among the large number of papers published by various authors on steroid analysis, we have recently presented a paper[1]

dealing with the measurement of 1α,25(OH)2-vitamin D3, a secosteroid which normally presents several challenging features to the mass spectrometrist. The low level of observed

ionization means a low intrinsic sensitivity at the mass spectrometer level which, when coupled with a very low concentration in the plasma, creates difficulties in obtaining

adequate functional sensitivity. As well, attempts to improve sensitivity by derivatization lead to longer and more complex sample preparation steps and reductions in instrumental

throughput.

This application note describes a novel LC/MS/MS method for the determination of dihydroxyvitamin D3 and D2 (DHVD3 and

DHVD2) from extracted human serum samples. The key features of the method are a 2D chromatographic separation followed by MRM detection of the DHVD species as their lithium adducts.

The use of the lithium DHVD adduct allows for low levels of detection without the use of derivatizing agents thereby greatly simplifying the analytical workflow.

Key Features of the Method

• The sensitive and selective Multiple Reaction Monitoring (MRM) scan function on the AB SCIEX Triple Quad™ 5500 and QTRAP® 5500 provides the highest level of sensitivity

over a wide linear dynamic range.

• The use of dual Phenomenex Onyx Monolithic C-18 columns enhances the separation of complex biological matrices while

providing extremely low back pressures. This allows the use of conventional LC equipment and standard fittings throughout the system.

• Sample clean-up is performed using a POROS R1/10 column providing direct in-line sample clean-up from the protein precipitated sample. Minimal sample clean-up increases

analytical throughput.

• Preparative and analytical column flows are regulated by use of a Valco 10-port valve assembly, allowing the use of

different mobile phase compositions from the dual binary LC pump configuration.

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Experimental Conditions

Serum and plasma samples were processed using a simple acetonitrile protein precipitation step followed by centrifugation.

The extraction procedure is described in Table 1.

For the measurement of 1α,25(OH)2-vitamin D2 and 1α,25(OH)2-vitamin D3, the layout of the HPLC system is shown in Figure 1.

The basis of the 2D-LC method is the “clean-up” of a large

sample volume (100 µL of protein precipitated sample) by way of a perfusion column (POROS R1/10, 4.6 x 50 mm, Applied Biosystems) with a binary gradient from the “loading” pump at a

high flow rate of 1.25 mL/min. The 10-port switching valve diverts the POROS effluent to waste. During this time the analytical column (2 x Onyx C18, 3 x 100 mm – Phenomenex) is

equilibrated at a low flow rate (0.10 mL/min) with a solution containing 0.5 mM Lithium acetate from the “separation” pump.

As the target material begins to elute from the POROS column, the 10-port valve is switched and the target analytes are flushed on to the analytical column as the flow from the “loading” pump is

reduced to 0.30 mL/min. After all of the target material has transferred to the analytical column, the 10-port valve is again switched to the waste position and the “separation” pump

gradient and flow increased to achieve separation on the analytical column.

The AB SCIEX API 5000™, Triple Quad™ 5500, or QTRAP®

5500 LC/MS/MS system is operated with the Turbo V™ source in the electrospray mode. The targeted lithiated ions are monitored in the Multiple Reaction Monitoring (MRM) mode

exploiting the transition m/z 423/369 for the 1α,25(OH)2-vitamin D3 and 435/399 for the 1α,25(OH)2-vitamin D2 . Transitions m/z 429/393 and m/z 441/405 are used for the 1α,25(OH)2-vitamin

D3-d6 and 1α,25(OH)2-vitamin D2-d6 internal standards, respectively.

Figure 2 shows the attainable performance for real-life samples

containing 57 pg/mL of dihydroxyvitamin D3 and 25 pg/mL of dihydroxyvitamin D2 analyzed on the AB SCIEX QTRAP® 5500 system. Under the conditions described an LOQ of

approximately 15 pg/mL (36 pmol/L) for 1α,25(OH)2-vitamin D3 is readily achieved. The useful linear range is from 0 to 250 pg/mL, and is shown in the calibration curve for 1α,25(OH)2-vitamin D3

(Figure 3).

Table 1. DHVD extraction procedure

Aliquot 200 µL Sample/Standard/QC into a 1.5mL polypropylene centrifuge tube

Add 20 µL of Internal Standard (DHVD3-d6 and DHVD2-d6 at 100 ng/mL)

Add 400 µL of acetonitrile to each tube and vortex mix for at least 30 seconds to precipitate proteins

Let stand for 10 minutes in water/ice bath

Centrifuge samples at 14000 rpm for 10 minutes

Transfer clean supernatant to HPLC autosampler vial

Inject 100 µL onto HPLC-MS/MS system

Figure 1. Schematic of HPLC set-up. The sample is e luted from the POROS column with a binary gradient from the “loadi ng” pump. The target analytes are flushed on to the analytical co lumn using a second binary gradient from the “separation” pump.

