Embed Size (px)
Citation preview
ON HPLC COLUMNS
Vitamins
No.TI121E
TI121E-CF13
High Speed Fat-Soluble Vitamins Analysis
Conventional ODS columns struggle to separate Vitamin D2 and D3 when fat-soluble vitamins are analyzed solely with methanol.
In this separation example as well, the conventional 250mm column could not completely separate D2 and D3. Cadenza CD-C18 150 x 4.6mm column, however, completely separates these vitamins. Cadenza's high molecular recognition in the stationary phase contributes to this separation of derivatives.
Cadenza offers even more benefits. Analysis time and solvent consumption are 60% of normal levels. Moreover, the overall sensitivity improves. By using Cadenza CD-C18 instead of the conventional 250mm column, you can save time and solvent while realized greater sensitivity.
MeOH, 1.0 mL/min, 37 °CUV at 280 nm2 uL inj (0.1-1.6 mg/mL)
Vitamin K3
Vitamin A
O
O
CH3
CH3
CH3CH3
CH3 CH3CH2OH
Vitamin A acetate
CH3
CH3CH3
CH3 CH3O
O
CH3
Vitamin D2
Vitamin D3
HO
CH2
HH
CH3
CH3H3CH3C
H
Vitamin E
OCH2CH2CH2CH
CH3
CH3
CH3H3C
HO
CH3
CH3
3
Vitamin K1
CH2CH2CH2CH
CH3
CH3
3O
O
CH2CH C
CH3CH3
HO
CH2
HH
CH3
CH3
CH3
H3CH3C
H
3
1
2
4
5
6
7
5.1 MPa
Rs(5/4) = 1.5
min0 10 20
mAU
0
50
100
12
34
5 6 7
min0 5 10
mAU
0
50
100
1
2
3
4
56 7
Rs(5/4) = 2.0
6.7 MPa
Achieve Higher Speeds with Cadenza 150mm than a Conventional 250mm column
Conventional ODS column250 x 4.6 mm
No.TI122E
TI122E-CF13
High Speed Analysis of Water-Soluble Vitamins
High-Speed / High-Sensitivity / Low Solvent Use
3 mM Na-heptanesulfonate, 0.05%TEA, 1% AcOH / ACN = 85 /151.0 mL/min, 37 °C, UV at 254 nm2uL inj. (0.1 - 0.4 mg/mL)
In the reverse phase of aqueous vitamins containing ascorbic acid, it is generally ion versus chromatography.
Under the same analytical conditions as a conventional column, Cadenza CD-C18 offers the same separation with half the column length. Furthermore, run time and solvent use is cut by 50%. Peak detection sensitivity also improves dramatically.
By switching to Cadenza CD-C18, you can easily realize time and solvent savings with improved sensitivity.
Cadenza is the perfect column for quality management departments seeking to cut costs and development departments hoping to improve development speed.
O O
OHHO
CHOCH2
H OH
L-ascorbic acid
nicotinamideN
C
O
NH2
pyridoxineN
CH2OHCH2OH
H3C
HO
thiamine
riboflavin
N
N
N
HN
O
OH3C
H3C
CH2CH CHCH2OH
OH
CH
OH OH
CH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
1
2
3
4
5
75 x 4.6 mm
5.3 MPa
5.9 MPa
min0 2 4
mAU
0
50
100
150
200
1
2
3
4
5
min0 2 4 6 8
mAU
0
50
100
150
200
12
34
5
Conventional 5um ODS Column150 x 4.6 mm
No.TI166E
TI166E-DJ01
High-efficiency separation by a 3um silica particle
The Unison series of columns provides authoritative results in normal phase separation using its silica stationary phase. This high level of performance is in addition to its extraordinary resolution, higher than conventional 5um columns. The example above shows a normal phase separation of the tocopherol isomers. Beta- and gamma-tocopherol are position isomers, compounds characterized as difficult to separate. Unison UK-Silica 150 x 4.6mm, however, provides excellent separation in under 10 minutes. By using the 3um silica Unison column, you can improve your efficiency and analysis speed compared with conventional 5um, 25cm columns.The Unison UK-Silica offers optimal solutions for specialty research areas including natural chemistry and organic combinatorial chemistry.
