[Application Note]

INT RODUCT ION

Several recent studies have shown that vitamin D deficiency

is common in adults and children in many parts of the world.

In addition to the well known effects of vitamin D deficiency such

as calcium malabsorption associated with rickets, osteoporosis

and osteomalacia, there is now growing evidence that vitamin

D deficiency may increase the risk of certain cancers and play

a role in many other diseases.1,2 Vitamin D exists in two forms;

vitamin D3 (D3) which is produced in the skin on exposure to

sunlight and vitamin D2 (D2) which is the plant derivative and is

found in many supplementation products. Vitamin D is metabo-

lised in the liver to form 25-hydroxyvitamin D, [25(OH)D] which

is further metabolised in the kidney to form the active metabolite

1,25-dihydroxyvitamin D. The measurement of 25(OH)D is

accepted as the clinical indicator of vitamin D status.3

The assessment of vitamin D status is important in the diag-

nosis of vitamin D deficiency and monitoring supplementation

therapy. More recently, LC/MS/MS has gained popularity

over other methods such as the competitive binding assay,

immunoassay and HPLC to quantify 25(OH)D2 and 25(OH)D3

in an attempt to improve the quality of the assay and reduce

costs. The major issue with immunoassays is that they cannot

differentiate between 25(OH)D2 & 25(OH)D3 and instead, rely

on the cross-reactivity of the antibody with 25(OH)D2 to measure

total 25(OH)D concentration. If that cross-reactivity is less than

100% then D2 therapy may not be monitored effectively.4

Figure 1: System configuration of Waters ACQUITY UPLC / TQD

T H E ANA LYSIS OF 25 -H YD ROX Y V ITAMIN D IN S E RUM USING U P LC /MS/MS

Lisa J Calton, Scott D Gillingwater, Gareth W Hammond, Donald P Cooper

Clinical Business Operations Group, Waters Corporation, Atlas, Park, Manchester, UK

EX PERIMENTAL

A Waters® ACQUITY® Tandem Quadrupole Detector (TQD) coupled to

an ACQUITY UPLC® (Waters Corporation, Manchester, UK) was used

for all analyses. The full system configuration is shown in Figure 1.

The instrument was operated in positive electrospray ionisation mode

using MassLynx™ 4.1 software with auto data processing by the

QuanLynx™ Application Manager.

The compound-dependent cone voltage was optimized to maximise

the abundance of the precursor ion entering the source and

selected to pass through the first quadrupole to the collision cell.

Collision-induced dissociation was facilitated by argon and collision

energy to produce characteristic product ions. Using this information

a specific Multiple Reaction Monitoring (MRM) experiment was

created as shown in Table 1.

LC Conditions

LC System: Waters ACQUITY UPLC System

Column: ACQUITY UPLC BEH C8 Column

2.1 x 50 mm, 1.7 μm

Column Temp: 45 ˚C

Flow Rate: 400 μL/min.

Mobile Phase A: 2mM ammonium acetate + 0.1%

formic acid in water

Mobile Phase B: 2mM ammonium acetate + 0.1%

formic acid in methanol

Gradient: Hold 2min 73%B, 73-98%B in 1.5min

MS Conditions

MS System: Waters ACQUITY TQD

Ionization Mode: ESI Positive

Capillary Voltage: 2.5 kV

Cone Voltage: 24 V

Desolvation Temp: 400 ˚C

Desolvation Gas: 900 L/Hr

Source Temp: 120 ˚C

Collision Gas Flow: 7.10x10-3mbar

[Application Note]

Compound MRM Dwell (secs)

Cone Voltage(V)

Collision Energy(eV)

25(OH)D3 401.35 >159.1 0.05 24 28

25(OH)D3* 401.35 >383.3 0.05 24 10

d6-25(OH)D3 407.35 >159.1 0.05 24 28

25(OH)D2 413.35 >83.1 0.05 24 22

25(OH)D2* 413.35 >395.3 0.05 24 10

Table 1: The tuning parameters used when monitoring for 25(OH)D2 and 25(OH)D3 and the internal standard. * denotes optional qualifier ion

