Gilliland & dowell: Journal of aoaC international Vol. 95, no. 3, 2012 1

INFANT FORMULA AND ADULT NUTRITIONALS

Simultaneous Determination of Vitamins D2 and D3 by LC-MS/MS in Infant Formula and Adult Nutritionals: First Action 2011.13

DonalD l. GillilanD, Charles K. BlaCK, James e. Denison, and Charles T. seipelT Abbott Nutrition, 3300 Stelzer Rd, Nutritional Research Center, Columbus, OH 43219 Dawn Dowell

AOAC INTERNATIONAL, 481 N. Frederick Ave, Gaithersburg, MD 20877-2417

During the “Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting” held on June 29, 2011, an Expert Review Panel (ERP) on behalf of AOAC INTERNATIONAL adopted the method “Simultaneous Determination of Vitamins D2 and D3 by LC-MS/MS in Infant Formula and Adult Nutritionals” as an AOAC Official First Action method. Vitamins D2 and D3 are extracted from the sample using pentane–ether; the extract is collected and dried under nitrogen. Vitamin D is separated from interfering compounds using UPLC, and quantitated using tandem mass spectrometry (MS/MS). Preliminary data showed the intermediate precision ranged from 3.34–8.05% and an accuracy range of 98.5–111% over the samples tested for vitamin D3. For vitamin D2, the intermediate precision ranged from 2.37–5.45% and accuracy ranged from 96.4–104% over the four matrixes evaluated. The analytical range for the method is bounded by the concentrations of the working standards, 21–270 ng/mL, and is equivalent to 0.168–2.16 mcg/100 g in ready-to-feed product. The practical method quantitation limit is 0.168 mcg/100 g product with method detection limit of 60 ng/100 g product. The ERP reviewed the data and determined that the performance characteristics of the method met the standard method performance requirements, and therefore the method was granted First Action status.

based on available validation data, to methods that meet the requirements of standard method performance requirements (SMPRs) established by the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). The ERP-approved methods remain in First Action status for approximately 2 years. During this time, additional information will be gathered by laboratories using the method. The performance of the methods will be monitored by the ERPs, and when the 2 year period is over, a determination will be made by the ERP on whether the method can be recommended to the AOAC Official Methods Board for Final Action (1).

Vitamins D2 and D3

Vitamins D2 and D3 are an essential part of bone health. The majority of dietary vitamins D2 and D3 comes from fortified foods such as milk products (2). Because of the need to assure the levels of supplements in food products are accurate, testing methods are needed that are user-friendly, less time-consuming, and accurate. Because of this need, the method “Simultaneous Determination of Vitamins D2 and D3 in Infant Formula and Adult Nutritionals by LC-MS/MS” was submitted for consideration for approval under AOAC’s new path to Official Methods^SM . The method was reviewed by an ERP at the Mid-Year Meeting and approved as Official First Action Method^SM 2011.13 .

AOAC Official Method 2011.13 Vitamins D2 and D3 in Infant Formula and Adult Nutritionals

LC-MS/MS First Action 2011

n March 28, 2011, a new path for selected methods to obtain Official First Action status was approved by the OAOAC Board of Directors. Under the new process, expert review panels (ERPs) grant Official First Action status,

Submitted for publication April 6, 2012.

The method was approved by the Expert Review Panel on Infant Formula and Adult Nutritionals as First Action. See “Standards News,” (2011) Inside Laboratory Management , July/August issue.

The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit for purpose and are critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author. Corresponding author’s e-mail: don.gilliland@abbott.com DOI: 10.5740/jaoacint.CS2011_13

(Applicable to the determination of vitamins D2 and D3 in infant formula and adult/pediatric nutritional formula by LC-MS/MS.)

Caution: Refer to the Material Safety Data Sheets (MSDS) of chemicals prior to use and follow the suggested personal protective equipment.

A. Principle

The test sample is weighed directly into a 50 mL glass centrifuge tube and inoculated with a stable isotope labeled internal standard. The sample is saponified at 75°C for 30 min with potassium hydroxide to break up the product matrix and facilitate analyte extraction. Vitamin D is extracted using a pentane–ether solvent, which is collected and dried under nitrogen. The dried extract is reconstituted and filtered prior to

2 Gilliland & dowell: Journal of aoaC international Vol. 95, no. 3, 2012

Table 2011.13A. Volumes required for working standard solutions

( 2 ) Phosphoric acid .—85%.

• ( 3 ) Ammonium formate .—≥99.0%.

• ( 4 ) Iso-octane .—≥99.0%.

Std	ISTD volume, µL	SIIS volume, µL	Volume MeOH, µL	Volume water, µL	Final volume, mL
WS1	45	75	1780	100	2
WS2	90	75	1740	100	2
WS3	150	75	1680	100	2
WS4	500	75	1326	100	2
WS5	1000	75	825	100	2

analysis. Vitamin D is separated from interfering compounds using UPLC and quantitated using tandem mass spectrometry. Elevated temperature saponification allows sample preparation to be completed in less than 3 h. Instrument analysis time is 12 min/sample.