Unison UK-Silica, 150 x 4.6 mmhexane / THF = 97 / 3
1.0 mL/min, 37 °C, 295 nm
Improved recognition for isomers
OCH2CH2CH2CH
CH3
CH3
CH3H3C
HO
CH3
CH3
3
OCH2CH2CH2CH
CH3
CH3
CH3HO
CH3
CH3
3
OCH2CH2CH2CH
CH3
CH3H3C
HO
CH3
CH3
3
OCH2CH2CH2CH
CH3
CH3HO
CH3
CH3
3
tocopherols
0 5 10 min
No.TI014E
TI014E-AA10
Cadenza CD-C18methanol37 °CUV at 290 nm
Tocol and Tocopherol
1 2
min0 1 2
mAU
0
25
50
75
100
1 2
min0 1 2
mAU
0
25
50
75
100
1 2
min0 1 2
mAU
0
25
50
75
100
12
tocopherol
O CH2CH2CH2CH
CH3
CH3
CH3H3C
HO
CH3
CH3
3
O CH2CH2CH2CH
CH3HO
CH3
CH3
3
tocol
0.4 mL/min, 3.0 MPa0.1 mg/mL, 2 uL
1.0 mL/min, 3.0 MPa0.5 mg/mL, 1 uL
2.0 mL/min, 2.6 MPa0.5 mg/mL, 2 uL
No.TI027E
TI027E-AB16
Cadenza CD-C18, 50 x 4.6 mmA: 0.3%TFA B: methanol0-40%B (0-7min)40-100%B (7-7.5min)100%B (7.5-15min)0.8 mL/min, 37 °CUV at 280 nm, 3.4 MPa
Vitamin preparation
1
2
3
4
5
7
6
benzoic acid
riboflavin monophosphatesodium salt
nicotinamide
L-ascorbic acid
pyridoxine
caffeine
Vitamin E acetate
N
CO
NH2
O O
OHHO
CHOCH2
H OH
N
CH2OHCH2OH
H3C
HO
NN
N
NO
O
CH3
CH3
H3C
H3C
CH
OH
CH2CH
NH
O
ON
NN
OH
H3C
CHCH2
OH
OHONaP
O
O
COOH
O CH2CH2CH2CH
CH3
CH3
CH3H3C
O
CH3
CH3
3
CO
H3C
1
2
3 4 5 6
7
min0 5 10 15
mAU
0
20
40
60
No.TI060E
TI060E-AL20
Cadenza CD-C18, 75 x 3.0 mmmethanol / 20mM H3PO4 = 20 / 80 0.4 mL/min, 7.2 MPa37 °C, UV at 290 nmfolic acid 10 ug
Folic acid
min0 2 4 6 8 10
mAU
0.0
2.5
5.0
7.5
10.0
1
HNCHC
ONH2N
CH2NHN
N
N C
C
O
CH2CH2OHOH
OH
O
No.TI128E
TI128E-CF11
Unison, 75 x 4.6 mm10mM DBA-Acetate / ACN = 85 / 151.0 mL/min, 37 °C, UV at 260 nmPQQ 1 µg ( 2µL )
PQQ ( pyrroloquinoline quinone )
0 2 4 6 8 10min
0
100
200
mAU
Unison UK-Phenyl6.8 MPa
Unison UK-C86.8 MPa
Unison UK-C187.8 MPa
NHN
C OHCHO
C OH
OO
OO
O
No.TI141E
TI141E-CL17
20mM ammonium acetate + 10mM dibutylammonium acetate37 °C, 260 nm, 0.5 - 1.5 µg/µL
Ascorbic Acid Derivative
1 2
3
1
1
2
2
3
3
0 5 10 15min
0
100
200
mAU
Unison UK-C1875 x 4.6 mm
1 mL/min8 MPa, 0.4 µL
Unison UK-C1875 x 3 mm0.43 mL/min
6 MPa, 0.2 µL
Unison US-C18150 x 4.6 mm
1 mL/min7 MPa, 0.4 µL
O O
OHHO
CHOCH2
H OH
L-ascorbic acid
1
L-ascorbic acid 2-glucoside
HO
CH2OH
OH
OH
O
OO
OHO
CH
OH
CH2OH2
N
CO
NH2
nicotinamide
3
No.TI154E
TI154E-DD08
Unison UK-Silica, 150 x 4.6mm50mM ammonium acetate / acetonitrile = 10 / 90
0.8mL/min, 30 °C, 260 nm, 2 µg
Ascorbic Acid Related Compounds
L-ascorbic acid
1
L-ascorbic acid 2-glucoside
HO
CH2OH
OH
OH
O
OO
OHO
CH
OH
CH2OH2 3
isoascorbic acid
OO
OHHO
CHOCH2
H OH
1
23
0 5 10 15 20 min
0
50
100
mAU
OHHO
CHOCH2
H OHO O
No.TI155E
TI155E-DD08
10mM dibutylammonium acetate0.8mL/min, 30 °C, 260 nm, 2 µg
Ascorbic Acid Related Compounds
Unison UK-C1875 x 4.6 mm
6 MPa
Unison US-C18150 x 4.6 mm
5 MPa
L-ascorbic acid
1
L-ascorbic acid 2-glucoside
HO
CH2OH
OH
OH
O
OO
OHO
CH
OH
CH2OH2 3
isoascorbic acid
OO
OHHO
CHOCH2
H OH
0 5 10 15 20min
0
100
200
mAU
1
1
2
2
3
3
Rs = 2.54
Rs = 2.53
OHHO
CHOCH2
H OHO O
No.TI156E
TI156E-DD19
75 x 4.6 mm50 mM HCOONH4 / acetonitrile= 98 / 21 mL/min, 37 °C, 260 nm, 0.2µL
Thiamine HCl (Vitamin B1) Injection Solution
0 2 4 6 8 min
0
200
mAU
thiamineCH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
Unison UK-Phenyl
Unison UK-C8
Unison UK-C18Cadenza CL-C18
No.TI157E
TI157E-DD20
75 x 4.6 mm50 mM HCOONH41 mL/min, 37 °C260 nm, 0.5µL
Pyridoxine HCl (Vitamin B6) Injection Solution
pyridoxineN
CH2OHCH2OH
H3C
HO
Unison UK-Phenyl
Unison UK-C8
Unison UK-C18
Cadenza CL-C18
