APPLICATION NOTE 70755
Two-dimensional HPLC determination of
water-soluble vitamins in a nutritional drink
Authors
Dai Zhenyu, Chen Jing, Xu Qun,
and Jeffrey Rohrer, Thermo Fisher
Scientific, Shanghai, People’s
Republic of China;
Keywords
Food Analysis, Food Quality,
Two-Dimensional HPLC, Acclaim
PolarAdvantage Column, Acclaim
120 C18 Column, Hypersil GOLD
Phenyl Column
1
Goal
To develop an efficient high-performance liquid chromatography (HPLC)
method for simple and sensitive determination of water-soluble vitamins in
a complex multivitamin/mineral drink. Target analytes are B group vitamins,
including thiamine (VB1), riboflavin (VB2), nicotinamide (VB3), pantothenic acid
(VB5), pyridoxine (VB6), biotin (VB7), and cyanocobalamin (VB12), and ascorbic
acid (VC).
Introduction
Vitamins are a well-known group of compounds that are essential for
human health. They can be classified into two main groups: water- and fat-
soluble. With the exception of VB6 and VB12, water-soluble vitamins are not
stored in the body. Thus, if one’s dietary vitamin intake is insufficient,
a vitamin supplement should be added to the diet.
The vitamin supplement can be in tablet form, a clear vitamin-enhanced
functional drink, vitamin-enhanced milk, or a nontransparent multivitamin/
mineral nutritional drink with additions of other substances (e.g., fruit
extracts) that make it more complex than clear products. To ensure that
these products contain the labeled amounts of vitamins, a number of
reliable quality control assays are available.1,2 For a vitamin tablet or a clear
functional drink, the analysis is relatively simple and a routine
HPLC method (e.g., a C18 column with UV detection)
is satisfactory for quantifying the vitamins.3
 Some samples, however, have too many additional
components to allow a routine HPLC vitamin-
quantification method. Vitamin-enhanced milk and a
nontransparent multivitamin/mineral nutritional drink—
referred to as a multivitamin nutritional drink throughout
the rest of this study—are two such samples. In addition
to vitamins, these samples may also supply amino acids,
minerals, coenzyme Q10, the compounds contained in
grape extracts, and more. These additional compounds
interfere with the separation of vitamins, making
quantification difficult. Therefore, a simple and sensitive
two-dimensional HPLC (2D-HPLC) method is needed to
quantify vitamins in these complex samples.
Equipment and Software
• The Thermo Scientific™ UltiMateTM 3000 Dual-
Gradient Rapid Separation (RS) LC system was
used, which includes:
––Thermo Scientific™ UltiMateTM 3000 Series SRD-
3600 Integrated Solvent and Degasser Rack |
(P/N 5035.9230)
––Thermo Scientific™ UltiMateTM 3000 DPG-3600RS
Dual-Gradient RS Pump (P/N 5040.0066)
––Thermo Scientific™ UltiMateTM 3000 WPS-3000TRS
Thermostatted Split-Loop Autosampler
(P/N 5840.0020)
––Thermo Scientific™ UltiMateTM 3000 TCC-3000RS
Rapid Separation Thermostatted Column
Compartment (P/N 5730.000) with 2p-6p valves
––Thermo Scientific™ UltiMate 3000 DAD-3000RS
TM
Diode Array Detector (P/N 5082.0020), equipped
with analytical flow cell, 13 µL, SST (P/N 6082.0100)
––Mixer for 800 μL Mixing Volume (P/N 6040.5750)
• Thermo Scientific™ Chromeleon™ Chromatography
Data System (CDS) software version 7.1 or higher
• Thermo Scientific™ Orion™ 2-Star pH
Benchtop Meter
Reagents and Standards
• Deionized (DI) water, 18.2 MΩ.cm resistivity
• Acetonitrile (CH3CN) for HPLC (Fisher Scientific
P/N AC610010040)
• Potassium Phosphate Monobasic (KH2PO4) (Fisher
Scientific P/N P286-1)
• o-Phosphoric Acid (H3PO4), 85%, (Fisher Scientific
P/N A260-500)
• Products from the National Institute for the Control of
Pharmaceutical and Biological Products, Beijing, China:
–– VB1, VB2, VB3, VB5, VB6, VB7, VB12, VC
Preparation of Standard Solutions
To prepare water-soluble vitamin standards of V B1, VB3,
VB5, VB6, VB7, VB12, and VC, weigh 10 mg of the vitamin
powder and add DI water to 10 mL in a volumetric flask
to make stock solutions of 1.0 mg/mL for each vitamin.
Make a fresh preparation of VC daily, due to its limited
stability. Also, because of the limited solubility of VB2
in water, decrease the concentration of the VB2 stock
solution to 0.03 mg/mL. Weigh 3 mg of V B2 into
100 mL DI water to address the solubility issue. If a 10
mL volumetric flask was used, 0.3 mg of V B2 would have
to be weighed, but that would exceed the precision
range of the balance.
Add 500 μL of VB2 stock solution, 100 μL each of VB5 and
VB7 stock solutions, and 50 μL each of VB1, VB3, VB6, VB12,
and VC stock solutions to a 1 mL sample vial. Bring the
final volume to 1 mL by adding 50 μL of DI water to make
the mixed stock standard solution. In this mixed stock
standard solution, the concentration of VB2 will be
15 μg/mL, the concentrations of VB5 and VB7 will be
100 μg/mL, and the concentration of the other vitamins
will be 50 μg/mL.

Table 1. Preparation of mixed working standard solutions of water-soluble vitamins.

