Determination of Soy Isoflavones in Foods and Dietary Supplements

Determination of Soy Isoflavones in Foods
and Dietary Supplements by UPLC
Christopher J. Hudalla and Kenneth J. Fountain
Waters Corporation, 34 Maple St., Milford, MA, USA
A P P L I C AT I O N B E N E F I T S
■■
Accurate determination of soy isoflavone
content in foods and dietary supplements
■■
Alternate selectivity to
traditional C18 methods
■■
Significant time savings relative
to currently accepted methods
WAT E R S S O LU T I O N S
ACQUITY UPLC® System
ACQUITY® SQD System
XSelect™ HSS Cyano, XSelect HSS Cyano
XP, and ACQUITY UPLC HSS Cyano
Columns
INT RODUC T ION
Isoflavones are a class of plant-derived compounds, produced almost exclusively by
members of the Fabaceae family, that have been shown to have estrogenic activity in
mammals. The major source of isoflavones in the human diet comes from soybeans,
in which genistein and daidzein are the predominant components. Isoflavones
remain the subject of many scientific studies with accepted methods established
by organizations such as AOAC1 and USP2, utilizing traditional reversed-phase C18
columns. Recently, the National Institute of Standards and Technology (NIST) has
developed a suite of soy-based candidate Standard Reference Materials (cSRMs)
for the certification of soy isoflavones in foods and dietary supplements.
For the analysis of those standards, they have developed a method utilizing
a 60-minute gradient on a 5 µm cyano column which enables the separation
of the soy isoflavones from other components not resolved using the standard C18
methods.3 Using these conditions, they have demonstrated resolution of the three
main soy isoflavones and their glycosides (structures shown in Figure 1), as well
as the acetyl and malonyl conjugates of the glycosides. Since the conjugates
are somewhat unstable in solution, they have added an additional sample
preparation step to hydrolyze these conjugates to their associated glycoside,
providing a more accurate, reproducible determination of total isoflavone content.
Optimization of this method utilizing the XSelect HSS Cyano XP 2.5 µm Column
provides a significant reduction in analysis time by traditional HPLC. Further
optimization of this method to UPLC® with the ACQUITY UPLC HSS Cyano 1.8 µm
Column provides additional savings in time, while maintaining the resolution for
accurate determination of soy isoflavones.
ACQUITY UPLC Columns Calculator
GHP Acrodisc ® Minispike Filters
KEY WORDS
Soy, isoflavone, UPLC, XP, infant
formula, method development,
method transfer
1
E X P E R IM E N TA L
UPLC Conditions
System:
H
ACQUITY SQD with
PDA detector
Column:
ACQUITY UPLC HSS Cyano,
2.1 x 50 mm, 1.8 µm,
part number 186005986
Mobile Phase A:
0.1% formic acid in water
Mobile Phase B:
0.1% formic acid in
acetonitrile
Column Temp.:
30 °C
Gradient:
10% (B) for 0.36 min,
10-30% (B) in 3.6 min, hold
at 30% (B) for 0.36 min,
re-equilibrate at 10% (B) for
1.8 min between injections
Flow Rate:
0.58 mL/min
Detection:
UV at 260 nm
Injection Volume: 3 µL
Strong Needle Wash:
50/50 acetonitrile/water
Weak Needle Wash:
10/90 acetonitrile/water
genistein
genistin
daidzein
daidzin
glycitein
glycitin
H
H
O
isoflavones
O
glycosides
Figure 1. Structures of the three main soy isoflavones (genistein, daidzein, and glycitein) and
their corresponding glycosides (genistin, daidzin, and glycitin).
SAMPLE PREPARATION
Standard Solution: Prepared from daidzin (25 ppm), glycitin (25 ppm), genistin
(15 ppm), daidzein (25 ppm), glycitein (25 ppm), and genistein (15 ppm) using
10/90 acetonitrile/water diluent.
Samples:
These UPLC conditions were scaled directly from
the 5 µm HPLC method using the ACQUITY UPLC
Columns Calculator. The calculator can be used to
scale these conditions back to the HPLC conditions,
for both the 5 µm and 2.5 µm materials.
MS Conditions
MS System:
Waters SQD
Ionization Mode:
ESI positive
Acquisition Range:
Single Ion Recording (SIR)
Capillary Voltage:
3.19 kV
Cone Voltage:
50 V
Desolvation Gas:
600 L/hr
Cone Gas:
0 L/hr
Source Temp.:
100 °C
Desolvation Temp.:
350 °C
2
Candidate Standard Reference Materials were obtained from the National Institute
of Standards and Technology. Each sample was weighed into 12 ml centrifuge tubes
(Table 1). For each tube, 4 mL of 80/20 methanol/water was added followed by
sonication for 1 hour. Tubes were centrifuged for 2 minutes at 3000 rpm. A 2 mL
aliquot of supernatant was collected from each tube and filtered using a 0.45 µm
GHP syringe filter prior to analysis. The remainder of the sample in each tube was
hydrolyzed using 150 µL of 2N sodium hydroxide. After mixing for 10 minutes, the
solutions were neutralized with 50 µL of glacial acetic acid. The sample was again
centrifuged for 5 minutes at 3000 rpm, with the collected supernatant filtered
using a 0.45 µm GHP syringe filter prior to analysis.