0 2 4 6 8 min
0
500
250
mA
U
No.TI159E
TI159E-DD26
Unison UK-Silica, 250 x 2 mmwater / acetonitrile / TFA = 5 / 95 / 0.50.2 mL/min (5MPa), 37 °C, 260 nm
Water-Soluble Vitamins
1 2 3 4
1
23
4
0 5 10 15min
0
20
40
60
80
100
mA
U
pyridoxine
N
CH2OHCH2OH
H3C
HO
nicotinamide
N
C
O
NH2
thiamineriboflavin
N
NH
N
N
O
OH3C
H3C
CH2CH CHCH2OH
OH
CH
OH OH
CH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
No.TI160E
TI160E-DE07
75 x 4.6 mm1% acetic acid / acetonitrile = 40 / 601 mL/min, 37 °C, 260 nm, 1 µg
Riboflavin Tetrabutyrate Tablet
0 2 4 6 8 10min
0
100
200
mAU
Unison UK-C18, UK003
Unison UK-C8, UK803
Unison UK-Phenyl, UKP03
Cadenza CD-C18, CD003
Cadenza CL-C18, CL003
riboflavin tetrabutyrate
N
NH
N
N
O
OH3C
H3C
CH2CH CHCH2OCOC3H7
OCOC3H7CH
OCOC3H7
OCOC3H7
No.TI162E
TI162E-DE24
Unison UK-Silica, 150 x 3 mmhexane / THF = 95 / 50.5 mL/min (4MPa), 37 °C, 295 nmtocopherol nicotinate 5µg
Cadenza CD-C18, 150 x 3 mmmethanol, 0.5 mL/min (6MPa)37 °C, 295 nmtocopherol nicotinate 5µg
Tocopherol Nicotinate Capsule
0 5 10 15min
0
50
100
mAU
O CH2CH2CH2CH
CH3
CH3
CH3H3C
CH3
CH3
3
N
CO
O
0 5 10 15min
0
100
200
mAU
No.TI163E
TI163E-DE24
Unison UK-C18, 75 x 3 mmA: water / heptafluorobutyric acid = 100 / 0.1 B: acetonitrile 10 - 40%B (0 - 4min)40 - 100%B (4 - 4.5min)100%B (4.5 - 10min)0.5 mL/min, 37 °C, 260 nm, 0.24 - 2.4µg(2µL)
Simultaneous Analysis Of Water-soluble And Fat-soluble Vitamins
1
2
3
4
5
6
1
2
3
4 5
6
0 5 10 min
0
100
200
mAU
CH3
CH3CH3
CH3 CH3CH2OH
HO
CH2
HH
CH3
CH3
CH3
H3CH3C
H
nicotinamideN
C
O
NH2
pyridoxineN
CH2OHCH2OH
H3C
HO
thiamine
retinol
ergocalciferol
riboflavin
N
NH
N
N
O
OH3C
H3C
CH2CH CHCH2OH
OH
CH
OH OH
CH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
No.TI164E
TI164E-DE27
Unison UK-Silica, 150 x 4.6 mm50mM ammonium acetate / acetonitrile = 10 / 901 mL/min, 37 °C, 210 nm, 2µL(1-2µg)
75 x 4.6 mm20mM NaH2PO4 + 10mM dibutylammonium acetate1 mL/min, 37 °C, 210 nm, 2µL(0.5-1µg)
Calcium Pantothenate and Ascorbic Acid
1
2
L-ascorbic acidOHHO
CHOCH2
H OH
OO
HOCH2
H3C
H3CCHOH
C CO
OHNHCH2CH2 CO
(1/2Ca2)
calcium pantothenate
1
1
1
1
2
0 5 10 15 20 25min
0
20
40
60
mAU
Cadenza CL-C18
Unison UK-C18
Unison UK-C8
Unison UK-Phenyl
1 2
0 5 10 15min
0
10
20
30mAU
Unison UK-Silica
No.TI175E
TI175E-DG08
Unison UK-C18, 75 x 4.6 mmA: water / formic acid = 100 / 0.1B: acetonitrile / formic acid = 100 / 0.10%B(0-3min), 0-30%B(3-8min)1 mL/min (6MPa), 37 °C, 260 nm
Vitamin B12 Related Conpounds
Co+NN
NN
CH3
CH3CH3
H3CH3C
H3C
R CH2CH2CONH2
CH2CH2CONH2
NH2COCH2CH2NH2COCH2
NH2COCH2
CH2CONH2CH3 CH3
CH3
CH3
N
N
HO H
CH2OHOH
HH
C
H3C
O O
O-
O
PCH2CH2CONHCH2
H
R = -CN cyanocobalamin = -OH hydroxocobalamin
0 5 10 min
0
100
200
mAU
benz
ylal
chol
cyan
ocob
alam
inhy
drox
ocob
alam
in
No.TI195E
TI195E-DI27
Unison UK-Silica150 x 3 mm
hexane / IPA = 998 / 20.5 mL/min, 37 deg.C
270 nm, 2ug (2uL)
75 x 3 mm0.5 mL/min, 37 deg.C
270 nm, 2ug (2uL)
Coenzyme Q10
0 2 4 6 8 10min
0
500
1000mAU
0 2 4 6 8 10 min
0
200
400
mAU
acetonitrile / tetrahydrofuran= 75 / 25
water / acetonitrile= 2 / 98
acetonitrile / tetrahydrofuran= 75 / 25
acetonitrile / tetrahydrofuran= 75 / 25
O
O
H3CO
H3CO CH3
CH3H
10
No.TI207E
TI207E-DL01
Unison UK-C8, 75 x 3 mm0.1% formic acid / acetonitrile / isopropanol = 1 / 5 / 50.8 mL/min, 10 uL(50 fg/uL)API4000: APCI, MRM Positive, Q1/Q3 : 863.5 / 197
LC-MS Application for Coenzyme Q10
Courtesy of J.Watanabe, TAKARA BIO INC.