Calibrators and QC’s

A single calibrator and bi-level QC (Chromsystems, Munich, Germany)

were prepared as per the manufacture’s instructions. A low QC was

prepared by pooling human serum and adding a known concentration of

25(OH)D2 and 25(OH)D3. The final concentrations of the low, medi-

um and high QC samples were 19, 27 and 84ng/mL for 25(OH)D2 and

13, 29 and 89ng/mL for 25(OH)D3 respectively. To assess linearity,

calibrators were prepared in mammalian serum over the concentra-

tion range 1-100ng/mL for 25(OH)D2 and 25(OH)D3. The final

concentration was adjusted after the stock solutions were scanned at

264nm.

Sample Preparation

Serum (150μL) was pipetted into a 2mL microcentrifuge

tube (Anachem), 10μL of the internal standard (250ng/mL

hexa-deuterated 25(OH)D3, Synthetica AS, in 80% MeOH/20%

IPA) was added and vortex mixed (10s). 0.2M ZnSO4 (150μL) was

added and vortex mixed (10s) to enhance the response. Methanol

(300μL) was added and mixed (10s) to precipitate proteins present

in the serum. Hexane (750μL) was added to extract the 25(OH)

D. Following mixing for 30secs the sample was centrifuged for 5

mins at 13,000rpm. The hexane layer was removed and placed

into Waters maximum recovery vials and evaporated to dryness

under nitrogen at 50°C. The samples were reconstituted in 75μL

of 70% methanol in water and 20μL was injected using the load

ahead feature of the ACQUITY Sample Manager onto the UPLC/

MS/MS system, giving an injection-to-injection time of 6 minutes.

Ion Suppression

A pooled human serum sample containing low levels of

25(OH)D2 and 25(OH)D3 was processed and the extract analyzed

with post-column addition of the analytes, each at 100ng/mL at

a flow rate of 10μL/min using the integral sample fluidics of the

ACQUITY TQD.

RESULTS

Linearity

The data were processed using QuanLynx™ quantification

software, using the ApexTrack™ integration algorithm. The linear-

ity of the assay was determined by adding a known concentration

of 25(OH)D2 and 25(OH)D3 to the serum over the concentration

range 2.5-100ng/mL. The coefficient of determination (R2) for

25(OH)D3 was >0.999 (Figure 2) and the calculated

concentrations for the calibrators were all within ±4% of the

assigned values. The coefficient of determination (R2) for

25(OH)D2 was >0.997 (Figure 3) and the calculated

concentrations for the calibrators were all within ±10% of the

assigned values.

Figure 2: Serum calibration curve for 25(OH)D3

Compound name: 25(OH)D3 Correlation coefficient: r = 0.999973, r^2 = 0.999946Calibration curve: 0.074922 * x + 0.00256404Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ng/mL0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

[Application Note]

Figure 3: Serum calibration curve for 25(OH)D2.

Accuracy

The accuracy of the assay was determined by the

analysis of external quality control samples from DEQAS

(www.deqas.org). The Chromsystems single point calibrator was

used and a calibration line constructed through zero to calculate

the DEQAS sample concentrations. Passing-Bablok linear

regression (Microsoft Office Excel 2003 with Add-In Analyse-It

version 1.73) was used to compare the Waters 25(OH)D3 results

with the DEQAS LC/MS method mean. All results were within

±11.5% deviation of the expected value (Figure 4).

Figure 4: Passing-Bablok linear regression analysis comparing the Waters 25(OH)D3 results to DEQAS LC/MS method mean

Precision

The intra-assay precision was determined by extracting and

analysing five replicates of the low , medium and high QC

samples. The coefficient of variation (CV) for 25(OH)D over

the three levels were calculated. The inter-assay precision was

determined over five consecutive days using the low, medium and

high QC samples. The results are shown in Table 2.

