tn-0111 applications€¦ · 200 rpm under room temperature for 1 hour 4) add 5 stml di water to 1...
TRANSCRIPT
Xianrong (Jenny) Wei Senior ScientistJenny is a Senior Scientist in the Phenomenex PhenoLogix applications laboratory.
TN-0111
For additional technical notes, visit www.phenomenex.com Page 1 of 8
APPLICATIONS
Vitamin D3 Pre-Vitamin D3
Vitamin D2 Pre-Vitamin D2
Determination of Vitamin D2/D3 and Pre-D2/D3 in Pet Food by LC/MS/MSXianrong (Jenny) Wei, Matthew Trass, Allen Misa, Scott Krepich and Sean OrlowiczPhenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
The same challenges in determining Vitamin D2/D3 in foods and dietary supplements, such as matrix interferences, isomer conversions, and light sensitivity persist in pet food matrices. In this technical note, we describe an LC/MS/MS method with a simplified saponification and extraction procedure suitable for a variety of pet food samples.
IntroductionVitamin D is an essential fat soluble vitamin, most known for its requirement in bone formation in animals amongst myriad other likely beneficial functions. Vitamin D3 is typically produced natu-rally with exposure to sunlight, and may need to be supplemented in diets if not adequately exposed (both humans and animals, par-ticularly under modern farming conditions). In animals, the con-sensus is that Vitamin D3 is the most biologically active form, and thus needs to be distinguished apart from Vitamin D2, periodically sourced from some plants and mushrooms. The isomeric forms, pre-vitamin D2 and pre-vitamin D3, are also important to quantify accurately without disrupting their natural equilibrium to accurately assess their source.
Experimental ConditionsReagent and chemicals All solvents and reagents were HPLC or analytical grade. Reference standards of Vitamin D2/D3 and its isotope internal standard of Vitamin D2-d3 and Vitamin D3-d3 were purchased from Cerilliant®; Pre-vitamin D2/D3 were from Toronto Research Chemicals. Samples from Purina®, Dog Chow®.
Figure 1. Vitamin D and Pre-Vitamin D Structures
Equipment and materialsAgilent® 1260 SL pumps and autosampler were used along with a SCIEX 4500™, positive polarity, APCI for detection.
Sample PreparationMatrix: Purina, Dog Chow (made with real chicken, for small dog), dry dog food.
Samples: Blank (n=1), Blank/IS (n=1), 4 Standards at concentration of STD (D2/D3): (Pre-D2/Pre-D3) 0/0 µg/mL, 0.5/0.25 µg/mL, 1/0.5 µg/mL, 5/2.5 µg/mL (n=1), and QC1 at 1.0/0.5 µg/mL, QC2 at 4/2 µg/mL (n=3 at each QC level).
Two stock solutions were prepared in 40 % ethanol at Conc. 40/40 µg/mL for Vitamin D2/D3, and 20/20 µg/mL for Pre-D2/Pre-D3
Saponification/Pre-extraction/LLE Extraction Procedure:
1) Weight ~2 g blended dog food powder + 0.2 g ascorbic acid + 0.2 g pyrogllic acid into 1st 50 mL centrifuge tube, mix, spike with corresponding standard stock solution to make STDs and QCs.