API4000 (TAKARA BIO INC. )
Coenzyme Q10 Mw. 863.36
O
OCH3
MeO
MeO
HCH3
10
Max. 131.6 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5Time, min
0
20
40
60
80
100
120
1322.04
500 fg
CoenzymeQ10 Result.rdb (CoenzymeQ10): "Linear" Regression ("1 / x" weighting): y = 6.66e+003 x + 17.3 (r = 0.9998)
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
r = 0.9998
Concentration, ng/mL0 20 40 60 80 100 120 140 160 180 200
No.TI225E
TI225E-EB25
Unison UK-C18, acetonitrile / THF = 80 / 20
Unison UK-Phenyl, water / acetonitrile = 15 / 85
Unison UK-C8, water / THF = 30 / 70
Cadenza CD-C18, acetonitrile / THF = 80 / 20
75 x 4.6 mm,1 mL/min, 280 nm37 degC, 2 uL (0.8-1.6ug)
Fat-Soluble Vitamins
1
2
3
1
2
3
mA
U
0 2 4 6 8 min
0
100
alpha-tocopherol
beta-carotene
coenzyme Q10
OCH2CH2CH2CH
CH3
CH3
CH3H3C
HO
CH3
CH3
3
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
O
O
H3CO
H3CO CH3
CH3H
10
No.TI227E
TI227E-HF30
ELSD application - taurine and water-soluble vitamins
Courtesy of Mr. Y.Sakai, M&S Instruments Trading Inc., Japan
0
100
200
mV
1 2 3 4 min 5
1
23
45 6 7
SO
OOHCH2 CH2H2N
taurine thiamine (B1)
CH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
pyridoxine (B6)
N
CH2OHCH2OH
H3C
HO
nicotinamide (B3)
N
C
O
NH2
Co+NN
NN
CH3
CH3CH3
H3CH3C
H3C
CH2CH2CONH2
CH2CH2CONH2
NH2COCH2CH2NH2COCH2
NH2COCH2
CH2CONH2CH3 CH3
CH3
CH3
N
N
HO H
CH2OHOH
HH
C
H3C
O O
O-
O
PCH2CH2CONHCH2
H
CN
cyanocobalamin (B12)riboflavin (B2)
N
NH
N
N
O
OH3C
H3C
CH2CH CHCH2OH
OH
CH
OH OH
NN
N
NO
O
CH3
CH3
H3C
caffeine
Unison UK-C18, 75 x 4.6 mmA: 5mM HCOONH4B: methanol10-50%B (0-2min), 50%B (2-5min)1 mL/min, 10uL(500ng), ELSD(SofTA)
1 2
3
4
567
No.TI228E
TI228E-HG04
Chlorophyll and Carotenoid
Courtesy of K. Nakanishi, Tokushima Prefectural Industrial Technology Center, Japan
Unison UK-C18, 100 x 4.6 mmA: methanol / water = 90 / 10, B: ethyl acetate0-100%B (0-7min), 100%B (7min - ), 1 mL/min, ambient, VIS 450 nmacetone extracts from Brassica campestris
0 5 10 min
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
chlorophyll a
chlorophyll b
N
N
N
NMg
COOCH3
C
CH
CH2
CH2
CH3H3C
H3C
H2C CH3
CH2CH3
C O
C
O
O
CH3 CH3
CH3
CH3 CH3
N
N
N
NMg
COOCH3
C
CH
CH2
CH2
CH3H3C
H3C
H2C CHO
CH2CH3
C O
C
O
O
CH3 CH3
CH3
CH3 CH3
1
2
3
1
2
3
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3HO
OH
lutein (xanthophyll) 4
4
-carotene
No.TI237E
TI237E-ED19
Unison UK-Silica, 250 x 4.6 mmhexane / ethyl acetate / tetrahydrofuran = 97 / 2 / 11 mL/min, 37 deg.C, 280 nm, 2 uL(0.2-3ug)
Fat-Soluble Vitamins
1
2
3
4
5
6
7
1
2
3 4,5
6
7
mA
U
0 5 10 min
0
40
Vitamin K3
Vitamin A
O
O
CH3
CH3
CH3CH3
CH3 CH3CH2OH
Vitamin A acetate
CH3
CH3CH3
CH3 CH3O
O
CH3
Vitamin E
OCH2CH2CH2CH
CH3
CH3
CH3H3C
HO
CH3
CH3
3
Vitamin K1
CH2CH2CH2CH
CH3
CH3
3O
O
CH2CH C
CH3CH3
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