B. Apparatus

• ( a ) Column .—Waters HSST 3 1.7 μm, 2.1 × 150 mm (Waters

• Corp., Milford, MA) or equivalent.

• ( b ) Liquid chromatograph .—Waters Acquity UPLC Binary

• Solvent Manager capable of 15 000 psi or equivalent.

• ( c ) Detector .—Waters Quattro Premier XE, Xevo triple

• quadrupole mass spectrometer, or equivalent.

• ( d ) Injector .—Waters Acquity sample manager with

• integrated column oven or equivalent.

• ( e ) Nitrogen generator .—Peak Scientific (Billerica, MA)

• Model NM30LA or equivalent.

• ( f ) Data system. —Waters MassLynx, latest revision or

• equivalent.

• ( g ) Centrifuge tubes .—Glass, 50 mL with Teflon-lined screw

• cap.

• ( h ) Centrifuge .

• ( i ) Water bath .—Capable of 75 ± 2°C.

• ( j ) Nitrogen source .—Purity >99.7% for solvent extract

• evaporation.

• ( k ) Vortex mixer .

• ( l ) Evapo-Rac Evaporator .—Cole-Parmer (Vernon Hills,

• IL), Model N-01610-35 or equivalent.

• ( m ) Balance.— Readable to at least 0.001 g. (Mettler-Toledo

• XS-204, Columbus, OH, or equivalent).

• ( n ) Syringe filters .—PTFE 0.45 μm.

• ( o ) Pipets .—100, 250, and 1000 μL adjustable.

• ( p ) Pipet tips .

C. Reagents

D. Standards

( a ) Vitamin D3 .—USP (Rockville, MD) reference standard No. 1310 (cholecalciferol = vitamin D3). Purity = 40 000 IU/mg. (Consult current USP literature for current Lot No.)

( b ) Trideuterated vitamin D3, 2H3-D3 .—Iso-Sciences (King of Prussia, PA), Cat. No. 3077, ≥97% purity and 97% incorporation of stable-isotope incorporation.

( c ) Vitamin D2 .—USP reference standard No. 1239005 (ergocalciferol = vitamin D2). Purity = 40 000 IU/mg. (Consult current USP literature for current Lot No.)

( d ) Trideuterated vitamin D2,² H3-D2 .—IsoSciences, Cat. No. 5014, ≥98.0% purity and >97% stable-isotope incorporation.

E. Solutions Preparation

( a ) Mobile phase.— ( 1 ) Mobile phase A .—2 mM ammonium formate (aqueous). Quantitatively transfer 126 (±5) mg ammonium formate into a 1 L volumetric flask and add approximately 5 mL laboratory water to dissolve and fill to volume with laboratory water.

( 2 ) Mobile phase B .—2 mM ammonium formate in methanol. Quantitatively transfer 126 (±5) mg ammonium formate into a 1 L volumetric flask and add approximately 5 mL laboratory water to dissolve and fill to volume with laboratory HPLC grade methanol.

( 3 ) Extraction solution .—Ether–pentane (20 + 80).

( b ) Vitamin D3 stock standard (approximately 48 000 ng/mL) .—Aliquot a portion of the solution into glass vials with Teflon lined screw caps. Store at <–10°C. Expiration: 8 weeks.

• ( 1 ) Weigh 24 (±1) mg vitamin D3.

• ( 2 ) Transfer into a 500 mL volumetric flask.

• ( 3 ) Dissolve and bring to volume using iso-octane.

• ( c ) Vitamin D2 stock standard (approximately

• 48 000 ng/mL) .—Aliquot a portion of the solution into glass vials with Teflon lined screw caps. Store at <–10°C. Expiration: 8 weeks.

• ( 1 ) Weigh 24 (±1) mg vitamin D2.

• ( 2 ) Transfer into a 500 mL volumetric flask.

• ( 3 ) Dissolve and bring to volume using iso-octane.

• ( d ) Vitamin D2/D3 mixed intermediate standard (ISTD;

• approximately 540 ng/mL) .—Prepare fresh each day. Expiration: 48 h.

( 1 ) Fill a 50 mL volumetric flask with approximately 25 mL LC-MS grade methanol.

• ( a ) Solvents.— ( 1 ) Pentane .—HPLC grade.

• ( 2 ) Ethyl ether .—Anhydrous. Refer to the appropriate

• MSDS and follow the precautions as substance is hazardous.

• ( 3 ) Acetonitrile .—HPLC grade.

• ( 4 ) Acetone .—HPLC grade.

• ( 5 ) Methanol .—HPLC grade (for sample preparation).

• ( 6 ) Methanol .—LC-MS grade (for LC-MS/MS mobile

• phase preparation).

• ( 7 ) Sodium ascorbate .—Sodium L-ascorbate, crystalline,

• ≥98%.

• ( 8 ) Distilled or deionized laboratory water .—Purified water,

• USP; ≥18Mohm/cm.

• ( b ) Solutions.— ( 1 ) Potassium hydroxide .—45%.