Mixed Working Standard Solution 1 2 3 4 5 6

Volume of Mixed Stock Standard Solution for a 10 mL
0.02 0.1 0.2 1.0 2.0 10
Preparation (mL)
VB2 0.03 0.15 0.3 1.5 3.0 15
Concentration of Each
VB5, VB7 0.2 1.0 2.0 10 20 100
Vitamin (mg/L)
VB1, VB3, VB6, VB12, VC 0.1 0.5 1.0 5.0 10 50

2
 Table 2. Labeled values of the multivitamin nutritional drink.
Amount Per Amount Per
Ingredient Ingredient
Serving* Serving
Total Carbohydrate 9g Zinc (as gluconate) 15 mg
Vitamin A 10000 IU** Selenium (as L-selenium methione) 100 mcg
Vitamin C 1000 mg Copper (as gluconate) 1 mg
Vitamin D3 200 IU Manganese (as gluconate) 5 mg
Vitamin E 200 IU Chromium (as amino acid chelate) 200 mcg
Vitamin K1 300 mcg Potassium (as citrate) 100 mg
Vitamin B1 30 mg Choline (as bitartrate) 30 mg
Vitamin B2 30 mg Inositol 30 mg
Vitamin B3 30 mg Boron (as amino acid chelate) 1 mg
Vitamin B5 150 mg Amino Acid Complex (proprietary formula) 125 mg
Vitamin B6 30 mg Grape Seed Extract 25 mg
Vitamin B7 300 mcg Coenzyme Q-10 5 mg
Vitamin B12 500 mcg Dimethyl Glycine 25 mg
Folate 400 mcg Paba 30 mg
Calcium 600 mg Citrus Bioflavonoids 13 mg
Phosphorus 80 mg Glucolactone 150 mg
Iron (as gluconate) 4 mg Plant-Derived Minerals 600 mg
Magnesium (as citrate, gluconate) 300 mg
* Serving size: 28.4 mL **International units (IU), mass depending on substance potency

For the preparation of mixed working standard solutions Conditions

for calibration, add the appropriate volume of the mixed First Dimension
stock standard solution into 10 mL glass vials and bring Columns: For water-soluble vitamins except for
to 10 mL with DI water. See Table 1 for details. VB12, Thermo Scientific™ Acclaim™
PolarAdvantage™ (PA), 5 μm Analytical,
Sample Preparation 4.6 × 250 mm (P/N 061321)
A multivitamin nutritional drink supplemented with VB1,
VB2, VB3, VB5, VB6, VB7, VB12, and VC was provided by a For VB12, Thermo Scientific™ Hypersil
customer. The drink also contained some fat-soluble GOLD™ Phenyl Analytical HPLC, 5 μm,
vitamins, minerals, amino acid complex, grape seed 4.6 × 150 mm (P/N 25905-154630)
extract, coenzyme Q10, and other ingredients added to
meet daily nutritional needs. Table 2 lists the Mobile Phase: A. 25 mM phosphate buffer
sample components. (dissolve ~3.4 g KH2PO4 in 1 L water
and adjust the pH to 3.0 with H3PO4)
Dilute the drink sample with DI water if necessary and B. CH3CN
filter through a 0.45 µm filter. To determine if the sample Gradient: See Table 3
needs to be diluted, compare the labeled values to the Flow Rate: 0.8 mL/min
calibration ranges in this study. Store the sample in a Inj. Volume: 10 μL
brown bottle at 4 °C before analysis. Temperature: 25 °C
Detection: UV, absorbance at 210 and 254 nm
Second Dimension
Column: Acclaim 120 C18, 5 μm Analytical,
4.6 × 150 mm (P/N 059148)
Mobile Phase: Same as used in the First Dimension
Flow Rate: 0.8 mL/min
Temperature: 25 °C
Detection: UV absorbance at 210, 245, 268,
and 291 nm
3
These conditions apply to Figures 3 through 7.
 Table 3. Gradient program and valve switching.

First Dimension (Right Pump) Valve Switching Second Dimension (Left Pump)
Flow %A Flow %A
Right Left
Time Rate (25 mM %B Time Time Rate (25 mM %B
Valve Valve
(min) (mL/ Phosphate (CH3CN) (min) (min) (mL/ Phosphate (CH3CN)
Position Position
min) Buffer, pH 3.0) min) Buffer, pH 3.0)
0 0.8 100 0 0 6_1 1_2 0 0.8 100 0
5 0.8 100 0 3.94 1_2 6_1 14 0.8 100 0
12.5 0.8 65.0 35.0 4.3 6_1 1_2 23.5 0.8 50 50
13.0 0.8 20.0 80.0 5.32 1_2 6_1 24 0.8 100 0
21.0 0.8 20.0 80.0 5.89 6_1 1_2 30 0.8 100 0
21.5 0.8 100 0 7.8 1_2 1_2 — — — —
30 0.8 100 0 8.35 6_1 6_1 — — — —
— — — — 13.12 1_2 1_2 — — — —
— — — — 13.28 6_1 6_1 — — — —
— — — — 14.61 1_2 1_2 — — — —
— — — — 14.82 6_1 6_1 — — — —
— — — — 15.42 1_2 1_2 — — — —
— — — — 15.60 6_1 6_1 — — — —
— — — — 15.97 1_2 1_2 — — — —
— — — — 16.12 6_1 6_1 — — — —
— — — — 28.5 6_1 1_2 — — — —