beta-carotene
coenzyme Q10
O
O
H3CO
H3CO CH3
CH3H
10
No.TI242E
TI242E-EF29
Cadenza CD-C18, 150 x 4.6 mmA: water / methanol = 50 / 50, B: tetrahydrofuran40 - 80 %B ( 0-25min), 0.8 mL/min, 37 deg.C, 470 nm, 5uL (0.1-20 ug)
Carotinoids
1
2
3
4
1
2
3
4
mA
U
0 10 20 min
0
50
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
beta-carotene
canthaxanthincoenzyme Q10
O
O
H3CO
H3CO CH3
CH3H
10
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
O O
astaxanthin
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
O O
HO
OH
No.TI268E
TI268E-FE12
Water-Soluble Vitamins
1
2
3
4
5
6
7
8
9
O O
OHHO
CHOCH2
H OH
L-ascorbic acid
nicotinamideN
C
O
NH2
nicotinic acid
pyridoxamine
pyridoxal
orotic acid
p-Aminobenzoic acid
N
C
O
OH
pyridoxineN
CH2OHCH2OH
H3C
HO
N
CH2OHCH2NH2
H3C
HO
thiamine
CH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
COOHN
HN
O
OH
N
CH2OH
H3C
HOC
O
H
OCH2N OH
1 2
3 456 7 8 9
1
2
34567
8 9
min0 2 4 6 8 10 12 14
mA
U
0
60
20 mM H3PO41.0 mL/min, 37 deg.C 260 nm
No.TI269E
TI269E-FE12
A: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile1 mL/min (5MPa), 37 deg.C, 254 nm2uL (0.2-5 ug)
Water-Soluble Vitamins
1
2
3
4
5
6
7
8
1 2
3
4
5 6
7 8
1 2 3 4 5
6 7 8
mA
U
0 5 10 15 min
0
200
O O
OHHO
CHOCH2
H OH
L-ascorbic acid
nicotinic acid
pyridoxal
N
C
O
OH
pyridoxineN
CH2OHCH2OH
H3C
HO
thiamine
riboflavin
N
NH
N
N
O
OH3C
H3C
CH2CH CHCH2OH
OH
CH
OH OH
CH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
N
CH2OH
H3C
HOC
O
H
folic acid
HNCHC
ONH2N
CH2NHN
N
N C
C
O
CH2CH2OHOH
OH
O
NN
N
NO
O
CH3
CH3
H3C
caffeine
0-30%B (0-7.5 min)
0-30%B (0-15 min)
No.TI307E
TI307E-GC23
Tocopherols
1
2
3
4
Unison UK-Amino, 250 x 4.6 mmhexane / ethyl acetate / acetic acid = 80 / 20 / 0.11 mL/min (6 MPa), 37 deg.C, 295 nm, 2 uL (2 ug)
1
2
3 4
mA
U
0 5 10 min
0
50
OCH2CH2CH2CH
CH3
CH3
CH3H3C
HO
CH3
CH3
3
OCH2CH2CH2CH
CH3
CH3
CH3HO
CH3
CH3
3
OCH2CH2CH2CH
CH3
CH3H3C
HO
CH3
CH3
3
OCH2CH2CH2CH
CH3
CH3HO
CH3
CH3
3
No.TI309E
TI309E-GD11
Unison UK-Amino, 100 x 4.6 mm1 mL/min, 37 deg.C, 260 nm
Water-Soluble Vitamins
1
2
3
4
5
6
7
mA
U 1
2
34
5
6
1
2
34
56
7
0 10 20 min
0
200
400gradient elutionA: ACN / acetic acid = 100 / 5B: water / acetic acid = 100 / 52-70 %B (0-10min)
isocratic elutionACN / water / acetic acid = 90 / 10 / 5
O O
OHHO
CHOCH2
H OH
L-ascorbic acid
nicotinamideN
C
O
NH2
nicotinic acidN
C
O
OH
thiamine
riboflavin
N
NH
N
N
O
OH3C
H3C
CH2CH CHCH2OH
OH
CH
OH OH
CH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
Co+NN
NN
CH3
CH3CH3
H3CH3C
H3C
CNCH2CH2CONH2
CH2CH2CONH2
NH2COCH2CH2NH2COCH2
NH2COCH2
CH2CONH2CH3 CH3
CH3
CH3
N
N
HO H
CH2OHOH
HH
C
H3C
O O
O-
O
PCH2CH2CONHCH2
H
cyanocobalamin
Folic acid
HNCHC
ONH2N
CH2NHN
N
N C
C
O
CH2CH2OHOH
OH
O
No.TI312E
TI312E-GD13
LC-MS/MS Application of Water Soluble Vitamins
Courtesy of Dr. Pei Chen, USDA, ARS, BHNRC, Food Composition Lab - Beltsville, MD, USA
