Analysis of Vitamin D in Foods by Ultra Pressure Liquid Chromatography with Mass Spectrometry Detection (UPLC-MS/MS) Definition: This is an UPLC-MS/MS method for the analysis of Vitamin D2 (ergocalciferol) and Vitamin D3 (cholecalciferol) in food matrices. Scope: This method is applicable to all foods. Range: Limits of Detection & Quantitation are discussed on page 19. Principle: A ground, homogenized sample is mixed with an internal standard solution containing isotopically labeled vitamins D2 and D3, with ethanol, and with potassium hydroxide. The mixture is heated to ~95°C and refluxed under nitrogen. After saponification and mixing, the samples are diluted with water and cooled to room temperature. Samples are quantitatively transferred to a separatory funnel using 40% ethanol in water, followed by liquid-liquid extraction into n-heptane. Extraneous materials present in the n-heptane fraction are removed by performing a sequential liquid-liquid extraction with dilute potassium hydroxide in water, 40% ethanol in water, and water, respectively. The n-heptane fraction is filtered through sodium sulfate to remove residual water and evaporated to dryness using a rotary vacuum system. The sample is reconstituted in methyl ethyl ketone and isopropyl alcohol and interferences removed by elution through a disposable solid phase extraction (SPE) silica gel column. The eluant from the SPE columns are evaporated to dryness and reconstituted in methanol and the samples assayed using a methanol and ammonium formate gradient on an UPLC-MS/MS system equipped with a C18 column. Vitamin D is reported as the quantity of vitamin D2 and D3 present. SPSFAM-INGR-04 Based from Call for Methods 03-09-2012
Transcript
Analysis of Vitamin D in Foods by Ultra Pressure Liquid Chromatography with Mass Spectrometry Detection (UPLC-MS/MS)
Definition: This is an UPLC-MS/MS method for the analysis of Vitamin D2 (ergocalciferol) and Vitamin D3 (cholecalciferol) in food matrices. Scope: This method is applicable to all foods. Range: Limits of Detection & Quantitation are discussed on page 19. Principle: A ground, homogenized sample is mixed with an internal standard solution containing isotopically labeled vitamins D2 and D3, with ethanol, and with potassium hydroxide. The mixture is heated to ~95°C and refluxed under nitrogen. After saponification and mixing, the samples are diluted with water and cooled to room temperature. Samples are quantitatively transferred to a separatory funnel using 40% ethanol in water, followed by liquid-liquid extraction into n-heptane. Extraneous materials present in the n-heptane fraction are removed by performing a sequential liquid-liquid extraction with dilute potassium hydroxide in water, 40% ethanol in water, and water, respectively. The n-heptane fraction is filtered through sodium sulfate to remove residual water and evaporated to dryness using a rotary vacuum system. The sample is reconstituted in methyl ethyl ketone and isopropyl alcohol and interferences removed by elution through a disposable solid phase extraction (SPE) silica gel column. The eluant from the SPE columns are evaporated to dryness and reconstituted in methanol and the samples assayed using a methanol and ammonium formate gradient on an UPLC-MS/MS system equipped with a C18 column. Vitamin D is reported as the quantity of vitamin D2 and D3 present.
SPSFAM-INGR-04 Based from Call for Methods 03-09-2012
Chemicals (equivalent chemicals can be used):
1. n-Heptane, HPLC grade VWR International LLC JT Baker Catalog #JT9177-03
Apparatus (equivalent instruments can be used): Note: Reflux condensers and saponification apparatus are not listed. 1. Analytical Balance (4 decimal places) Mettler Toledo balance AB204
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37. VWR® PTFE Magnetic Stirbar retriever VWR International LLC Catalog # 89026-264 38. 3 mL B-D Disposable syringes VWR International LLC Catalog # 301073
Reagents: 1. Ethanol (40% v/v)
a. Dispense 800 mL 200 proof ethanol into a 2 L volumetric flask. b. Fill to volume with Nanopure deionized water and transfer contents to a bottle
dispenser. c. Stable for three months stored at room temperature.