2) Add 100 µL of IS stock mix (10/10 µg/mL of Vit D2-d3/Vit D3-d3) and 8 mL ethanol to all samples except blank, mix/shake for the pre-extraction at 200 rpm for 15 min
3) Add 2 mL of 45% KOH to all samples, mix/shake/incubate at 200 rpm under room temperature for 1 hour
4) Add 5 mL DI water to 1st 50 mL centrifuge tube, shake at 200 rpm for 15 min, centrifuge at 4500 rpm for 5 min, transfer upper supernatant (dark brown color) to 2nd 50 mL centrifuge tube
5) Add 5 mL 40% ethanol to 1st 50 mL centrifuge tube, shake at 200 rpm for 15 min, centrifuge at 4500 rpm for 5 min, transfer/combine upper supernatant (dark brown color) to corresponding 2nd 50 mL centrifuge tube, discard 1st 50 mL centrifuge tube
6) Add 6 mL heptane to 2nd 50 mL centrifuge tube, shake at 200 rpm for 15 min, centrifuge at 4500 rpm for 5 min, transfer upper supernatant (light yellow color) to 100 X 16 mm glass tube by plastic transfer pipettes, dry samples in TurboVap at 30°C under N2 for 20 min
7) Add 6 mL heptane to 2nd 50 mL centrifuge tube again, shake at 200 rpm for 15 min, centrifuge at 4500 rpm for 5 min, transfer upper supernatant (light yellow color) to corresponding 100 X 16 mm glass tube by plastic transfer pipettes, discard 2nd 50 mL centrifuge tube, dry samples in TurboVap at 30°C under N2 about 10-15 min until liquid volume remaining is ~3 mL (DO NOT DRY DOWN ALL LIQUID VOLUME)
CUR: 20
TEM: 350
GS1: 70
GS2: 0
CAD: 8
NC: 2
DP 60
Sample ID Recovery (%)
Vitamin D2 38.0
Vitamin D3 33.7
Pre-Vitamin D2 30.0
Pre-Vitamin D3 31.4
Vitamin D2 Vitamin D3 Pre-Vitamin D2 Pre-Vitamin D3
Sample ID Conc. Found (µg/mL) - QC 1
Norminal Conc. (µg/mL) 1 1 0.5 0.5
QC 1 1 1.08 0.979 0.439 0.490
QC 1 2 1.1 1.08 0.483 0.495
QC 1 3 0.936 0.917 0.526 0.514
Mean Conc. Found (µg/mL) 1.039 0.992 0.48 0.500
STDV 0.089 0.082 0.044 0.013
CV% 8.61 8.3 9.01 2.53
Accuracy (%) 103.9 99.2 96.5 99.9
n= 3 3 3 3
Sample ID Conc. Found (µg/mL) - QC 2
Norminal Conc. (µg/mL) 4 4 2 2
QC 2 1 4.05 4.50 1.75 2.15
QC 2 2 4.31 4.16 1.83 2.25
QC 2 3 4.12 4.11 1.81 2.22
Mean Conc. Found (µg/mL) 4.16 4.26 1.80 2.21
STDV 0.135 0.212 0.042 0.051
CV% 3.23 4.99 2.32 2.33
Accuracy (%) 104 106 89.8 110
n= 3 3 3 3
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APPLICATIONS
Dry down sample at 30 °C under N2, then reconstitute the sample tube with 300 µL of 40% ethanol in water mix.
Transfer samples into amber autosampler vial with 300 µL glass insert. Inject on LC/MS/MS system.
LC/MS/MS ConditionsColumn: Kinetex® 2.6 µm F5
Dimensions: 30 X 2.1 mmPart No.: 00A-4723-AN
Mobile Phase: A: 0.1 % Formic Acid in WaterB: 0.1 % Formic Acid in Methanol
Gradient: Time (min) % B 0 806 806.01 1008 1008.01 809.5 80
Flow Rate: 300 µL/minInjection Volume: 10-25 µL
Temperature: AmbientDetection: MS/MS (APCI+) (SCIEX 4500™)
HPLC System: Agilent® 1100 HPLC with Quaternary PumpAnalytes: 1. Vitamin D2
2. Pre-Vitamin D3
3. Vitamin D3
4. Pre-Vitamin D3
ID Q1 Mass (Da) Q3 Mass (Da) Dwell (msec) Param CE CXP
D2 1 397.3 69.1 50 CE 20 15
D2 2* 397.3 106.9 50 CE 20 15
D3 1* 385.3 259.1 50 CE 21 15
D3 2 385.3 107.1 50 CE 35 15
D2-d3 1 400.2 271.2 50 CE 25 13
D2-d3 2* 400.2 68.9 50 CE 28 13
D3-d3 1* 388.2 259.1 50 CE 28 13
D3-d3 2 388.2 110 50 CE 28 13
Pre-D2 1 397.3 69 50 CE 37 13
Pre-D2 2* 397.3 158.7 50 CE 35 13
Pre-D3 1* 385.2 259.2 50 CE 19 13
Pre-D3 2 385.2 106.9 50 CE 24 13
* Quantitation mass transition
Table 3. Assay Recovery
Table 4. Accuracy and Precision
Table 2. Mass Transitions
Table 1. Instrument Parameters
Recovery calculation includes pre-treatment, LLE and SPE extractions, resulting in average recovery for each step of near 70% for all analytes.