Column: Acclaim PA1,Column: Acclaim

5 µm Analytical, 4.6PA1, 5 µm
× 250 mmAnalytical, 4.6 × 250 mm
Results and Discussion Column:Phase:
Mobile Acclaim
A. 25 mM PA1,
Column:
Mobile5 µm
phosphate Analytical,
Phase: Acclaim
A.
buffer 25 4.6
mM PA1,
(dissolve ×phosphate
250
5 µm
~3.4 mmgAnalytical,
KHbuffer in 4.6
1L×
PO4 (dissolve 250
~3.4mm
water g KH2PO4 in 1
2
Conventional HPLC Method for the Column:
Mobile Phase: Acclaim
A. and
25 mM PA1,
Column:
Mobile
adjust 5 µm
phosphate
pH Analytical,
Phase:
to 3.0 Acclaim
buffer
A. and
with 25H34.6 PA1,
(dissolve
mM )×phosphate
adjust
PO 4
250
5 µm
~3.4
pH mm
to Analytical,
g3.0
KHbuffer
2
H34.6
1 L4)×
PO4 (dissolve
with in PO 250
~3.4mm
water g KH2PO4 in 1
Column:
Mobile Phase: Acclaim
A. and
B. 25 3mM
CH PA1,
Column:
Mobile
adjust
CN 5 µm
phosphate
pH Analytical,
Phase:
to 3.0 Acclaim
buffer
A.
with 25
B. and
CHH334.6
PO
CN PA1,
(dissolve
mM )×
adjust 250
5
pHµm
phosphate
~3.4 mm
to g Analytical,
KH
3.0 PO
buffer
with in
H 4.6
1
POL
(dissolve )× 250
water
~3.4mm
g KH2PO4 in 1
Determination of Vitamins Mobile Phase: A. 25
B. and
4 2 4 3 4
Gradient: CH CN,mM
CH
3 3
CN Mobile
phosphate
adjust pHPhase:
Gradient:
0–5 min, 3.0buffer
to0%; A.
with
CHand
B.
12.5 25
(dissolve
CHCN,
min,
3 33
mM
H PO
CN0–5) phosphate
adjust
35%;
4
~3.4
pH
min,
13–21g3.0
to0%;KH PO4 min,
buffer
with
12.5
min,
2
in
(dissolve
H3PO1 L35%;
4
)water
~3.4
13–21 2
PO4 in 1
g KHmin,
Currently, there is no U.S. Pharmacopeia (USP) method Gradient: CHand
B.
80%;CHCN,
3 3
adjust
CN0–5
21.5–30 pH
min,
Gradient: to
min, 3.0
0%;
0% with
B.
12.5
CH
80%; and
CHH
min,
CN,
3 33
PO0–5)
adjust
CN 35%;
21.5–30
4
pH
min,to
13–21 3.0
0%;
min, with
min,
0%12.5 H
min,
3
PO )
35%;
4
13–21 min,
for the separation of a mixture containing all eight water- Gradient:
Flow Rate: B.
CH
80%;
0.8 CHCN,CN0–5
Flowmin,
Gradient:
21.5–30
mL/min
3 3
min,
Rate:0%;0% B.
12.5
CH
80%;
0.8 CH
min,
CN,
3 3
CN0–5
35%;min,
21.5–30
mL/min 13–21
0%;0%
min, min,
12.5 min, 35%; 13–21 min,
Gradient:
FlowVolume:
Inj. Rate: CH
80%;
0.8
10 CN, 0–5
21.5–30
mL/min
µL min,
Gradient:
min,
FlowVolume:
Inj. Rate:0%;0%12.5
CH
80%;
0.8
10 min,
CN, 0–5
mL/min
µL 35%;min,
21.5–30 13–21
0%;0%
min, min,
12.5 min, 35%; 13–21 min,
soluble vitamins. A USP method for individual vitamins is Flow Rate:
Inj. Volume:
Temperature: 80%;
0.8
10
25
3
µL
°C 21.5–30
mL/minFlow min, 0% 25
Rate:
Inj. Volume:
Temperature: 80%;
0.8
10
3
µL
°C 21.5–30 min, 0%
mL/min
complicated (i.e., sodium perchlorate, phosphoric acid, Flow
Inj. Rate:
Volume:
Temperature:
Detection: 0.8
10 mL/min
µL Flow
Inj.
25 absorbance
UV °C Rate:
Volume:
Temperature:
Detection:
at 210 nm 0.8
10
25 µL
UV mL/min at 210 nm
°C
absorbance
Inj. Volume:
Temperature:
Detection:
Samples: 10
25
A. µL
°C
UVStandard Inj.
absorbance Volume:
Temperature:
Detection:
Samples:at 210 nm
mixture 10
25 µL
°C
UVMultivitamin
B.A. absorbance
Standard at 210 nm
mixture
nutritional B.drink
Multivitamin nutritional drink
dimethyl sulfoxide, acetonitrile, and water are needed for
Temperature:
Detection:
Samples: 25
UV °C
A. Standard Temperature:
absorbance
Detection:
Samples:at 210 nm
mixture B.25
UV °C
absorbance
A.Multivitamin
Standard at 210 nm
mixture
nutritional B.drink
Multivitamin nutritional drink
biotin determination) and involves an ion-pairing agent Detection:
Samples:
Peaks: UV
1. absorbance
A. VStandard
B1
Detection:
Samples:
Peaks:
50 at 210 nm
mixture
mg/L 5.UV
B.1. absorbance
A.VMultivitamin
VStandard
B5B1
mixture
50 mg/L
100 at 210 nm
nutritional B.drink
5. VMultivitamin
B5
100 nutritional drink
to retain hydrophilic vitamins.4 Due to the irreversible Samples:
Peaks: A. VStandard
1.
2. B1
C
Samples:
50 mixture
Peaks:
mg/L B.
5.
6.A.
1.
2.VMultivitamin
VStandard
B5C
B12B1
100
50 mixture
nutritional
mg/L B.
5.
6. drink
VMultivitamin
B5
B12
50 nutritional drink
100
Peaks: 1.
2. VB3
3. Peaks:
50 mg/L 6.1.
5.
7. VB3
2.VB2
3. 100
50 mg/L
15 5.
6. VB2
7. 100
50
15
impact of the ion-pairing agent on column performance, Peaks: 1.
2. VB6
3.
4.
B1
C
Peaks:
50 mg/L 5.
6.
7.
8.1.
2.
3.
4.V
B5
B12
V
B1
C
50
15
100 mg/L 5.
6.
7.
8. V
B5
B12
50
15
100
B1
C
B3 B5
B12
B2
B7 B1
C
B3
B6 B5
B12
B2
B7
extensive research was conducted to search for 500
2.
3.
4. VCB3
B6
50
500
6.3.
7.
8.2. VCB3
4.VB2
B12
B7 B6
50
15
100 6. VB2
7.
8. B12
B7
50
15
100
3.
4. VB3 50 7.
8.3.
4.V V 15
50
100 7.
8. V 15
100
methods without ion-pairing agents and with the use of a 500 B6
500 B2
B7B3
B6 B2
B7
4. VB6 50 8.4.VB7
VB6 100 50 8. VB7 100
simpler mobile phase. 500 500
500 500