Table 1. Samples used for analysis
Soy flour (cSRM)
Soy tablet (cSRM)
Soy protein isolate (cSRM)
Soy protein concentrate (cSRM)
Soy-based infant formula
(commercially-available)
UPLC Analysis of Soy Isoflavones using the ACQUITY UPLC HSS Cyano
Weight
104.2 mg
111.9 mg
365.6 mg
973.7 mg
1119.2 mg
R E S U LT S A N D D I S C U S S I O N
Based on the method presented by NIST, we developed a method using the soy standard solution with an XSelect
HSS Cyano, 4.6 x 150 mm, 5 µm Column, resulting in chromatography with excellent resolution of the soy
isoflavones and glycosides. To maximize productivity of the HPLC system, this method was transferred, using the
ACQUITY UPLC Columns Calculator, to an XSelect HSS Cyano XP, 4.6 x 75 mm, 2.5 µm Column. This resulted in
an HPLC method with a 76% reduction in run time, relative to the 5 µm column. To achieve the greatest
benefit, the method was then transferred to UPLC using an ACQUITY UPLC HSS Cyano, 2.1 x 50 mm, 1.8 µm
Column. Figure 2 shows the chromatography of the isoflavone standards under HPLC and UPLC conditions.
These chromatograms demonstrate the ability to scale between different column configurations by maintaining
the same ratio of column length to particle size (L/d p), resulting in similar chromatography but with a significant
decrease in analysis time (~88% decrease in run time for the UPLC column relative to the 5 µm HPLC column).
HPLC
1
2
4
3
5
6
5 µm
10
12
14
16
18
20
22
24
26
28
30
32
34
6.5
7.0
7.5
8.0
36 min
Compounds (ppm)
1. Daidzin (25)
2. Glycitin (25)
3. Genistin (15)
4. Daidzein (25)
5. Glycitein (25)
6. Genistein (15)
2.5 µm
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
8.5 min
UPLC
1.8 µm
1.0
4.2 min
Figure 2. HPLC and UPLC separations (UV) of soy isoflavone standards on cyano columns: XSelect HSS Cyano, 4.6 x 150 mm, 5 µm
(top); XSelect HSS Cyano XP, 4.6 x 75 mm, 2.5 µm (middle); and ACQUITY UPLC HSS Cyano, 2.1 x 50 mm, 1.8 µm (bottom). The
gradient profiles, flow rates, and injection volumes were scaled for each method using the ACQUITY UPLC Columns Calculator. The
flow rates were 1.0 mL/min, 2.0 mL/min, and 0.58 mL/min, respectively.
The UPLC method was used to analyze each of the candidate SRM extracts, both before and after hydrolysis of
the glycoside conjugates (Figure 3). The identity of the peaks in the UV chromatograms were assigned based
on retention times and m/z values from the LC/MS analyses (Soy Flour cSRM example shown in Figure 4). The
disappearance of the glycoside conjugate peaks after sample treatment with sodium hydroxide confirms the
hydrolysis of these conjugates was successful and complete, which allows for simplified determination of the total
isoflavone content.
UPLC Analysis of Soy Isoflavones using the ACQUITY UPLC HSS Cyano
3
Compounds
1. Daidzin
2. Glycitin
3. Genistin
4. Malonyl daidzin
5. Malonyl glycitin
6. Acetyl daidzin
7. Acetyl glycitin
8. Malonyl genistin
Soy Flour
8
0.40
3
0.20
5
2
Extracted
0.40
4
1
0.00
0.60
AU
AU
Soy Tablet
3
0.60
9. Daidzein
10. Glycitein
11. Acetyl genistin
12. Genistein
67
12
3
1
4 min
2
3
4 min
3
0.02
8
AU
AU
12
Soy Protein Concentrate
12
3
0.20
0.10
6 7 10 11
9
Hydrolyzed
Soy Protein Isolate
0.30
4
Extracted
0.00
2
2
0.20
11
Hydrolyzed
1
1
9
2
Extracted
5
6 7
10
11
8
4
2
11
6
12
Extracted
Hydrolyzed
0.00
1
0.01
4
1
0.00
1
2
3
Hydrolyzed
4 min
1
3
2
4 min
Figure 3. UPLC analysis of extracted (top chromatograms) and hydrolyzed (bottom chromatograms) candidate Standard
Reference Materials (cSRMs).