Solid Phase Extraction (SPE) Procedures
SPE Cartridge: Strata® NH2 (55 µm, 70A), 2 g/12 mL Giga Tubes
Part No.: 8B-S009-KDG
Condition: 2X 6 mL Methylene chloride (DCM)
Equilibration: 2X 6 mL Heptane: Hexane 50:50
Load: ~3 mL supernatant from LLE (procedure step 7)
Wash: 2X 5 mL 10% DCM in Hexane
Dry: 4-5 min
Elute: 2x 6 mL Methylene chloride (DCM) into 10 X 16 mm glass tube
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APPLICATIONS
Figure 2. Representative of chromatograms of neat solution (500 ng/mL)
Figure 3. Representative a chromatogram of STD 1 (0.5/0.25 µg/mL) in matrix
Ap
p ID
240
21A
pp
ID 2
4022
Ap
p ID
240
23
Ap
p ID
240
18A
pp
ID 2
4019
Ap
p ID
240
20
Vitamin D2Vitamin D2 (0.5 µg/mL)
Vitamin D3 Vitamin D3 (0.5 µg/mL)
TIC TIC
Pre- Vitamin D3
Pre-Vitamin D3
(0.25 µg/mL)
Pre- Vitamin D2
Pre-Vitamin D2
(0.25 µg/mL)
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 6.5 7.0 min0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
Inte
nsity
, cps
24019
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 6.5 7.0 min
1.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e51.6e51.7e51.8e51.9e52.0e52.1e52.2e5
Inte
nsity
, cp
s
24018
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 6.5 7.0 min0.0
1.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e51.6e51.7e51.8e51.9e52.0e52.1e5
Inte
nsity
, cps
24020
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 6.5 7.00.0
1000.02000.03000.04000.05000.06000.07000.08000.09000.0
1.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e4
Inte
nsity
, cp
s
min
24022
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 6.5 7.00.0
1000.02000.03000.04000.05000.06000.07000.08000.09000.01.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e4
Inte
nsity
, cps
min
24021
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 6.5 7.00
200400600800
1000120014001600180020002200240026002800300032003400360038004000
Inte
nsity
, cp
s
min
24023
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APPLICATIONSFigure 4. Representative a chromatogram of Matrix Blank
Figure 5. Representative a chromatogram of STD 3 (5/2.5 µg/mL) in matrix
Ap
p ID
240
27A
pp
ID 2
4028
Ap
p ID
240
29
Ap
p ID
240
24A
pp
ID 2
4025
Ap
p ID
240
26
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 6.5 7.00.0
1000.02000.03000.04000.05000.06000.07000.08000.09000.0
1.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e41.8e41.9e42.0e4
Inte
nsity
, cp
s
min
24024
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 6.5 7.00.0
2000.04000.06000.08000.0
1.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e4
Inte
nsity
, cp
s
min
24027
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 6.5 7.00.0
1000.02000.03000.04000.05000.06000.07000.08000.09000.0
1.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e41.8e41.9e42.0e4
Inte
nsity
, cp
s
min
24025
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 6.5 7.00.0
1000.02000.03000.04000.05000.06000.07000.08000.09000.0
1.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e41.8e41.9e42.0e42.1e42.2e4
Inte
nsity
, cp
s
min
24028
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 6.5 7.00
100200300400500600700800900
10001100120013001400150016001700180019002000
min
24026
Inte
nsity
, cp
s
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 6.5 7.0 min0.0
2000.04000.06000.08000.0
1.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e4
Inte
nsity