Table 2011.13B. Approximate sample weights

Sample type	Sample size, g
Ready to feed	12
Concentrated liquidsa	6
Powder productsb	12

• a Reconstitute powder samples by weighing 25 g into 200 g laboratory water and mix thoroughly to generate a homogenous mixture prior to sampling for analysis.

b Concentrated liquids are diluted 1:1 with laboratory water after weighing into sample tube.

Gilliland & dowell: Journal of aoaC international Vol. 95, no. 3, 2012 3

Table 2011.13C. Chromatographic parameters

Instrument parameter		Waters Acquity UP	LC
Mobile phase A		2 mM ammonium format	e in water
Mobile phase B		2 mM ammonium formate	in methanol
Flow rate		500 µL/min
Flow rate into MS		500 µL/min
Column		Waters HSST 3 1.7 µm, 2.	1×150 mm
Column temperature		60°C
Injection volume		20 µL
Injection type		Full loop
Pre-sample air boundary		Automatic
Post-sample air boundary		Automatic
Sample temperature		Ambient
Injector wash parameters
Weak wash solvent		Water
Weak wash volume		1000 µL
Strong wash solvent		Methanol
Strong wash volume		600 µL
	Gra	dient method Gradient detail
Time, min	% A	% B	Curve
0.0	10	90	1
3.0	10	90	6
6.0	0	100	7
8.0	0	100	6
8.5	10	90	6
12.0	10	90	6

Table 2011.13D. Mass spectrometer (Quattro Premier/Xevo) operating conditions

Instrument parameter	Setting
Tune page file name
Ionization mode	APCI+
Corona	5.0 µA
Cone voltage	20
Source temperature	130ºC
Desolvation temperature	500ºC
Desolvation gas flow l	1100 L/h
Cone gas flow	50 L/h
Analyzer cell pressure	~2.0 e–5
Collision cell pressure	~2.0 e–3
Low mass 1 resolution	10.0
Low mass 2 resolution	10.0
Ion energy 1	0.5
Entrance lens	–5
Collision energy	15
Exit lens	1
High mass 1 resolution	10
High mass 2 resolution	10
Ion energy 2	1.0
Multiplier	710

( b ) Add 75 μL SIIS and vortex for 10 s to mix.

( c ) Saponification .—( 1 ) Add about 0.4 ± 0.1 g sodium ascorbate and vortex for a minimum of 10 s.

( 2 ) Add 6 ± 0.3 mL HPLC grade methanol and vortex for 15 s.

( 2 ) Transfer 560 μL of each stock standard solution into the 50 mL volumetric flask.

( 3 ) Dilute to 50 mL with LC-MS grade methanol.

( e ) Trideuterated vitamin D2/D3,² H3-D2 /² H3-D3, stableisotope labeled internal standard solution (SIIS; approximately 5000 ng/mL) .—The prepared solution is transferred into singleuse vials and tightly sealed. Store at –70°C. Solution can be used indefinitely until noticeable degradation of the solution concentration is observed.

( 1 ) Quantitatively transfer the entire contents each of an ampule of trideuterated D2 and D3 (1 mg) to a 200 mL volumetric flask using LC-MS grade methanol to aid in dissolution of the standards as well as to wet the flask. Dilute to 200 mL with HPLC grade methanol once the contents of both ampules have been completely transferred to the flask.

( f ) Working standards .—Prepare fresh on day of analysis. Expiration: 48 h.

( 1 ) Add 100 µL water, 75 µL SIIS mixture, the specified volume of native standard mix (ISTD), and methanol per Table 2011.13A .

( 2 ) Cap and mix well.

( 3 ) Transfer to an amber autosampler vial and cap.

F. Procedure—Sample Preparation

( a ) Accurately weigh designated sample size into a 50 mL centrifuge tube ( see Table 2011.13B ).

( 3 ) Add 4 ± 0.3 mL of 45% potassium hydroxide solution and immediately vortex for a minimum of 20 s to thoroughly mix contents.

( 4 ) Place tubes in a preheated water bath at 75 ± 2°C for a minimum of 30 min making sure to vortex for approximately 5 s at approximately 10 and 20 min intervals.

( 5 ) After 30 min, remove the tubes from the water bath and promptly place into an ice bath for a minimum of 30 min to bring them rapidly to room temperature.

( d ) Liquid–liquid extraction .—( 1 ) Add 5 ± 0.3 mL acetonitrile to each tube, cap, and vortex at a moderate speed for a minimum of 5 s.

( 2 ) Add 22 ± 1 mL extraction solvent (20 + 80, ether– pentane) and shake in a wide, semicircular arc across the front of the body 20 times. Invert the tubes with each stroke.

( 3 ) Briefly centrifuge at moderate speed (approximately 300 × g for 1.5 min) to complete layer separation.

( 4 ) Draw off the clear ether–pentane and transfer the top layer to a 50 mL conical centrifuge tube, leaving behind a few milliliters of the ether–pentane mixture.

( 5 ) Evaporate to dryness the transferred ether–pentane layer in a 40 ± 4°C water bath, using a flow of nitrogen over the sample.

( 6 ) Remove the centrifuge tubes from the water bath as soon as evaporation is complete. Observe the extract as it should