503
100
519
Soy Flour cSRM
t R(min)m/z Compound
1.52417Daidzin
1.69 447Glycitin
2.12 433Genistin
2.30
503
Malonyl daidzin
2.42
533
Malonyl glycitin
2.65
459
Acetyl daidzin
2.79
489
Acetyl glycitin
2.86
519
Malonyl genistin
2.92255Daidzein
3.07285Glycitein
3.31
475
Acetyl genistin
3.81271Genistein
433
m/z values
%
417
533
459
475
489
447
271
285
0
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
Figure 4. ESI + LC/MS confirmation of peak identity for the Soy Flour cSRM, using single ion recording (SIR).
4
UPLC Analysis of Soy Isoflavones using the ACQUITY UPLC HSS Cyano
4.4 min
Because of their biological activity and the pervasiveness of soy-based food products, isoflavones have been
the focus of many nutritional studies. Some studies advocate the nutritional benefit of isoflavones, asserting
that consumption of soy products leads to decreased menopausal symptoms and lowers the risk of certain
cancers.4 Other researchers have reached alternate conclusions, with one such study of the isoflavone genistin
on mice resulting in significant thymic and immune abnormalities, raising concerns for the use of soy-based
infant formulas.5 Because of these concerns, understanding the isoflavone profiles of food products is of critical
interest. Using the current method, a sample of commercially-available soy-based infant formula was prepared
and analyzed by both LC/UV and LC/MS. Analysis of the extracted sample revealed, in addition to the isoflavones
and their glycosides, only a minor concentration of the malonyl genistin conjugate. After hydrolysis, all evidence
of the conjugate is gone, revealing only the isoflavones and their glycosides (Figure 5). Using this method, the
concentrations of soy isoflavones can be evaluated and monitored, facilitating fundamental research, as well as
the quality control of food and nutraceutical consumer products.
Hydrolyzed Infant Formula
Compounds
1. Daidzin
2. Glycitin
3. Genistin
4. Malonyl daidzin
5. Malonyl glycitin
6. Acetyl daidzin
7. Acetyl glycitin
8. Malonyl genistin
9. Daidzein
10. Glycitein
11. Acetyl genistin
12. Genistein
3
1
100
2
a
b
%
12
9
UV (260 nm)
10
MS (TIC)
c
m/z (447 + 285)
d
m/z (417 + 255)
e
0
m/z (433 + 271)
1
2
3
4
5 min
Figure 5. UPLC analysis of isoflavones in a commercially-available, soy-based infant formula (after hydrolysis);
a) UV [260 nm], b) MS - Total Ion Chromatogram [TIC], c) extracted ion chromatogram for m/z 447 + 285 [glycitin
and glycitein], d) extracted ion chromatogram for m/z 417 + 255 [daidzin and daidzein], and e) extracted ion
chromatogram for m/z 433 + 271 [genistin and genistein].
UPLC Analysis of Soy Isoflavones using the ACQUITY UPLC HSS Cyano
5
CONCLUSIONS
REFERENCES
Analysis of soy isoflavones using the HSS Cyano stationary phase
offers complimentary selectivity to traditional C18 based methods.
Hydrolysis of the glycoside conjugates simplifies the resulting
chromatography and the determination of total isoflavone content
of a sample. The availability of the HSS Cyano phase in 5-, 3.5-,
2.5-, and 1.7-µm particle sizes facilitates scaling between different
instrument platforms (HPLC and UPLC ). Method conditions can
be easily scaled for different column configurations using the
ACQUITY UPLC Columns Calculator. Scaling methods to utilize the
XSelect HSS Cyano XP 2.5 µm Columns maximizes productivity
with existing HPLC instrumentation. Additional method transfer to
UPLC conditions offers the greatest savings in time and resources.
1. Collison, M. W., AOAC Official Method 2008.03, Total Soy
Isoflavones in Dietary Supplements, Supplement Ingredients,
and Soy Foods, J AOAC Int., 2008, 91(3), 489-500.
2. USP Monograph. Soy Isoflavones, USP34-NF29 [1236].
The United States Pharmacopeial Convention, official from
December 1, 2011.
3. Bedner, M., Gradl, M. K., Arce-Osuna, M., Phillips, M. M.,
Rimmer, C. A., Sander, L. C., Sharpless, K. E., Albert, K., Poster:
HPLC Conference, Budapest, Hungary, 2011.
4. Berdanier, C.D., Dwyer, J.T., and Feldman, E.B., Handbook of
Nutrition and Food, 2nd Ed., CRC Press, 2007.
5. Cooke, P.S., Yellayi, S., Naaz, A., Szewczykowski, M.A., Sato,
T., Woods, J.A., Chang, J., Segre, M., Allred, C.D., and Helferich,
W.G., 2002, PNAS, 99, 7616-7621.
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©2012 Waters Corporation. Produced in the U.S.A.
August 2012 720004253EN IH-PDF
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