, cp
s
24029
Note: No Vitamin D2 or Previtamin D2
Vitamin D3
TIC TIC
Pre-Vitamin D3
Vitamin D2
(5 µg/mL)
Vitamin D3
(5 µg/mL)
Pre-Vitamin D2
(2.5 µg/mL)
Pre-Vitamin D3
(2.5 µg/mL)
TN-0111
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APPLICATIONSFigure 6. Minimized matrix effect after SPE extraction
Figure 7. Interference peaks after analytes
Ap
p ID
240
30A
pp
ID 2
4031
Ap
p ID
240
32
Interference peaks
Vitamin D3
Vitamin D3
TIC
Blank matrix after LLE
TIC
Blank matrix after LLE and SPE
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 6.50.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
24030
min
Inte
nsity
, cp
s
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 6.5 7.0 min0.0
1000.02000.03000.04000.05000.06000.07000.08000.09000.0
1.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e41.8e41.9e42.0e4
Inte
nsity
, cp
s
24031
Pre-Vitamin D2
(2.5 µg/mL)
Pre-Vitamin D3
(2.5 µg/mL)
0 1 2 3 4 5 6 7 8 9 10 11 min0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e524032
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APPLICATIONS
Results and DiscussionQuantifying Vitamin D in complex food matrices is filled with a range of challenges, from isomer conversion to light sensitivity and saponification and extraction efficiency.
Samples were marginally protected from light with amber vials and dim lighting, and was completed in 1 hour, room temperature sa-ponification was performed to minimize conversion to the pre-D forms for a more accurate quantification. After a double liquid-liq-uid extraction, a normal phase SPE protocol using Strata® NH2 cartridge was used to further remove some matrix components (Figure 6.).
Near baseline resolution of vitamin D and pre-vitamin D (Figure 2.) was achieved with a fast run-time on a short (30 x 2.1mm) Kinetex® 2.6 µm F5, leveraging the polar and aromatic selectivity of the pen-tafluorophenyl ligand. The mass transitions of the analytes were listed in Table 2. The LC gradient was extended an additional two minutes (6.01-8 min in gradient table) with 100% organic solvent to elute some matrix components that might otherwise interfere in subsequent injections (Figure 7.).
Quantification was done using a standard addition curve, extrapo-lating from a back calculated blank sample. Accuracy and preci-sion were evaluated at two QC levels (n=3); accuracy 99.2-106 % with CV % of 3.23-8.61 % for parent compounds of the vitamin D2/
Figure 10. Lab photos in SPE extraction procedures
Wash step after sample loading
After elution
Figure 8. Representative curve of Vitamin D2 and Pre-Vitamin D2
Figure 9. Representative curve of Vitamin D3 and Pre-Vitamin D3
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Analyte Conc. / IS Conc.
0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.02.12.22.32.4
Ana
lyte
Are
a /
IS A
rea
Vitamin D2 curve range: 0 – 5 µg/2g
r =0.9987
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5
Analyte Conc. / IS Conc.
0.000.050.100.150.200.250.300.350.400.450.500.550.600.650.700.750.800.850.87
Ana
lyte
Are
a /
IS A
rea
r =0.9950
Pre-Vitamin D2 curve range: 0 – 2.5 µg/2g
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Analyte Conc. / IS Conc.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.55.7
Ana
lyte
Are
a /
IS A
rea
r =0.9999
Vitamin D3 curve range: 0 – 5 µg/2g
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5
Analyte Conc. / IS Conc.
