LC/MS/MS Quantitative Analysis of Fat Soluble Vitamins in Serum
Lauren Frick, Rory Doyle
Agilent Technologies, Inc, Lexington, MA and Wilmington, DE
MSACL 2015
Poster No.- 31
Introduction
Experimental
The major fat soluble vitamins include Vitamin A (Retinol, Retinal and Retinoic acid),
Carotenoids (Alpha, Beta- and Gamma-Carotene and Beta-Cryptoxanthin), Vitamin D
(25-Hydroxy-Vitamin D2 and D3- Not tested), Vitamin E (Alpha-, Beta- Gamma- and
Delta-Tocopherol and Alpha-, Beta-, Gamma-, and Delta-Tocotrienol), Vitamin K (K1Phylloquinone, K2-Menaquinone and K3-Menadione) as well as Coenzyme Q10.
Therefore in this case, we developed LC/MS/MS analytical methods and evaluated
various columns and
solvent
combinations in order to demonstrate the
chromatographic separation, detection and quantification of the fat soluble vitamins in
blood. The sample preparation was a simple liquid-liquid extraction and the
methodologies were developed on an Agilent 1260 HPLC and 6460 Mass Spectrometer
with a 9.5 minute analytical gradient method in positive ionization mode.
Retinol
Retinal
Retinoic Acid
Beta-Carotene
Beta-Cryptoxanthin
Alpha-Tocopherol
Beta-Tocopherol
Gamma-Tocopherol
Delta-Tocopherol
Alpha-Tocotrienol
Beta-Tocotrienol
Gamma-Tocotrienol
Reagents
Ethyl Acetate:
Hexane:
Methanol:
Honeywell
Honeywell
Honeywell
Results and Discussion
Formic Acid:
Ammonium Formate:
Sigma-Aldrich
Sigma-Aldrich
Table 2: Sensitivity
Sample Preparation
• 500 µL of serum sample, calibrators, controls was taken and 50 µL of ISTD at 1000
ng/mL followed by 450 µL of Methanol were added to each and vortexed briefly
• 2000 µL of HPLC grade mixture of Ethyl Acetate:Hexane (1:1) was added to each
tube and vortexed for 1 min prior to centrifugation for 10 minutes at 13000 rpm
• Organic layer (Upper) was transferred to another clean tube and then dried down
under nitrogen at room temperature
• Samples, calibrators, controls and blanks were reconstituted in 120 µl 75%
Methanol:25% Water and transferred to an MS vial or 96 well plate for analysis
All in-house calibrators were prepared in Vitamin D Free Serum (Golden West
Biological, Inc.).
Method
HPLC ConditionsAgilent 1260 Infinity HPLC series binary pump, well plate, thermostatted column
compartment
Column:
Agilent Technologies Poroshell 120 PFP,
2.1 x 100 mm, 2.7 µm
Column Temperature:
4 oC
Injection Volume:
5 µL
Autosampler Temperature: 4 oC
Needle Wash:
Flush port (50%Methanol:50%Water) 10 seconds
Mobile Phase A:
0.1% Formic Acid + 5 mM Ammonium Formate in Water
Mobile Phase B:
0.1% Formic Acid in Methanol
Flow Rate:
0.5 ml/min
Gradient:
0 min- 30%A:70%B
8.0 min- 2%A:98%B
Run/Stop time:
9.5 min/1.5 min
K1-Phylloquinone
MS ConditionsAgilent 6460 Triple Quadruple Mass Spectrometer- Dynamic MRM
Ion mode:
Agilent Jet Stream Positive Mode
Gas Temperature:
250 oC
Gas Flow:
8 L/min
Nebulizer:
45 psi