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00Time0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
Nist-1000x-02 6: MRM of 2 Channels ES+ 245 > 227
8.04e316.75
16.10 17.67
Nist-1000x-02 6: MRM of 2 Channels ES+ 377 > 243
2.33e516.33
Nist-1000x-02 5: MRM of 2 Channels ES+ 442.2 > 295
7.37e315.42
12.13 14.9112.87 13.68
Nist-1000x-02 5: MRM of 2 Channels ES+ 220 > 90
5.06e514.69
Nist-1000x-02 3: MRM of 2 Channels ES+ 123 > 80
1.08e65.37
Nist-1000x-02 3: MRM of 2 Channels ES+ 170 > 152
1.03e64.65
Nist-1000x-02 2: MRM of 1 Channel ES+ 177 > 141
1.45e53.61
Nist-1000x-02 1: MRM of 1 Channel ES+ TIC
1.77e52.26
Unison UK-C18, 250 x 2 mmA: 0.1% HCOOH in waterB: 0.1% HCOOH in acetonitrile0%B (0-5min), 0-50%B (5-15min), 50-95%B (15-17min), 0%B (17-25min) 0.25mL/min, ambient, 5uLESI-MS, MRM Positive
No.TI317E
TI317E-GD24
Ascorbic Acid and Erythorbic Acid
0 10 20 min
50
Unison UK-Amino150 x 2 mmacetonitrile /water /acetic acid = 80 / 20 / 20.2mL/min (5MPa), 37deg.C, 260nm, 1uL(1.3ug)
mAU
0 10 20 min
50mAU
Unison UK-Amino250 x 2 mmwater /acetic acid = 100 /0.50.2mL/min (14MPa), 37deg.C, 260nm, 0.2uL(0.5ug)
L-ascorbic acid(Vitamin C)
OHHO
CHOCH2
H OH
OO
isoascorbic acid(erythorbic acid)
OO
OHHO
CHOCH2
H OH
1 2
12
1 2
anion exchange
normal phase
No.TI330E
TI330E-GF18
Unison UK-Amino, 250 x 4.6 mmhexane / ethanol / acetic acid = 100 / 0.2 / 0.51 mL/min (4MPa), 37 deg.C, 280 nm1 uL (0.5-5ug)
Fat-Soluble Vitamins
1
2
3
4
5
6
7
1
2
3
4
5,6
7
mA
U
0 5 10 15min
0
100Vitamin K3
Vitamin A
O
O
CH3
CH3
CH3CH3
CH3 CH3CH2OH
Vitamin A acetate
CH3
CH3CH3
CH3 CH3O
O
CH3
Vitamin D2
Vitamin D3HO
CH2
HH
CH3
CH3H3CH3C
H
Vitamin E
OCH2CH2CH2CH
CH3
CH3
CH3H3C
HO
CH3
CH3
3
Vitamin K1
CH2CH2CH2CH
CH3
CH3
3O
O
CH2CH C
CH3CH3
HO
CH2
HH
CH3
CH3
CH3
H3CH3C
H
No.TI341E
TI341E-GG23
Dehydroascorbic Acid and Related Compunds
Unison UK-Amino150 x 2 mmacetonitrile /water /acetic acid = 80 / 20 / 50.2mL/min (5MPa), 37deg.C, ELSD, 1uL(2-4ug)
0
50mAU
Unison UK-Amino150 x 2 mmwater /acetic acid = 100 /0.50.2mL/min (9MPa), 37deg.C, ELSD, 1uL(2-4ug)
L-ascorbic acid(Vitamin C)
OHHO
CHOCH2
H OH
OO
isoascorbic acid(erythorbic acid)
OO
OHHO
CHOCH2
H OH
3
1
2
0 5 10 min
0
200mV
15
0 10 min
0
50mV
5 15
dehydroascorbic acid1 2
3
1
2
3
unkn
own
unkn
own
anion exchange
normal phase
O OCHOCH2
H OH
OO
No.TI359E
TI359E-GH23
Unison UK-Amino, 250 x 4.6 mmtetrahydrofuran / water / acetic acid = 100 / 2 / 11 mL/min (9MPa), 37 deg.C, 260 nm, 1 uL (1ug)
Vitamin B6
1 2
pyridoxal pyridoxine
N
CH2OHCH2OH
H3C
HO
N
CH2OH
H3C
HOC
O
H
mA
U
1
2
0 2 4 6 8 min
0
20
No.TI394E
TI394E-HB18
Nicotinic Acid
1
nicotinic acidN
CO
OH
Unison UK-Amino, 100 x 2 mmA: acetonitrile / HCOOH = 100/0.1B: water / HCOOH = 100 / 0.120-60 %B(0-8min), 0.2 mL/min37 deg.C, 260 nm, 0.4 uL(0.8 ug)