Recently, acidic or neutral phosphate buffer/organic

solvent mobile phases have been used to separate mAU mAU
mAU mAU
vitamins in an extract of a multivitamin tablet and mAU mAU
in vitamin-enhanced functional drinks that are less mAU mAU
complex than the one investigated here.1 For complex 6 6

samples, such as certain multivitamin nutritional B 3 4B 3 6 4 6

B 3 4 B 3 6 4 6
drinks, the additional supplements can interfere with B 1 3 4B 1 53 6 74 5
6 7
vitamin separation and, ultimately, their detection by UV B
A 1 23 4AB 1 253 74 8 5 78
0 A 1 2 0 A 1 25 78 5 78
absorbance. Figure 1 shows that the use of a typical 0 A 1 2 0 A 1 25 78 78
–500 –50 5
HPLC method for a multi-vitamin nutritional drink results 0 A 52 0 0 A 10 52 15 8 10 20 15 8 25 20
–500 –500 Minutes
0 5 0 10 Minutes
5 15 10 20 15 25 20
in the water-soluble vitamin peaks being hard or even –50
0 5
–50
0 10 Minutes
5 15 10 Minutes
20 15 25 20
Figure 1. A standard–50
–50 mixture (A) and a multivitamin nutritional
Minutes Minutes drink
impossible to quantify due to the large number of 0 5 0 10 5 15 10 20 15 25 20
4 sample (B) using a conventional HPLC
Minutesmethod. Minutes
 UV-absorbing interfering peaks. Obviously, Peaks 1–3
cannot be quantified. Peaks 5–8 can be detected in the
sample, but due to all the additional peaks, the vitamin
peaks cannot be precisely quantified. Comparison of the
UV spectra of the standard and the sample confirmed
that Peaks 5–8 were not pure enough for quantification.
The 2D-HPLC Method
Two-dimensional HPLC has been used to achieve
efficient separation of complex samples. Not only
do two columns with different chemistries provide
additional separation power, but the use of heart-cutting
technology in on-line 2D-HPLC also simplifies the
separation in the second dimension. The work shown
here uses 2D chromatography to determine water-
soluble vitamins in a complex sample.
Figure 2 shows the configuration of the 2D-HPLC system
used for this study. After simple sample preparation (filter
the sample, then dilute with DI water if necessary), inject
the sample into the first dimension and partially separate
it using an Acclaim PA column. The in-line UV detector
determines where the water-soluble vitamins elute. An
injection of the mixed standard determines the start and
end times for each vitamin peak. Use these values to
switch the valves.
Dual-gradient Autosampler
pump
Left pump Right pump
750 µL
mixer
Left
2 1
3 6
4 5
Waste UV
Column 2
Detector 2
Figure 2. Configuration of the 2D-HPLC system.
Switch the right valve to the 1_2 position to individually
transfer the vitamin peaks to the flow path of the
second column. For early eluting vitamin peaks, switch
the left valve to the 1_6 position so that these vitamin
fractions are directly transferred to the second column.
For late eluting vitamin peaks, switch the left valve to
the 1_2 position to put the 750 μL mixer in line. In this
5
configuration, the water mobile phase in the second
dimension will dilute the acetonitrile from the first-
dimension mobile phase. This enables the second-
dimension column to trap the vitamin peaks.
Note: When the right valve is switched to the 1_2 position, UV
Detector 1 will be connected between the two columns. However,
the backpressure of the second-dimension column may exceed the
pressure limit of the UV Detector 1 flow cell. Thus, choose the proper
particle size and length of the second-dimension column to keep the
backpressure below the pressure limit of the flow cell. The pressure
limit command in the instrument method can also be set to stop the
flow in any situation when backpressure may become too high.
Development of the 2D-HPLC Method
Valve switching
To achieve optimal resolution and peak shape in
2D-HPLC applications, the critical part of method
development is the transfer of first-dimension peaks
to the second-dimension column. Ideally, the first-
dimension mobile phase being cut and transferred with
target peaks to the second dimension will be same
as the second-dimension mobile phase at the time of
transfer. But in reality, the two mobile phases usually
will have different concentrations of acetonitrile or other
organic solvent.
Figure 3, Chromatogram A shows that Peaks 5, 7, and
8 disappear when they are directly transferred to the
second dimension. Obviously, the late eluting peaks from
the first column are contained in too high a concentration
Column 1
of acetonitrile to be retained on the second column. The
traditional approach to this problem is to provide water
UV
to dilute the acetonitrile in the transferred fraction with
Detector 1
a tertiary pump so the fraction can be retained on the
Right trap column ahead of the second column.5 However, the
need for an additional pump limits this application.
2 1
3 6 Waste
4 5
Thermo Scientific Application Note (AN) 1023
demonstrated an alternative way to solve this problem.6
Briefly, a 750 μL mixer was configured in line before the
eluted fraction was transferred to the second dimension.
Then the peak mobile phase was mixed extensively with
the second-dimension starting mobile phase (water
phase) before the second-dimension separation.
In this study, the authors initially applied the AN 1023
approach for all target peaks. It worked well for late
eluting peaks, which had high concentrations of
acetonitrile, but the early eluting peaks broadened
(Figure 3, Chromatogram B). After conducting additional
experiments, the authors discovered that early
 Detection: UV absorbance
Detection: UV absorbance
Detection: at 245
at 245 UV nm
nm absorbance
Detection: at 245UVnm absorbance at 245 nm
eluting vitamin peaks broadened due to their ValveValve Position:
Position:
Detection: See See
TableTable
Valve
UV absorbance 3at 245 nm
3 Position:
Detection: See Table
Valve
UV absorbance 3Position:
Detection: at 245See
UVnm Table 3 at 245 nm
absorbance
physiochemical characteristics. These peaks cannot be ValveSamples:
Samples:
Position: A. A. Standard
Samples:
SeeStandard
Table mixtureA.
3 mixture
Valve Position: SeeStandard
Samples:
Table mixture A.
Valve 3Position: SeeStandard
Table 3 mixture
Samples: B. B. Multivitamin
A. Multivitamin
Standard
Samples: nutritional
B.
nutritional
mixture A. drink drink mixture
Multivitamin
Standard
Samples: nutritional
B.
A. Multivitamin
drink mixture
Standard nutritional drink
trapped at the head of the second column, even with B. Multivitamin nutritionalB. drink
Multivitamin nutritional
B. Multivitamin
drink nutritional drink
Peaks:
Peaks: 1. VB1
1. VB1 50 mg/L 3.
Peaks:
50 mg/L 3.
1. VVB3 V 50 mg/L
Peaks: 50 5. VB125. VVB12
1. 3. VB3 5050
50 mg/L 5. VB12 3. VB350 50 5. VB12
a 100% water mobile phase. Thus, the configuration Peaks: 2. 2. VCPeaks:
1. VCB1 50
50 mg/L 2.
4.
3.
B1 B3
4.
1. VVB6 V 50 mg/L
Peaks: 50 6. 6.
5. VB22. 3.
B1
VVCB1
1. 4. VB3
15
50 15
5050
mg/L 5.6. VB12
4.
3. VB315 50
50 6. VB12
5.
B1 B6
C
B3 B12 B2 B6 B2 B6 B2
was modified so that early eluting peaks are directly 200 200 2. V C
50
200 2.
4. VV200
B6
C
50 6. V 2.
B2
V
4. 15
C
V B6
5050 6. V 4.
B2
V 15 50
B6
6. VB2
transferred to the second column, whereas late eluting 200 200 200