Sheath Gas Temperature:
350 oC
Sheath Gas Flow:
11 L/min
Capillary Voltage:
3000V
Nozzle Voltage:
500V
Q1/Q2 Resolution:
Wide/Unit
Delta EMV:
+400V
Cell Accelerator Voltage:
2
Compound
Rt
Min
MRM
Transitions
LOD
LOD
Compound
(ng/ml)
LOD
LOQ
Retinol
50
100
α-Tocotrienol
0.5
1
Retinal
10
25
β-Tocotrienol
2.5
5
Retinoic Acid
Interference
γ-Tocotrienol
2.5
5
α/β−Carotene
5
10
δ-Tocotrienol
0.25
0.5
β-Cryptoxanthin
1
2.5
K1
0.1
0.25
α-Tocopherol
1
2.5
K2
0.5
1
β-Tocopherol
0.5
1
K3
5
10
γ-Tocopherol
1
2.5
Coenzyme Q10
2.5
5
δ-Tocopherol
10
25
Table 3: Chromatograms
Cpd 1: K3: +ESI MRM Frag=112.0V CF=0.000 DF=0.000 [email protected] (173.1000 -> 104.9000) 100 ngml-FSV.d
x10 5
1
0.881
0
Cpd 2: Retinol: +ESI MRM Frag=112.0V CF=0.000 DF=0.000 [email protected] (269.2000 -> 93.0000) 100 ngml-FSV.d
x10 6
1
2.874
0
Cpd 3: Retinoic Acid: +ESI MRM Frag=87.0V CF=0.000 DF=0.000 [email protected] (301.2000 -> 91.0000) 100 ngml-FSV.d
x10 5
1
3.071
2.712
0
Cpd 4: Retinal: +ESI MRM Frag=87.0V CF=0.000 DF=0.000 [email protected] (285.2000 -> 160.9000) 100 ngml-FSV.d
x10 6
1
3.430
3.017
0
3.749
Cpd 6: Delta-Tocotrienol: +ESI MRM Frag=122.0V CF=0.000 DF=0.000 [email protected] (397.3000 -> 136.9000) 100 ngml-FSV.d
x10 5
1
4.575
0
Cpd 7: Gamma-Tocotrienol: +ESI MRM Frag=117.0V CF=0.000 DF=0.000 [email protected] (411.3000 -> 150.9000) 100 ngml-FSV.d
x10 6
1
5.116
0
Cpd 8: Beta-Tocotrienol: +ESI MRM Frag=132.0V CF=0.000 DF=0.000 [email protected] (411.3000 -> 150.9000) 100 ngml-FSV.d
x10 6
Table1: MRM Acquisition table (*Quantifier Ion)
Delta-Tocotrienol
Compound
(ng/ml)
1
Fragmentation
Voltage
5.116
0
Collision
Energy
Cpd 9: Alpha-Tocotrienol: +ESI MRM Frag=122.0V CF=0.000 DF=0.000 [email protected] (425.3000 -> 164.9000) 100 ngml-FSV.d
x10 6
1
5.420
0
Retinol
2.87
269.2 – 93*/41.2
112
20/56
Retinol-D5
2.85
274.2 – 98.2
114
20
Retinal
3.54
285.2 -175.1*/160.9
87
8/4
Cpd 10: Delta-Tocopherol: +ESI MRM Frag=112.0V CF=0.000 DF=0.000 [email protected] (403.4000 -> 136.9000) 100 ngml-FSV.d
x10 5
1
5.694
0
Cpd 11: Beta-Tocopherol: +ESI MRM Frag=102.0V CF=0.000 DF=0.000 [email protected] (417.4000 -> 150.9000) 100 ngml-FSV.d
x10 6
1
6.132
0
K2-Menaquinone
K3- Menadione
Coenzyme Q10
Experimental
Reagents, Standards, Calibrators and Controls
Standard/Calibrators
Retinol:
Sigma-Aldrich
Retinol-D5- Santa Cruz Biotech
Retinal:
Sigma-Aldrich
Retinoic Acid:
Cayman Chemicals
Beta-Carotene:
Sigma-Aldrich
Beta-Cryptoxanthin:
Sigma-Aldrich
Alpha-Tocopherol:
Isosciences
Alpha-Tocopherol-D6: Isosciences
Beta-Tocopherol:
Sigma-Aldrich
Gamma-Tocopherol:
Cerilliant
Delta-Tocopherol
Sigma-Aldrich
Alpha-Tocotrienol:
Cayman Chemicals
Beta-Tocotrienol:
Cayman Chemicals
Gamma-Tocotrienol:
Cayman Chemicals
Delta-Tocotrienol:
Cayman Chemicals
Vitamin K1:
Isosciences
Vitamin K1-D7:
Isosciences
Vitamin K2
Sigma-Aldrich
Vitamin K3:
Isosciences
Vitamin K3-D3:
Isosciences
Coenzyme Q10:
Isosciences
Coenzyme Q10-D9: Isosciences
Retinoic Acid
3.09
301.2 – 91*/41.2
87
64/72
α/β−Carotene
8.23
537.5 – 445.3
177
10/36
β-Cryptoxanthin
7.32
553.4 – 461.3/118.9*
156
12/40
Cpd 12: Gamma-Tocopherol: +ESI MRM Frag=107.0V CF=0.000 DF=0.000 [email protected] (417.4000 -> 150.9000) 100 ngml-FSV.d
x10 6
1
6.132
0
Cpd 13: K2: +ESI MRM Frag=122.0V CF=0.000 DF=0.000 [email protected] (445.3000 -> 187.1000) 100 ngml-FSV.d
x10 5
1
6.322
0
α-Tocopherol
6.39
431.4 – 164.9
102
16
α-Tocopherol-D6
6.38
437.4 – 170.9
102
16
β-Tocopherol
6.01
417.4 – 150.9
102
12
Cpd 15: Alpha-Tocopherol: +ESI MRM Frag=102.0V CF=0.000 DF=0.000 [email protected] (431.4000 -> 164.9000) 100 ngml-FSV.d
x10 6
1
6.395
0
Cpd 17: K1: +ESI MRM Frag=157.0V CF=0.000 DF=0.000 [email protected] (451.4000 -> 186.8000) 100 ngml-FSV.d
x10 6
1
7.267
0
γ-Tocopherol
6.12
417.4 – 150.9
107
24
δ-Tocopherol
5.69
403.4 – 136.9
112
24
α-Tocotrienol
5.42
425.3 – 164.9
122
24
Cpd 18: Beta-Cryptoxanthin: +ESI MRM Frag=156.0V CF=0.000 DF=0.000 [email protected] (553.4000 -> 461.3000) 100 ngml-FSV.d
x10 6
1
7.331
0
Cpd 20: Beta-Carotene: +ESI MRM Frag=185.0V CF=0.000 DF=0.000 [email protected] (537.5000 -> 445.3000) 100 ngml-FSV.d
x10 6
1
8.241
0
β-Tocotrienol
4.95
411.3 – 150.9
132
20
γ-Tocotrienol
5.12
411.2 – 150.9
122
24
δ-Tocotrienol
4.58
397.3 – 136.9
122
24
Vitamin K1
7.26
451.4 – 186.8
157
20
Vitamin K1-D7
7.26
458.4 – 193.9
162
24
Vitamin K2
6.31
445.3 – 187.1
122
16
Vitamin K3
0.88
173.1 – 104.9*/77.1
112
20/40
Vitamin K3-D3
0.88
176.1 – 105
112
24
Coenzyme Q10
8.61
863.7 – 196.9
245
40
Coenzyme Q10-D9
8.61
872.8 – 205.9
245
36
Cpd 22: Coenzyme Q10: +ESI MRM Frag=245.0V CF=0.000 DF=0.000 [email protected] (863.7000 -> 196.9000) 100 ngml-FSV.d
x10 5
1
8.614
0
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
5
5.2
5.4
5.6
5.8
6
6.2
6.4
6.6
6.8
7
7.2
7.4
7.6
7.8
8
8.2
8.4
8.6
8.8
9
Counts vs. Acquisition Time (min)
Conclusions
•
•
•
•
•
Baseline separation of the Fat Soluble Vitamins in 9.5 minutes with good LOD/LOQ in
positive mode was achieved except for Retinoic Acid and Retinol was present in the serum
matrix used.
Simple sample preparation in serum matrix but requires using a Fat Soluble Vitamin free
serum or 5% HAS/O.1M PBS solution for the calibrators.
Excellent linearity of calibration curves with acceptable accuracy, precision and
reproducibility in positive mode was achieved in all matrices <10% for %CV where possible
Further evaluate different sample preparation techniques and fat soluble vitamin free
serum matrixes available to determine which gives the best results while maintaining low
cost and ease of use.
Further evaluate the different Fat Soluble Vitamins per group method rather than a global
method for better analytical needs and results.
For research use only. Not to be used for diagnostic procedures