12

3

4

56

7

9

10

AUTOSAMPLER VALVE

MS/MS

Binary Pump / SeparationA = 0.5 mM LiAcetate in waterB = 0.5 mM LiAcetate in MeOH

10 Portswitching valve

POROScolumn

Dual C-18 columns

Binary Pump / LoadingA = H20

B = 25% ACN in MeOH

8

12

3

4

56

7

9

10

AUTOSAMPLER VALVE

AUTOSAMPLER VALVE

MS/MS

Binary Pump / SeparationA = 0.5 mM LiAcetate in waterB = 0.5 mM LiAcetate in MeOH

10 Portswitching valve

POROScolumn

Dual C-18 columns

Binary Pump / LoadingA = H20

B = 25% ACN in MeOH

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Figure 2. Extracted Ion Chromatogram (XIC) of DHVD3 and DHVD2. XIC of DHVD3 (57 pg/mL) and DHVD2 (25 pg/mL), with DHVD3-d6 internal standard, acquired on the AB SCIEX QTRAP® 5500.

XIC of +MRM (3 pairs): 423.3/369.3 Da ID: DHVD3-1 from Sample 36 (Spike 25 ppt) of Monolithic_new.wiff (Turbo Spray) Max. 1293.0 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e5

4.56 9.704.37 9.29 10.529.86 10.839.166.27 6.566.92 8.518.31

XIC of +MRM (3 pairs): 435.3/399.4 Da ID: DHVD2-1 from Sample 36 (Spike 25 ppt) of Monolithic_new.wiff (Turbo Spray), Smoothed, S... Max. 963.8 cps.

8.80 8.85 8.90 8.95 9.00 9.05 9.10 9.15 9.20 9.25 9.30 9.35 9.40 9.45 9.50 9.55 9.60 9.65 9.70 9.75 9.80 9.85 9.90Time, min

0

100

200

300

400

500

600

700

800

900

1000

1100

DHVD3

DHVD2

Internal StandardDHVD3-d6

XIC of +MRM (3 pairs): 423.3/369.3 Da ID: DHVD3-1 from Sample 36 (Spike 25 ppt) of Monolithic_new.wiff (Turbo Spray) Max. 1293.0 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e5

4.56 9.704.37 9.29 10.529.86 10.839.166.27 6.566.92 8.518.31

XIC of +MRM (3 pairs): 435.3/399.4 Da ID: DHVD2-1 from Sample 36 (Spike 25 ppt) of Monolithic_new.wiff (Turbo Spray), Smoothed, S... Max. 963.8 cps.

8.80 8.85 8.90 8.95 9.00 9.05 9.10 9.15 9.20 9.25 9.30 9.35 9.40 9.45 9.50 9.55 9.60 9.65 9.70 9.75 9.80 9.85 9.90Time, min

0

100

200

300

400

500

600

700

800

900

1000

1100

DHVD3

DHVD2

Internal StandardDHVD3-d6

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Conclusions

This method has been demonstrated to be robust and reproducible. Using this approach, 1α,25(OH)2-vitamin D2 and

1α,25(OH)2-vitamin D3 can be effectively separated from other matrix components with minimal interferences (e.g. ion suppression) in the ion source. With proper set up and

calibration an LOD as low as 10 pg/mL is achievable. The lithium adduct provides additional sensitivity which eliminates the time consuming requirement of derivatization and concomitant clean-

up which is a feature of many other analytical tandem MS procedures.

References 1. B. Casetta, I. Jans, J. Billen, D. Vanderschueren, R. Bouillon, European Journal of Mass Spectrometry 16 (2009) 81.

Figure 3. Calibration curve for DHVD3. Background corrected calibration curve from 0 to 250 pg/mL for DHVD3 in double charcoal stripped human serum.

090929_Test_10ppt_Blank_corrected.rdb (DHVD3-1): "Linear" Regression ("1 / x" weighting): y = 0.000359 x + 5.93e-005 (r = 0.7927)

0 20 40 60 80 100 120 140 160 180 200 220Analyte Conc. / IS Conc.

0.000

5.000e-3

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

0.085

0.089

For Rese arch Use Only. Not for use in diagnostic procedures .

© 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

Publication number: 0460310-01