Unison UK-Amino, 100 x 2 mmA: acetonitrileB: 20mM HCOONH450-90 %B(0-8min), 0.2 mL/min37 deg.C, 260 nm, 0.4 uL(0.8 ug)
1mA
U
0 2 4 6 8 min
0
100
1
mA
U
0 2 4 6 8 min
0
100
200
No.TI421E
TI421E-HH25
Unison UK-Amino, 150 x 2 mm�A: acetone / formic acid = 100 / 0.1�B: water / formic acid = 100 / 0.1�0 - 100%B (0-10min)0.2 mL/min (3MPa)37 deg.C, ELSD, 2 uL (2ug)
Unison UK-Silica, 150 x 2 mm�A: acetone / formic acid = 100 / 0.1�B: water / formic acid = 100 / 0.1�0 - 100%B (0-10min)0.2 mL/min (3MPa)37 deg.C, ELSD, 2 uL (2ug)
PQQ: pyrroloquinoline quinone (Normal Phase)
NHN
C OHCHO
CO
OO
OO
OH
mA
U
0 5 10 min
0
20
40
60
mA
U
0 5 10 min
0
10
20
PQQ ( pyrroloquinoline quinone )
No.TI464E
TI464E-HL10
LC-MS/MS Application for 25-Hydroxy Vitamin D3
Courtesy of Penn State Hershey Medical Center, USA
Cadenza CD-C18, 150 x 4.6 mm
A: water / methanol = 75 / 25B: acetonitrile / ethyl acetate = 70 / 3040%B (0-1min), 40-95%B (1-8min), 95%B (8-15min), 40%B (15-22min)0.7mL/min, ambient
Applied Biosystems 4000 Q trapIonization: APCI Positive modeLOQ: 1ng/mL
XIC of +MRM (1 pair): 383.3/211.1 amu from Sample 5 (fresh std 500ng/ml opt conditions_inj 20uL) of 102708 25-OH Vit D3 stds_Imtakt ... Max. 5.6e4 cps.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e410.37
10.877.00 11.1711.92
25-Hydroxy Vitamin D3(Calcifediol)
HO
CH2
HH
CH3
CH3H3CH3C
H
OH
No.TI465E
TI465E-HL18
150 x 4.6 mmmethanol1 mL/min (6-8MPa)37 deg.C, 470 nm2 uL (1ug)
Lycopene
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
lycopene
mA
U
0 10 20 30 40 min
0
100
No.TI492E
TI492E-IC19
Cadenza CW-C18, 150 x 3 mmA: water / trifluoroacetic acid = 100 / 0.1B: acetonitrile / trifluoroacetic acid = 100 / 0.073-30 %B (0-10min), 0.4 mL/min (9MPa), 37 deg.C210 nm, 1 uL (0.1-1.2ug, ethanol, water)
Water-soluble vitamins
1
2
3
4
folic acid
HNCHC
ONH2N
CH2NHN
N
N CC
O
CH2CH2OHOH
OH
O
HOCH2
H3C
H3CCHOH
C CO
OHNHCH2CH2 CO
(1/2Ca2)
calcium pantothenate
pyridoxine
N
CH2OHCH2OH
H3C
HO
riboflavin
N
NH
N
N
O
OH3C
H3C
CH2CH CHCH2OH
OH
CH
OH OH
12
3
4
mA
U
0 5 10 min
0
50
No.TI554E
TI554E-IL25
Alpha-carotene and beta-carotene
Courtesy of Mr Baburao. TR; Davos lifescience Pte Ltd, Singapore
1 2
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
150 x 4.6 mmmethanol, 1 mL/min (7-8 MPa), 37 deg.C, 470nm, 1 uL (0.5 ug)
0 10 20 30 40
0
100
200
mA
U
min
Cadenza CL-C18
Cadenza CD-C18
Unison UK-C18
1
2
1
2
1
2
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
alpha-carotene
CH3
CH3CH3
CH3 CH3
CH3
H3CH3C
CH3 CH3
beta-carotene
No.TI559E
TI559E-JA13
Tocopherols and tocotrienols
1
2
3
4
5
6
7
8
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
O
CH3
CH3
CH3H3C
HOCH3 CH3 CH3
CH3CH3
O
CH3
CH3HO
CH3 CH3 CH3
CH3
CH3
OCH3
H3C
HOCH3 CH3 CH3
CH3CH3
OCH3
HOCH3 CH3 CH3
CH3O
CH3
CH3
CH3H3C
HOCH3 CH3 CH3
CH3
CH3
O
CH3
CH3HO
CH3 CH3 CH3