Low QC Medium QC High QC

25(OH) 25(OH) 25(OH) 25(OH) 25(OH) 25(OH) D2 D3 D2 D3 D2 D3

Intra-assay % CV 5.6 7.5 8.0 3.9 5.1 6.2

Inter-assay % CV 8.9 6.3 9.2 5.5 7.2 5.8

Table 2: Summary of the intra and inter-day precision of the assay

Sensitivity

A chromatogram of the lowest extracted in-house serum

calibrator is shown in Figure 5. Each chromatogram is annotated

with compound name, peak-to-peak signal-to- noise ratio (SNR)

and the concentration. All Responses are above the limit of

detection (SNR 5:1) enabling severely deficient patients to be

detected (<6ng/mL).

Figure 5: Chromatograms to show the lowest calibrator and thesignal-to-noise measurement of each analyte

Compound name: 25(OH)D2 Correlation coefficient: r = 0.998969, r^2 = 0.997939Calibration curve: 0.176983 * x + 0.058252Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ng/mL0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

Scatter Plot with Passing & Bablok Fit

5

10

15

20

25

30

35

40

45

5 15 25 35 45

DEQAS 25(OH)D3

Wat

ers

25(O

H)D

3

Identity

Passing & Bablok (I) f it(0.06 + 1.03x)

95% CI bands

Time2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40

%

2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40

%

413.4 > 82.953.33e4

S/N:PtP=45.38

401.4 > 158.955.08e3

S/N:PtP=29.31

25(OH)D3 4ng/mL

25(OH)D2 8ng/mL

[Application Note]

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, ACQUITY UPLC, ACQUITY, UPLC, MassLynx, TargetLynx, Quattro Premier and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are acknowledged. This application is an example of an assay that can be performed using Waters systems. Complete method validation by the end user is required.

©2008 Waters Corporation. October 2008 720002748EN KK-PDF

Figure 6: Ion suppression profile for 25(OH)D, an expanded view is shown with the elution profile of 25(OH)D2 and 25(OH)D3

The use of a stable isotope labelled internal standard is required

to compensate for the variation in matrix effects observed between

individuals.5

DISCUSSION

A method for the UPLC/MS/MS analysis of 25(OH)D2 and

25(OH)D3 in serum has been developed. The methodology involves

a simple liquid-liquid extraction of the analytes from serum and the

MRM detection of each analyte using two transitions, quantifier and

qualifier ions. Additional confirmation of correctly identifying the

analyte is facilitated by monitoring the qualifier ion ratio. The assay

demonstrates good sensitivity with acceptable intra and inter-day

precision. Using this methodology it is feasible to manually process

and analyse up to 100 samples per day.

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

100

Time3.05 3.10 3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50

%

12

25(OH)D3 25(OH)D2 25(OH)D3 25(OH)D2

CONCLUSION

A method for the analysis of 25(OH)D2 and 25(OH)D3 in serum

has been developed with good linearity, sensitivity and precision.

This method consistently delivers reliable results as compared to

traditional immunoassays.

UPLC/MS/MS allows for the accurate and reliable measurement

of 25(OH)D2 and 25(OH)D3 in serum to prevent the misreporting

of the total Vitamin D concentration in patients who are receiving

D2 supplementation.

REFERENC ES

1. Gorham ED, Garland CF, Garland FC, Grant WB, Mohr SB, Lipkin M, et al. Optimal vitamin D status for colorectal cancer prevention: a quantitative meta-analysis. Am J Prev Med 2007;32:210–6.

2. Garland CF, Gorham ED, Mohr SB, Grant WB, Giovannucci EL, Lipkin M, et al. Vitamin D and prevention of breast cancer: pooled analysis. J Steroid Biochem Mol Biol 2007;103:708 –11.

3. Standing Committee on the Scientific Evaluation of Dietary Reference Intakes Institute of Medicine. DRI Dietary Reference Intakes for calcium phosphorus, magnesium, vitamin D and fluoride. National Academy Press, Washington, DC; 1997.

4. Hollis B. Editorial: The Determination of Circulating 25-Hydroxyvitamin D: No Easy Task. J Clin Endocrinol Metab, July 2004, 89(7):3149–3151.

5. Viswanathan CT. et al. Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays. Pharmaceutical Research 2007; 24(10):1962-1973.

[Application Note]

Ion Suppression

The ion suppression study demonstrated that 25(OH)D3 partially

elutes in a region of ion suppression, as shown in Figure 6.