peaks are transferred into the 750 μL mixer before the

second column. Figure 3, Chromatogram C shows that
the final configuration works well for all eight water- 5 5 6 6 5 6
soluble vitamins. 5 6 5 6

mAUmAU mAU mAU

mAU mAU mAU

Column: Acclaim PA1, 5 µm Analytical, 4.6 × 250 mm

Column: Acclaim PA1, Column:
Mobile 5 Phase:
µm Analytical,
Acclaim
A. 25 mM4.6PA1,
× 250
5 µm mm
phosphate Analytical, 4.6 × 250
buffer (dissolve ~3.4mm
g KH2PO4 in 1 L water
Column:
Mobile Phase: Acclaim
A. 25 mM PA1,
Column:
Mobile 5 Phase:
µm Analytical,
phosphate Acclaim
buffer
A. and
25 4.6PA1,
(dissolve
mM ×
adjust 250
5 µm mm
phosphate
~3.4
pH to Analytical,
g3.0
KH Hin34.6
1 L4)×water
PO4 (dissolve
buffer
with PO 250 g KH2PO4 in 1 B
~3.4mm B
L water B B
Mobile Phase: Acclaim
Column: A. and
25 mM Mobile
phosphate
adjust
PA1,
Column:5 Phase:
pH to 3.0
µm buffer
A.
with 25
B. and
CH
Analytical,
Acclaim(dissolve
H3mM
PO
CN
4.6 )
adjust
PA1,
×phosphate
~3.4
pH
250
5 to
µmmmg KH
3.0
2
PO
buffer
with
Analytical,Hin 1
PO
4.6L
(dissolve )
× water
~3.4
250 g
mm KH PO in 1 B
L water B2 23 3 B 2 3 5 156 6 2 3 5
4 2 4 3 4 2 4 1 1 2 1 2 5 2 5 6
Mobile Phase: A. B. and
CH3mM
25 adjust
CN pHPhase:
Gradient:
Mobile to 3.0buffer
phosphate with
CHand
B.
A. CH
CN,
25 PO
H3mM
CN0–5 ) min,
adjust pH to0%;
g3.0 with
12.5 Hin3PO
PO4min, L4)water
1 35%; 13–21
g KHmin, 3 3 16 3 6
3 (dissolve
4 phosphate
~3.4 KH
buffer (dissolve ~3.4 PO4 in 1 L water 1 1
2 2
4 4 4 4
Gradient: B. CH
CHandCN, CN
0–5
adjust min,
Gradient:
pH 0%;
to 3.0 B.
12.5
CH
80%;
with CH
min,
CN,
and
H CN
PO0–5
35%;
21.5–30
)
adjust min,
13–21
pH 0%;
min,
to min,
0%
3.012.5
with min,
H PO35%;
) 13–21 min,
Gradient:
Detection: CH
80%;
B.
3 3
CN,
CH 0–5
21.5–30
CNFlow
UV 3absorbance min,
Gradient: 0%;
min,
Rate: 0%12.5
CH
80%;
0.8
B.
at 210 nm 3 3
3 3
min,
CN,
CH
4
0–5
35%;
21.5–30
mL/min
CN min,
13–21
0%;
min, min,
0%12.5 min,
3 4
35%; 13–21 min, A A A A 4 4 4
Flow
Valve Rate:
Gradient: 80%;
0.8
See3CN,
Position: CH
3
21.5–30
mL/min
Table Flow
Inj.
0–5 min,
0%;0%
Rate:
3 Volume:
min,
Gradient: 80%;
0.83µL
10
12.5
CH 21.5–30
mL/min
min,
CN, 0–5
35%;min, min,
0%;0%
13–21 min,
12.5 min, 35%; 13–21 min, –50 –50A –50 A –50 A
Flow
Inj. Rate:
Volume:
Configurations: 0.8
10
80%; mL/min
µL
A. 750 Flow Rate:
Inj.mixer
21.5–30 Volume:
µLTemperature:
min, 0.8
10 °C
25
0%off-line
always 80%; mL/min
µL 21.5–30 min, 0% –50 0 0 5 5–50 0 10 10 5 –50 15 015 10 20 520 15 25 10 25 20 30 15
30 25 20
0 5 0 10 5 Minutes
Minutes
15 0 10 20 5 Minutes
15 25 10 20 Minutes
30 15 25 20
Inj.
Flow Volume:
Temperature:
Rate: 10
25
0.8 µL
°C
B. 750mL/min Inj.
Flow Volume:
Temperature:
µLDetection:
mixerRate: 10
25 µL
UV
0.8
always in-line °C
absorbance
mL/min at 210 nm Minutes Minutes Minutes
Temperature:
Detection:
Inj. Volume: 10 25
UV °C
µL
C. 750 Temperature:
absorbance
Detection:
µLSamples:
Inj. atoff-line
Volume:
mixer 210 nm 25
UV
A.
10 °C
absorbance
forStandard
µL
Peaks mixture
1–3, at 210for
in-line nm
B.Peaks
Multivitamin
4–8 nutritional drinkFigure 4. A standard mixture (A) and a multivitamin nutritional drink
Detection:
Samples:
Temperature: UV
25 absorbance
A. Standard
°C Detection:
Samples: at 210 nm
mixture
Temperature: UV
B.25 absorbance
A.Multivitamin
Standard
°C at 210 nm
mixture
nutritionalB.drink
Multivitamin nutritional drinksample (B) using an Acclaim PA column in the first dimension.
Samples:
Peaks:
Detection: A. VStandard
1.
UV B1
Samples:
mixture
Peaks:
50 mg/L
absorbance
Detection:
at 210 nm B.1.
A.VMultivitamin
5.
UV VStandard
100
absorbance
B5B1
mixture
50 mg/L nutritional
at 210 nm B.drink
5. VMultivitamin
B5
100 nutritional drink
Peaks:
Samples: 1.
A. VStandard
2. B1
C
Peaks:
50 mg/L
mixture 6.
Samples: B.1.
5.
2. VStandard
100
50 mg/L
A.VMultivitamin
B5C
B1
B12
nutritional5.
mixture 6. VMultivitamin
B.drink
B5
B12
100
50 nutritional drink
Peaks: 1.
3. VB1
2. CB3
Peaks:
50 mg/L 6.1.
2.VVB2
5.
3.
7. B5B1
B12
C
B3
100
15 mg/L
50 5.
6. VB2
7. B5
B12
100
50
15
Peaks: 2.
3.
4. VB1
1. C
B3
B6 Peaks:
50 mg/L 6.
2.
3.
7.
4.
8.
1.
5. VVB12
B2
B7
B5C
B3
B6
B1
15
50
100 mg/L 6.
7.
8.
5. V B12
B2
B7
B5
50
15
100
300 3. Detection: UV absorbance at 245 nm
A 2. VCB3
4. B6 500
50 6.3.
2.VVB2
7.
4.
8. 50
15
100
6
7.
6. VB2
8. 15
100
50
B7B3
B6
B12
C B7
B12 Valve Position: See Table 3
4. VB3
3. 50 4.VVB2
8.3.
7. 100
B6 45015 8. VB2
7. 100
15
500 B6 500 B7B3 B7 Samples: A. VB12 standard mixture (20 mg/L)
4. VB6 50 8.4.VVB7B6 10050 8. VB7 100 B. Multivitamin nutritional drink sample
500 500
500mAU 500 3
1 Peak: 1. VB12
2 50

–50 mAU
mAU
200 mAU
B 6
mAU mAU
mAU mAU
6
mAU
B 3 6 44 6
7
B 3 4B 3 64 5 6 mAU
B 3 4B 1 3 64 8 6 7
5
–50 B 1 3 4AB 1 253 47 5 78
250 A 1 2 0 A 1 25 78 5 78
0 AC 1 2 0 A 1 25 47 8 78 1
–50 6 5
0 2 00 A 52 8 10 15 8 20 B 25
–500 A –500 Minutes 1
0 5 0 310 5 15 10 20 15 25 20 25
–50 –50 Minutes
mAU0 5 1 0 10 5 15 10 7 Minutes
20 15 25 20 25
–50 –50 Minutes 510 Minutes
0 5 0 10 5 15 20 15 25 20 25
2 Minutes 8Minutes
A
–50
0 5 10 15 20 25 30
–25
Minutes 20 21 22 23 24 25
Minutes
Figure 3. Second-dimension analysis of the standard mixture with Figure 5. A VB12 standard (A) and a multivitamin nutritional drink
three different configurations. sample (B) using a Hypersil GOLD Phenyl column in the first
dimension.