CH3
CH3
OCH3
H3C
HOCH3 CH3 CH3
CH3
CH3
OCH3
HOCH3 CH3 CH3
CH3
150 x 4.6 mm, 1 mL/min , 37 deg.C, 295 nm, 2 uL (2ug)
Unison UK-Silicahexane / ethyl acetate / acetic acid= 97 / 3 / 0.13 MPa
Unison UK-Aminohexane / tetrahydrofuran / acetic acid= 91 / 9 / 0.14 MPa
0 10 200
100
200
mA
U
min
1 2
3 45
6 7 8
12
34
5 6 78
Courtesy of Mr Baburao. TR; Davos lifescience Pte Ltd, Singapore
No.TI600E
TI600E-JF08
Analysis of Water-soluble Vitamins using LCMS-2020
Imtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])North America Other Countries
Shimadzu LCMS-2020(ESI, positive/negative, SIM)
Scherzo SM-C18, 150 x 2 mmA: 5 mmol/L ammonium formate + 0.1% formic acid - waterB: acetonitrile0 - 55 %B (0-10 min), 0 %B(10.01-20 min)1,000 pg/uL, 1uL
pyridoxineN
CH2OHCH2OH
H3C
HO
thiamineCH2CH2OHN
N
H3C
CH2 N
S
+ CH3
NH2
nicotinic acidN
C
O
OH
nicotinamideN
C
O
NH2
HOCH2
H3C
H3CCHOH
C CO
OHNHCH2CH2 CO
pantothenic acid
Co+NN
NN
CH3
CH3CH3
H3CH3C
H3C
CH2CH2CONH2
CH2CH2CONH2
NH2COCH2CH2NH2COCH2
NH2COCH2
CH2CONH2CH3 CH3
CH3
CH3
N
N
HO H
CH2OHOH
HH
C
H3C
O O
O-
O
PCH2CH2CONHCH2
H
CN
cyanocobalamin
riboflavinN
NH
N
N
O
OH3C
H3C
CH2CH CHCH2OH
OH
CH
OH OH
folic acid
HNCHC
ONH2N
CH2NHN
N
N C
C
O
CH2CH2OHOH
OH
O
1
2
3
4
5
6
7
8 NHHN
S
O
H H
COOH
biotin
9
�0
�1�0�0�0�0�0
�2�0�0�0�0�0
�3�0�0�0�0�0
�4�0�0�0�0�0
�5�0�0�0�0�0
�6�0�0�0�0�0
�7�0�0�0�0�0
�8�0�0�0�0�0
�9�0�0�0�0�0
�1�0�0�0�0�0�0
�1�1�0�0�0�0�0
�1�2�0�0�0�0�0
�1�3�0�0�0�0�0
�1�4�0�0�0�0�0
�1�5�0�0�0�0�0
�1�6�0�0�0�0�0
�1�7�0�0�0�0�0
�1�8�0�0�0�0�0
12
3
4
5
67
8
9
(+):245.10(2.50)(+):377.20(4.00)(+):678.60(8.00)(+):220.20(2.50)(+):123.20(2.00)(+):124.20(4.00)(+):170.20(+):265.10
(-):440.10(18.00)
0.0 2.5 5.0 7.5 10.0 12.5 min
No.TI648E
TI648E-KC25
Data provided by Dr. Eric VERETTE, SEDERE S.A.S., FranceImtakt Corp./JAPAN ([email protected])Imtakt USA ([email protected])
North America Other Countries
Scherzo SM-C18, 150 x 2 mmA: H2O + 0.3% HCOOH
B: acetonitrile0%B (0-0.1 min)
0 - 30%B (0.1-10 min)30%B (10-11 min)
30 - 100%B (11- 12 min)100%B (12-26 min)
0.3 mL/min, 30°C2uL(DMSO soln.)
ELSD (SEDERE, France)SEDEX 90LT, 40°C, 3.5Bar
Simultaneous HPLC/LT-ELSD Analysis of Water- and Fat-Soluble Vitamins
Vitamin Mix (15 compounds)
0 5 10 15 20 25Minute
mV
123
4 5 678
9
10
11
1213
14
15
ng (o.c.) ResponseRTMinutes
LOD(S/N=3)%RSD (n=6)RT
25.8623.6123.2622.8920.8717.6410.7010.4910.179.927.321.941.441.140.92
15-K1: Phylloquinone14-E: a-Tocopherol13-D3: Cholecalciferol12-D2: Ergocalciferol11-K2: Menaquinone10-A: Retinol
9-B7: Biotin8-B2: Riboflavin7-B12: Cyanocobalamin6-B9: Folic acid5-B5: Pantothenic acid4-C: Ascorbic acid3-B6: Pyridoxine2-B8: myo-Inositol 1-B1: Thiamine
0.070.070.060.060.070.060.090.110.110.090.150.250.160.090.11
1.71.62.61.52.16.74.23.44.13.16.82.42.76.22.6
41.745.052.647.614.21.74.85.04.68.06.99.61.86.35.7