2. KOH (50% w/v)
a. Wear proper personal protective equipment, e.g. lab coat, nitrile gloves and face shield. (Use extreme caution, since this heats rapidly and can splatter.)
b. Dispense 250 mL of water into a 500 mL beaker chilled into an ice bath equipped with a stir bar and magnetic stirrer.
c. Weigh 250 g ± 0.1 g potassium hydroxide. d. Add in 25 g increments potassium hydroxide to the water until dissolved. e. Stable for two years stored at room temperature.
3. KOH (1M)
a. Weigh 56 ± 0.1 g potassium hydroxide and dispense carefully into a 1 L volumetric flask containing approximately 500 mL of deionized water.
b. Dissolve and mix by hand agitation. c. Fill to volume with Nanopure deionized water and transfer contents to a bottle
dispenser. d. Stable for one year stored at room temperature.
4. Mobile Phase A1: 2 mM NH4COOH
a. Weigh 0.126 g ± 0.001 g of ammonium formate and dispense into a 1 L volumetric flask containing approximately 100 mL of deionized water. Mix well.
b. Fill to volume with Nanopure deionized water, stopper and invert to mix well. c. Vacuum filter through a 0.45 µm nylon filter membrane. d. Stable for six months stored at room temperature.
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5. Mobile Phase B1: 2 mM NH4COOH/MeOH
a. Weigh 0.126 g ± 0.001 g of ammonium formate and dispense into a 1 L volumetric flask containing approximately 100 mL of methanol. Mix well.
b. Fill to volume with LC/MS reagent grade methanol, stopper and invert to mix well.
c. Vacuum filter through a 0.45 µm nylon filter membrane. d. Stable for six months stored at room temperature.
a. Dispense 800 mL LC/MS reagent grade methanol into a 1 L volumetric flask. b. Dispense 200 mL Nanopure deionized water into the 1 L volumetric flask. c. Stopper, invert to mix well. d. Vacuum filter through a 0.45 µm nylon filter membrane. e. Stable for six months stored at room temperature.
a. Dispense 900 mL Nanopure deionized water into a 1 L volumetric flask. b. Dispense 100 mL LC/MS reagent grade methanol into the volumetric flask. c. Stopper, invert to mix well. d. Vacuum filter through a 0.45 µm nylon filter membrane. e. Stable for six months stored at room temperature.
8. Internal Standard Stock Solution Vitamin D2 2H3 (0.1 mg/mL)
a. Dispense 1 ±0.1 mg vitamin D2 2H3 into a 10 mL volumetric flask. b. Fill to volume with methanol. c. Stopper, invert and shake to mix well. d. Briefly sonicate to insure dissolution of materials. e. Stable for one year stored at -20 °C.
9. Internal Standard Stock Solution Vitamin D3 2H3 (0.1 mg/mL)
a. Dispense 1 ±0.1 mg vitamin D3 2H3 into a 10 mL volumetric flask. b. Fill to volume with methanol. c. Stopper, invert and shake to mix well. d. Briefly sonicate to insure dissolution of materials. e. Stable for one year stored at -20 °C.
a. Dispense 200 mL isopropanol into a 1 L volumetric flask. b. Fill to volume with methyl ethyl ketone. c. Stopper and mix well by inversion. d. Stable for one year stored at room temperature.
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a. Dispense 2 mL of isopropanol into a 1 L volumetric flask. b. Fill to volume with methyl ethyl ketone. c. Stopper and mix well by inversion. d. Stable for one year stored at room temperature.
12. Vitamin D mixed Internal Standard Working Stock Solution (1000 ng/mL)
a. Dispense an aliquot of approximately 10 mL methanol into a 25 mL volumetric flask.
b. Dispense 250 µL of the Internal Standard Stock Solution Vitamin D2 2H3 (0.1 mg/mL) into the 25 mL volumetric flask.
c. Dispense 250 µL of the Internal Standard Stock Solution Vitamin D3 2H3 (0.1 mg/mL) into the 25 mL volumetric flask.
d. Fill to volume with methanol. e. Stopper, invert and shake to mix well. f. Stable for one year stored at -20 °C.