0.00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.8
Ana
lyte
Are
a /
IS A
rea
Pre-Vitamin D3 curve range: 0 – 2.5 µg/2g
r =0.9998
Ap
p ID
240
33A
pp
ID 2
4034
Ap
p ID
240
35A
pp
ID 2
4036
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APPLICATIONS
Strata® NH2/WAXFormat Sorbent Mass Part Number UnitTube
100 mg 8B-S009-EAK 1 mL (100/box) 200 mg 8B-S009-FBJ 3 mL (50/box) 500 mg 8B-S009-HBJ 3 mL (50/box) 500 mg 8B-S009-HCH 6 mL (30/box)
1 g 8B-S009-JCH 6 mL (30/box) Giga Tube
500 mg 8B-S009-HDG 12 mL (20/box)2 g 8B-S009-KDG 12 mL (20/box)5 g 8B-S009-LEG 20 mL (20/box)
10 g 8B-S009-MFF 60 mL (16/box)20 g 8B-S009-VFF 60 mL (16/box)
96-Well Plate
25 mg 8E-S009-CGB 2 Plates/Box50 mg 8E-S009-DGB 2 Plates/Box100 mg 8E-S009-EGB 2 Plates/Box
2.6 μm Minibore Columns (mm)SecurityGuard™
ULTRA Cartridges‡
Phase 50 x 2.1 100 x 2.1 150 x 2.1 3/pkF5 00B-4723-AN 00D-4723-AN 00F-4723-AN AJ0-9322
for 2.1 mm ID
2.6 μm MidBore™ Columns (mm)SecurityGuard
ULTRA Cartridges‡
Phase 50 x 3.0 100 x 3.0 150 x 3.0 3/pkF5 00B-4723-Y0 00D-4723-Y0 00F-4723-Y0 AJ0-9321
for 3.0 mm ID
2.6 μm Analytical Columns (mm)SecurityGuard
ULTRA Cartridges‡
Phase 50 x 4.6 100 x 4.6 150 x 4.6 3/pkF5 00B-4723-E0 00D-4723-E0 00F-4723-E0 AJ0-9320
for 4.6 mm ID
1.7 μm Minibore Columns (mm)SecurityGuard
ULTRA Cartridges‡
Phase 50 x 2.1 100 x 2.1 150 x 2.1 3/pkF5 00B-4722-AN 00D-4722-AN 00F-4722-AN AJ0-9322
for 2.1 mm ID
Ordering Information
Kinetex® Core-shell Columns
D3, and accuracy 89.8-110 % with CV % 2.32-9.01 % for pre-vi-tamin D2/D3 (Table 4.). The assay overall recovery was shown in Table 3. The dynamic curve range of Vitamin D2/D3 is 0 – 5 µg/2g and pre-vitamin D2/D3 is 0 -2.5 µg/2 g (Figures 8 and 9.). The blank matrix, STD 1 (0.5/0.25 µg/mL) and ULQ (5/2.5 µg/mL) are seen in Figures 3 – 5., respectively.
ConclusionIn this technical note, a quantitative method for the determination of Vitamins D2 and D3 and pre-vitamins D2 and D3 in dog food was developed with opportunities to explore in a wide range of food and pet food matrices.
If Strata SPE products do not perform as well or better than your current SPE product of similar phase, mass and size, return the product with comparative data within 45 days for a FULL REFUND.
If Kinetex core-shell columns do not provide at least an equivalent separation as compared to a competing column of the same phase, return the column with comparative data within 45 days for a FULL REFUND.
‡ SecurityGuard ULTRA cartridges require holder, Part No.: AJO-9000.
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APPLICATIONS
TN49
0101
17_W
www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at [email protected]
Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions.
Trademarks Kinetex and Strata are registered and SecurityGuard and Midbore are trademarks of Phe-nomenex. Sciex 4500 Triple Quad is a trademark of AB SCIEX Pte. Ltd. AB SCIEX™ is being used under license. TurboVap is a registered trademark of Biotage LLC. Cerilliant is a regis-tered trademark of Cerilliant Corporation and Purina and Dog Chow are registered trademarks of Nestle Purina. Agilent is a trademark of Agilent Technologies, Inc.
Disclaimer Phenomenex is in no way affiliated with Nestle or Cerilliant.
SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362 CAUTION: this patent only applies to the analytical-sized guard cartridge holder, and does not apply to SemiPrep, PREP or ULTRA holders, or to any cartridges.
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