6
 Choice of wavelength for the UV detector impurity in the drink sample, making quantification
Vitamins B1, B2, B6, and C were detected at 245, of VB12 impossible.
268, 291, and 245 nm, respectively, which are the
wavelengths of maximum UV absorbance for each. The authors searched for another column to use for
Vitamin B3 was detected at 268 nm, which is close the first dimension. The Hypersil GOLD Phenyl column
to its maximum UV absorbance. The maximum UV worked well for this purpose. Figure 4 shows that VB12
absorbance wavelengths of VB5 and VB7 are below was not detected at 245 nm when an Acclaim PA
200 nm; to minimize noise, both were detected at 210
nm. Vitamin B12 was detected with the 245 nm channel.
Detection: Detection:
UV absorbance at 210 nmUV absorbance at 210 nm
If desired, a fifth channel of 361 nm can be configured to Valve Position:
Detection: Table Valve
Seeabsorbance
UV 3 at Position:
210 nmSee
Detection: Table 3 at 210 nm
UV absorbance
detect VB12 at its absorbance maximum. Detection:
Samples:
Valve Position: UV
A_H,
See Samples:
absorbance
consecutive
Table Valve 210 nmA_H,
Detection:
3 at injections
Position: UV1–8
See consecutive
3 at injections
absorbance
Table 210 nm 1–8
Valve Position:
Samples:
Detection: Seeabsorbance
A_H,
UV Table Valve
3 at Position:
consecutive
Samples: injections
Detection:
210 nmUV See
A_H,
1–8 Table 3 at injections
consecutive
absorbance 210 nm 1–8
100 Samples:
Peaks:Position:
Valve A_H,
1.
SeeV
100B1
50
Table Peaks:
consecutive
Samples:
3mg/L
Valve Position:5. 1.
injections
VSee
B5
V
A_H,
1–8 50
100
Table
B1
mg/L
consecutive
3 5. VB51–8100
injections
Column choice and its impact on separation 100 Peaks:
Samples: 2.
1. VB1
A_H,
100C
50Samples:
consecutive 6. 2.
mg/L injections
Peaks: 5. VB1 consecutive
VA_H,
1. 1–8
B12 C
B5
100 6.
5. VB5
50 mg/L injections 50
1–8100
B12

100 Peaks: 1.
3.100
2. VCB1 mg/L
50Peaks: 5. 3.
1.
7. 2.
6. VB5 VCB1 100
15 mg/L
50 7. VB5
5.
6. 15
100
50
Several combinations of columns were tested. Previous Peaks: 2.
4.100
3.
1.
B3
VB1 50Peaks:
mg/L 7.
5. 4.
8. 2.
6. 3.
1.
B2 B3
B12
VB5 VB1 50 mg/L
15
100 8.
6.
B2
B12
7. VB5
5. 50
15
100
100 C
B6
B3 B7 B6
B12
B2 C
B3 B7
B12
B2
work suggested that good choices for most water- 3. VCB6
4.
2. B3
50 8.
6. 3.
7. 2.
4.
VB12VB6
B2 C
B7 B3
15
100
50 7. VB12
8.
6. B2
B7
15
100
50
4.
3. VB3 50 8. 4.
7. 3.
VB2 VB6 10015
50 8. VB2
7. 100
15
soluble vitamins are an Acclaim PA column used as the B6
4. VB6 50 8. 4.
B7 B3
VB7VB6 410050
B7
8. VB7 4100
first-dimension column and an Acclaim 120 C18 column 4 6 4 6
6 6
used as the second-dimension column.7 Most water- 4
6
4
6
4 4
soluble vitamins can be separated with good resolution 6 6
using this column combination, but VB12 coelutes with an 3 3
3 3
mAU mAU
3 3 7 7
Table 4. Reproducibility of retention time and peak area for mAU 1mAU 1 5
3 35 78 78
water-soluble vitamins. mAU 1mAU 1 5 5
mAU H 1mAU 2H 1 2 78 78
5 5
H 1 2 H 1 2 78 78
Water-Soluble Retention Peak Area G G 5 5
H 2H 2 8 8
G G
Vitamin Time RSD RSD H
G F 2FH
G
2
FG FG
VB1 0.05% 1.08 F
E E
F
E E
VB2 FED D
FE
0.02% 0.32
D D
C
ED C
ED
VB3 0.11% 0.62 C C
B
D B
D
VB5 0.03% 0.36 C
B C
B
A
C A
C
B B
VB6 0.03% 0.45 A
B
A
B
A A
VB7 0.02% 0.87 –40 A –40 A
–40 0 5 –40 010 515 10
20 1525 2030 25
VB12 0.02% 0.48 –40 0 5 –40 010 Minutes
515 10
20 Minutes
1525 2030 25
–40 0 6. Chromatogram
Figure 5 –40 010 overlaysMinutes
of 10
20 Minutes
515eight consecutive1525 2030 25
VC 0.1% 3.44 0 5 Minutes Minutes
injections of a mixture of010 515 vitamin
water-soluble 10
20 standards.
1525 2030 25
Minutes Minutes

Table 5. Calibration data and MDLs for the water-soluble vitamins.