13. Vitamin D2 Stock Solution (0.1 mg/mL)
a. Dispense 10 ±0.1 mg of vitamin D2 into a 100 mL volumetric flask. b. Fill to volume with 200 proof ethanol. c. Stopper and invert to mix well. d. Stable for one year stored at -20 °C.
14. Vitamin D3 Stock Solution (0.1 mg/mL)
a. Dispense 10 ±0.1 mg of vitamin D3 into a 100 mL volumetric flask. b. Fill to volume with 200 proof ethanol. c. Stopper and invert to mix well. d. Stable for one year stored at -20 °C.
15. Vitamin D mixed Standard Working Stock Solution (1000 ng/mL)
a. Dispense an aliquot of methanol into a 25 mL volumetric flask. b. Dispense 250 µLs of the Internal Standard Stock Solution Vitamin D2 2H3 (0.1
mg/mL) into the 25 mL volumetric flask. c. Dispense 250 µLs of the Internal Standard Stock Solution Vitamin D3 2H3 (0.1
mg/mL) into the 25 mL volumetric flask. d. Fill to volume with methanol. e. Stopper, invert and shake to mix well. f. Stable for one year stored at -20 °C.
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16. Vitamin D mixed Reference Standard (200 ng/mL)
a. Dispense 1 mL of Vitamin D mixed Internal Standard Working Stock Solution (1000 ng/mL) into a 5 mL volumetric flask.
b. Dispense 1 mL of Vitamin D mixed Standard Working Stock Solution (1000 ng/mL) into the 5 mL volumetric flask.
c. Fill to volume with methanol. d. Stopper, invert and shake to mix well. e. Aliquot in 1 mL increments into vials to use for instrument calibration. f. Prepare fresh with each analytical run.
Maintenance: Consult the respective laboratory equipment or analytical instrument operating manual for preventive and routine maintenance of the equipment utilized in this method: Mettler Analytical Balance GraLab Universal Timer Eppendorf pipette VWR Refrigerator/freezer unit Waters Acquity UPLC system Genevac Rocket Evaporators Waters Quattro Premier LC/MS/MS VWR Vortex Mixer Glas-Col separatory shaker system 6 x 500 mL heating mantle Vacuum manifold SPE 12 port Replace the Waters UPLC HSS C18 column as necessary when the peak symmetry deteriorates. Sample Preparation: 1. Homogenize samples in air-tight, dark containers stored at or below 8 °C. 2. Handling and storage of samples should be in accordance with good laboratory practices.
Allow refrigerated or frozen samples to come to room temperature before weighing. 3. All laboratory work should be performed under yellow or golden fluorescent lighting
conditions for vitamin analysis using either low actinic or covered clear glassware. 4. All laboratory work will be performed in a hood with the exception of the Genevac
Rocket evaporation systems, sample reconstitution with methanol, and sample analysis on the LC/MS/MS.
Safety Precautions: 1. Saponification Manifold
a. Verify the nitrogen back pressure at the second valve on the tank does not exceed 5 psi.
b. Verify that the nitrogen flow through the flow meter attached to the nitrogen line does not exceed 5 mL/minute.
c. Do not close the nitrogen outlet in the hood under any circumstances when the unit is in use.
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d. Do not close the nitrogen valve to a condenser if the condenser is attached to a 500 mL round bottom flask.
e. Do not adjust the water inlet valve to each condenser unit. Use the master valve to turn water on/off.
f. Attach empty round bottom flasks with condensers to manifold slots not occupied with samples instead of closing the individual nitrogen valves.
Potassium Hydroxide a. All laboratory work involving 50% w/v KOH should include wearing of a
laboratory coat, nitrile gloves, safety glasses, and face shield.
1. Turn on the 6 x 500 mL heating mantle to a setting of 1 on the heat control knob (~95°C) 10 minutes prior for intended use in order to allow it to reach temperature equilibrium.
Procedure for Sample Preparation of vitamin premix and food matrices:
2. Label an appropriate quantity of 500 mL round bottom flasks for use in the assay. Due to the limitation of the number of stations on the heating mantle, proceed in sets of six or less.
3. Place a 500 mL round bottom flask on the balance and tare it to zero. 4. Dispense an appropriate quantity of sample into a 500 mL round bottom flask based on
the table below as a guide. Record the weight to 0.001 g for food and 0.0001 g for vitamin d premix samples.
Vitamin D Samples Quantity (grams) RM 25 (RTE Cereal) 2-3
NIST 1849, Infant Nutritional Formula 1 Bread 4-5
Mushrooms 5 Eggs 2-3
Yogurt 20-25 Cheese 2-3
Fish 2-3 Butter 3-5 Milk 20-25
Premix < 0.005
5. Add a stir bar and a few boiling chips to each flask.
6. Dispense approximately 0.5 g ascorbic acid and 0.5 g pyrogallic acid to each boiling flask.
7. Dispense 200 µL of Vitamin D mixed Internal Standard Working Stock Solution (1000
ng/mL) to each boiling flask.
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8. Dispense 80 mL of 200 proof ethanol to each boiling flask using a bottle dispenser.
Rinse down the sides of the flasks when necessary to assure the sample congregates at the bottom of the flask.
9. Don a face shield and dispense 20 mL of 50% KOH to the first set of six (or less)
samples in boiling flasks. 10. Attach a connecting adapter to the neck of each boiling flask and connect the flask to the
reflux condenser on the Saponification Manifold. Turn the magnetic stirrers and cooling water on. Turn on the nitrogen gas at a flow rate such that a steady stream of bubbles will exit the tube immersed into water.
11. Reflux each sample for 15 minutes at ~95 °C.
12. After saponification, remove each boiling flask from the unit and add 50 mL Nanopure water. Mix the contents by gently swirling the boiling flask. Allow the sample to equilibrate to room temperature (place in a cold water bath if necessary to speed cooling).
13. Remove the glass adapter and magnetic stir bar from each boiling flask. Use 200 proof ethanol in a squirt bottle to rinse off the ground glass joint of the glass adapter and the stir bar into the boiling flask to prevent loss of material.
14. For each sample, set up a corresponding numbered separatory funnel on holding racks (500 mL on top rack, 250 mL on bottom rack). Insure stopcocks are closed for each separatory funnel and an appropriate screw cap is available.
15. Transfer the contents of the boiling flask to the corresponding labeled 500 mL separatory funnel. Dispense 50 mL 40% v/v ethanol solution into the boiling flask and mix well by swirling the flask. Transfer this into the 500 mL separatory funnel. Repeat this step.
16. Dispense 75 mL heptane into each boiling flask, swirl and transfer into its respective 500 mL separatory funnel. Seal funnel with twist cap, shake gently a few times, invert, and vent by adjustment of the stopcock. Shake for one additional minute by hand or on the separatory platform shaker (use rubber bands to secure funnels to shaker).
17. Place separatory funnels back onto the holding rack, loosen and/or remove twist cap, and allow phases to separate. Drain the aqueous phase (bottom layer) into the corresponding labeled round bottom flask.
18. Transfer the heptane phase remaining (upper layer) in the 500 mL separatory funnel into the corresponding 250 mL separatory funnel on the bottom row of the white holding racks.
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19. Close the stopcock of the 500 mL separatory funnel. Transfer the contents of the round bottom boiling flasks back into to its respective 500 mL separatory funnel.
20. Add another 75 mL heptane to the boiling flask, swirl and transfer to its respective 500 mL separatory funnel. Gently shake and vent funnel, and then shake vigorously for one minute.
21. Drain aqueous phase (bottom layer) into the boiling flask or a secondary waste container and dispose the aqueous phase into the appropriate hazardous waste drum. Boiling flasks are no longer needed.
22. Dispense the heptane phase from the 500 mL separatory funnel into its respective 250 mL separatory funnel. Rinse the interior walls of 500 mL separatory funnel with a squirt bottle containing 200 proof ethanol and drain the contents into the 250 mL separatory funnel. The 500 mL separatory funnels and caps are no longer needed.
23. Dispense 50 mL of 1 M KOH into each 250 mL separatory funnel. Cap and gently shake the funnels for approximately 30 seconds. Discard the aqueous phase (bottom layer) when the phases have separated.