Detection
Water-Soluble Range MDL
Wavelength Regression Equation r
Vitamin (µg/mL) (µg/mL)
(nm)
VB1 245 0.5–10 A=0.1755c-0.1080 0.9978 0.30
VB3 268 0.5–50 A=0.2385c+0.0276 0.9999 0.23
VB6 291 0.5–50 A=0.2918c+0.1237 0.9999 0.18
VB5 210 1.0–100 A=0.0462c+0.0161 0.9999 0.26
VB12 245 0.5–50 A=0.1026c+0.0153 0.9999 0.20
VB2 268 0.15–15 A=0.9618c-0.0423 0.9995 0.03
VB7 210 2.0–100 A=0.0101c 0.9994 1.5
VC 245 — — — —
Due to working standard instability, Vitamin C calibration and quantification were not provided.
7
 column was used in the first dimension. However, when
the Hypersil GOLD Phenyl column was used in the first
dimension, VB12 was detected (Figure 5). The authenticity
of the VB12 peak was confirmed by its UV spectrum.
Reproducibility, Linearity, and Detection Limits
Prior to sample analysis, reproducibility was estimated
by making eight replicate injections of water-soluble
vitamins. The RSDs for retention time and peak area
are shown in Table 4. An overlay of the eight injections
is shown in Figure 6.
Calibration linearity for the water-soluble vitamins
was investigated by making three replicate injections
of a mixed standard prepared at five or six different
concentrations. The external standard method was used
to calculate the calibration curve and quantify these
compounds in samples. Table 5 reports the data from
the calibration as calculated by Chromeleon CDS
software. The authors found linear calibration curves
for each vitamin over the ranges evaluated. The single-
sided Student’s t test method was used for estimating
method detection limits (MDL) for a 99% confidence
level. These data are reported in Table 5.

Detection: UV absorbance at 245 nm

Sample Analysis
Valve Position: See Table 3 Vitamins B7 and B12 were not detected in the
Sample: Multivitamin nutritional drink sample
multivitamin nutritional drink, possibly due to their
250 Peaks: 1. VB1 3. VB6 5. Unknown low concentration in the sample. Vitamin B3 was
2. VC 4. VB5 6. VB2
surprisingly not detected. Vitamin B3 was fully
2
recovered when spiked into the sample at its labeled
value; therefore, the method is capable of determining
VB3. The other water-soluble vitamins were detected
mAU
close to their labeled values.
3
Although VC in the working standard solutions is
4 6
1 5 quite unstable, its presence in the multivitamin
–10 nutritional drink appeared to be relatively stable. As
0 5 10 15 20 25 30
Minutes shown in Figure 7, VC is a major peak in the sample
chromatogram. Good recoveries of water-soluble
Figure 7. Second dimension of multivitamin nutritional drink sample vitamins in the spiked sample (Table 6) provided
(100-fold dilution).

Table 6. Analysis results of water-soluble vitamins in the multivitamin nutritional drink sample.

Labeled Detected Added Found Recovery

Analyte
(mg/mL)* (µg/mL) (µg/mL) (µg/mL) (%)

VB1 1 1.03 1 1.98 95

VB3 1 ND 1 0.96 96

VB6 1 1.1 1 2.2 110

VB5 5 4.51 2 6.03 76

VB12 0.015 ND 1 0.83 83

VB2 1 1.03 0.3 1.34 103

VB7 0.01 ND 2 2.30 115

VC 35 Detected — — —

*The sample is diluted 1000 times before analysis; thus, 1 mg/mL will be detected as 1 µg/mL.
Due to working standard instability, Vitamin C was not quantified. ND = not detected.

8
another positive indicator of method accuracy. This References
1. Moreno, P.; Salvado, V. Determination of Eight Water- and Fat-Soluble Vitamins
2D-HPLC method greatly simplifies the second-
in Multivitamin Pharmaceutical Formulations by High-Performance Liquid
dimension chromatogram, as shown in Figure 7. Chromatography. J. Chromatogr., A 2000, 870, 207–215.
2. Perveen, S.; Yasmin, A; Khan, K.M. Quantitative Simultaneous Estimation of Water
Conclusion Soluble Vitamins, Riboflavin, Pyridoxine, Cyanocobalamin and Folic Acid in Nutraceutical
Products by HPLC. The Open Analytical Chemistry Journal 2009, 3, 1–5.
Two-dimensional HPLC simplifies the determination of 3. Thermo Fisher Scientific Application Note 252: HPLC Assay of Water-Soluble Vitamins,
the vitamin content of a multivitamin nutritional drink, Fat-Soluble Vitamins, and a Preservative in Dry Syrup Multivitamin Formulation
a complex sample. Analysis of this complex sample 4. Vitamins/Dietary Supplements, The U.S. Pharmacopeia 35, NF 30, Washington, DC,
2008, pp 1592–1605.
requires only off-line filtration because the remainder
5. Thermo Fisher Scientific Application Note 287: Two-Dimensional HPLC Combined with
of the sample preparation is automated by the UltiMate On-Line SPE for Determination of Sudan Dyes I–IV in Chili Oil. Sunnyvale, CA, 2011.
3000 Dual-Gradient HPLC system and Chromeleon [Online] https://tools.thermofisher.com/content/sfs/brochures/111142-AN287-HPLC-
Sudan-Dyes-Chili-Oil-21Sept2011-LPN2919.pdf
CDS software. The application was successful for
6. Thermo Fisher Scientific Application Note AN72597: Determination of Sudan Dyes
determination of the B group vitamins in the presence I–IV in Curry Paste. Sunnyvale, CA, 2012.
of other nutritional additives. 7. Thermo Fisher Scientific Technical Note 72488: Determination of Water- and
Fat-Soluble Vitamins by HPLC. Sunnyvale, CA, 2010.
The method was not able to quantify Vitamin C,
although detected.

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