24. Dispense 50 mL of 40% ethanol v/v to each 250 mL separatory funnel. Cap and gently shake the funnel for approximately 30 seconds. Discard the aqueous phase (bottom layer) up to the emulsified layer when the phases have separated.
25. Dispense another 50 mL of 40% ethanol v/v to each 250 mL separatory funnel. Cap and gently shake the funnel for approximately 30 seconds. Discard the aqueous phase (bottom layer) up to the emulsified layer when the phases have separated.
26. Dispense 100 mL of water into each 250 mL separatory funnel. Cap and gently shake the
funnel for approximately 30 seconds. Discard the aqueous phase (bottom layer) when the phases have separated.
27. Obtain the appropriate quantity of evaporator flasks and add approximately 5 mg of BHT
(a few crystal grains) into each flask. 28. Assemble a sodium sulfate filter to trap water and residual solids for each sample by
placing a swab of glass wool into the bottom of a disposable funnel and adding a layer of anhydrous sodium sulfate on top.
29. Filter the heptane phase from each 250 mL separatory funnel through its respective
sodium sulfate filter into a respectively marked evaporator flask. 30. Quantitatively transfer any residual contents that may have adhered to the interior of the
250 mL separatory funnel with heptane using a squirt bottle into the respective sodium sulfate filter and evaporator flask.
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31. Place the evaporator flasks into the Genevac Rocket Evaporator (the maximum capacity is six flasks), with flasks positioned in parallel to each other for balance. Unoccupied positions are filled with blanking plugs or empty flasks.
Attach the inner rotor lid, close the outer main lid and start evaporation utilizing Method 02: Low BP, which takes approximately 50 minutes for completion.
32. Remove flasks from the evaporator and reconstitute the samples with 1 mL of 99.8:0.2
methyl ethyl ketone:isopropyl alcohol solution. Vortex mix well. 33. Prepare the silica gel solid phase extraction cartridges by placing the appropriate quantity
onto a vacuum manifold with closed stopcocks and clean disposable test tubes as the collection vessels.
34. Fill the cartridge reservoir (~5.5 mL) with 80:20 methyl ethyl ketone:isopropyl alcohol
solution. Open the stopcocks and apply the vacuum to draw the solution through at a slow and consistent rate, collecting the eluant into the test tube inside the vacuum manifold and stopping when the level is just above the top frit by use of the stopcock and vacuum adjustment. Do not dry the packing.
35. Fill the cartridge reservoir (~5.5 mL) with 99.8:0.2 methyl ethyl ketone:isopropyl alcohol
solution. Open the stopcocks and apply vacuum to draw solution through at a slow and consistent rate, collecting the eluant into the test tube inside the vacuum manifold and stopping when the level is just above the top frit by use of the stopcock and vacuum adjustment. Do not dry the packing.
36. Open the vacuum manifold, dispose of the wash solutions and return the glass disposable
test tubes to the unit. 37. Transfer the samples in the evaporation flask reconstituted with 1 mL of 99.8:0.2 methyl
ethyl ketone:isopropyl alcohol solution to the top of the SPE cartridge utilizing the extra long disposable transfer pipettes. Open the stopcocks and apply vacuum to draw solution through at a slow and consistent rate, collecting the eluant into the test tube inside the vacuum manifold and stopping at a level just above the top frit by use of the stopcock and vacuum adjustment. Do not dry the packing.
38. Add 1 mL of 99.8:0.2 methyl ethyl ketone:isopropyl alcohol solution to each evaporator flask. Vortex mix and transfer the contents to the top of the respective SPE cartridge utilizing the extra long disposable transfer pipettes. Evaporation flasks are no longer needed.
39. Open the stopcocks and apply vacuum to draw solution through at a slow and consistent
rate, collecting the eluant into the test tube inside the vacuum manifold and stopping at a
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level just above the top frit by use of the stopcock and vacuum adjustment (~2 mL eluant collected). Do not dry the packing.
40. When the receiving test tube becomes full, transfer the eluant collected into a properly
labeled clean evaporator flask containing ~5 mg of BHT (a few crystal grains). Use a squirt bottle containing methyl ethyl ketone (neat) to insure quantitative transfer from the test tube into the rotary evaporator flask.
41. Fill the cartridge reservoir (5 mL) with 99.8:0.2 methyl ethyl ketone:isopropyl alcohol solution. Open the stopcocks and apply vacuum to draw solutions through at a slow and consistent rate, collecting the eluant into the test tube inside the vacuum manifold and stopping at a level just above the top frit without drying by use of the stopcock and vacuum pressure. Do not dry packing. Repeat this step in a similar manner for a total of five iterations, so that a total of ~27 mL eluant is collected. Please note, a 16 x 125 mm disposable borosilicate test tube has a maximum volume of ~17 mL, e.g. the second 5 mL elution will require sample transfer from the test tube to a rotary evaporator flask (~12 mL).
42. After all the eluant has been collected (~27 mL), the test tube and solid phase cartridge
can be disposed of. 43. Transfer the flasks to the Genevac Rocket Evaporator utilizing Method 02: Low BP,
which takes approximately 50 minutes for completion. 44. Reconstitute the samples with 1 mL of methanol, vortex mix well. Transfer an aliquot
with a 9” disposable transfer pipet into a 3 mL disposable syringe equipped with a 13 mm syringe filter. Filter the sample into a HPLC vial containing an insert1
. Assay by LC/MS/MS.
1 It is recommended to save the remaining solution in a separate autosampler vial as a retain and store at 5ºC until the assay results have been reported.
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Parameter
UPLC Operating Conditions
Inlet Method Vitamin D Value
Column HSS C18 UPLC 2.1 x 100 mm, 1.8 μm Column Temperature 40°C Sample Temperature 14°C Sample Loop Full loop,
offline=1.0 minute Flow Rate 0.4 mL/minute Injection volume 20 μL Seal Wash 1.0 minute Run Time 6.0 minutes Wash Solvents Weak (600 μL) Strong (600 μL)
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1. Calculate the Response Factor for the vitamins in the Vitamin D Reference Standard solution with respect to the corresponding isotopically labeled internal standards.
Calculations:
𝑅𝑅𝑅𝑅 = 𝑃𝑃𝑃𝑃𝑎𝑎 𝑥𝑥 𝐶𝐶𝑖𝑖𝑖𝑖𝑃𝑃𝑃𝑃𝑖𝑖𝑖𝑖 𝑥𝑥 𝐶𝐶𝑎𝑎
RF = Response Factor PAa = Peak area of analyte Cis = Concentration of respective internal standard PAis = Peak area of respective internal standard Ca = Concentration of analyte
2. Calculate the Concentration (IU/100 g) of the vitamin in the samples. I think you should calculate in ng/100g, then have a 3rd equation for conversion to IU.
𝐶𝐶 =𝑃𝑃𝑃𝑃𝑎𝑎 𝑥𝑥 𝐶𝐶𝑖𝑖𝑖𝑖 𝑃𝑃𝑃𝑃𝑖𝑖𝑖𝑖 𝑥𝑥 𝑅𝑅𝑅𝑅𝑎𝑎
𝑥𝑥 1𝑊𝑊
𝑥𝑥10025
C = Concentration of analyte in sample IU/100 g PAa = Peak area of analyte in sample Cis = Concentration of respective internal standard added to sample (ng) PAis = Peak area of respective internal standard in sample RFa = Response Factor of analyte calculated from equation 1 above W = Weight of sample in grams 25 = Conversion factor from ng to IU, e.g. 25 ng=1 IU 100 = Conversion factor to IU/100 g
SPSFAM-INGR-04 Based from Call for Methods 03-09-2012
System Linearity Study: The linearity of Vitamin D2 and D3 was performed in the range of 0.005-50 ug/mL. The data demonstrates a linear response for the instrument over the concentration range studied.
y = 49718x + 3357.1R² = 0.9996
y = 80994x + 23542R² = 0.9977
0
1000000
2000000
3000000
4000000
5000000
0 10 20 30 40 50 60
Peak
Are
a Re
spon
se
ug/mL
Vitamin D2 & D3 Linear Regression
vitamin d2
Limit of Detection and Quantitation The limits of detection and quantitation of the assay were calculated based on the standard deviation (SD) of the slope response and the slope (S) of the calibration curve according to the formulas:
𝐿𝐿𝐿𝐿𝐿𝐿 = 3.3 𝑆𝑆𝐿𝐿𝑆𝑆
𝐿𝐿𝐿𝐿𝐿𝐿 = 10 𝑆𝑆𝐿𝐿𝑆𝑆
The LOD and LOQ for Infant Formula are calculated from the Injection Solution LOD & LOQ using a typical 1g sample weight and 1 mL final solution volume. Greater sensitivity can be attained by adjusting sampling weight and final solution volume.
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Precision The precision of the method was evaluated by the analysis of different food matrices in triplicate over a five day timeframe. Most foods contained Vitamin D3, with the exception of the irradiated mushrooms containing only Vitamin D2. The whole eggs contained both Vitamin D2 and D3. Vitamin D Precision IU/100 g Results AOAC Sector 1 2/6 4 4 5 5 5 7 9
% rsd 7.63 6.43 8.90 12.25 12.94 10.10 13.10 13.26 13.52 Accuracy Accuracy was performed on four matrices at four levels and analyzed according to the method and the % recovery calculated from the slope of the linear regression.
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The accuracy of the assay was also examined by a comparison of the mean LC/MS/MS precision results to the HPLC-UV analysis of the samples and the label claim of the products used in the validation study. Vitamin D IU/100 g
Robustness During the validation studies, two different lots and lengths of the Waters UPLC HSS C18 column were used (100 mm s/n 010738038154 12, and 50 mm s/n 011730124156 71) with no significant impact on the results obtained. Evaluation of an alternative stationary phase was performed on the Waters UPLC HSS T3 column (100 mm s/n 0116392741) resulting in poor peak symmetry and is not recommended. Various manufacturers and lots of materials used in mobile phase preparation and solid phase extraction cleanup were performed with no aberrations observed. Depending upon availability, the Waters Quattro Premier XE Micromass LC/MS/MS units2
2 UPLC and LC/MS/MS systems located in laboratory 8-123 and 10-105, respectively.
(s/n VAB 1100 and s/n 880) were used intermittently throughout the study. The sample solution stability was inherent to the validation study due to accessibility to the extraction equipment or the LC/MS/MS; finding samples stored post solid phase extraction in methanol at 5ºC in enclosed containers were stable for five days. No significant difference in Vitamin D content was observed in samples stored in the dark in an enclosed container of heptane containing BHT or dried down in an enclosed rotary evaporator flask after three days at room temperature, e.g. stoppered rocket flask in cabinet or left in the unit over the weekend. Various grain matrices were evaluated as Wheat Chex, Rice Chex, and Corn Chex finding no significant matrix interference present.
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Measurement Uncertainty The measurement uncertainty was calculated for the standard reference material by the combined use of the precision data (n=15) from the validation and the analytical competence data (n=18) obtained during technical transfer of the method according to the formula:
𝜇𝜇 = 𝑘𝑘𝑘𝑘 µ= measurement uncertainty, Vitamin D IU/ 100 g k= coverage factor of 2 obtained from Student’s T tables with respect to degrees of freedom σ= standard deviation of data points SRM25 F Measurement Uncertainty
Mean σ k µ235.6639 26.12749 2.04 53.30
Standard Reference Material(s): The GMI RM 25 (RTE Cereal) series is the recommended reference material for this method. RM Handling: 1. Label RM containers with date received.
2. Store RM containers in a desiccator.
3. Record date opened.
Frequency of RM Analysis: 1. Under normal circumstances, analyze the RM once every shift, or at least once every day
the analysis is conducted.
References: 1. Filling the AOAC Triangle with food matrix standard reference materials, Sharpless et al.
Anal Bioanal Chem, 2004 Mar, 378(5) 1161-7. 2. AOAC Method 995.05 Vitamin D in Infant Formulas and Enteral Products. 3. AOAC Method 992.25 Vitamin D3 in Ready-to-Feed milk based infant formulas. 4. AOAC Method 2002.05 Cholecalciferol (Vitamin D3) in selected foods.
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