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FOOD COMPOSITION AND ADDITIVES

Liquid Chromatographic Method for Determination of Vanillin


and Ethyl Vanillin in Imitation Vanilla Extract (Modification of
AOAC Official Method 990.25): Collaborative Study
KAHAN & KRUEGER: JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO. 3, 1997
SIDNEY KAHAN
Kahansultants, Inc., 66 Peachtree Lane, Roslyn Heights, NY 11577
DANA A. KRUEGER
Krueger Food Laboratories, Inc., 24 Blackstone St, Cambridge, MA 02139
Collaborators: R. Berger; A. Filandro, L.R. Hageman; T. Korpinski; S. Lin; N.E. Lally; M. Parrish; K.L. Schoen

A collaborative study of a method for analysis of


vanillin and ethyl vanillin in fortified and imitation
vanilla flavors was performed. The method, which
is an extension of AOAC Official Method 990.25, Vanillin, Vanillic Acid, p-Hydroxybenzaldehyde, and pHydroxybenzoic Acid in Vanilla Extract, Liquid
Chromatographic Method, involves reversed-phase
high-pressure liquid chromatography (LC) of the
sample on a C8 column with a watermethanolacetic acid (89 + 10 + 1) mobile phase and UV detection at 254 nm. The method yields good recoveries
of vanillin at 484 and 723 mg/100 mL and of ethyl
vanillin from 37 to 400 mg/100 mL. For vanillin, repeatability (r) values were 8 mg/100 mL at a level of
155 mg/100 mL, 12 mg/100 mL at a level of
484 mg/100 mL, and 31 mg/100 mL at a level of
723 mg/100 mL. Reproducibility (R) values were
20 mg/100 mL, 55 mg/100 mL, and 137 mg/100 mL
over the same range. For ethyl vanillin, r values
were 2.4 mg/100 mL at a level of 37 mg/100 mL,
3.2 mg/100 mL at a level of 74 mg/100 mL, and
8.6 mg/100 mL at a level of 180 mg/100 mL. R values were 6.4 mg/100 mL, 5.4 mg/100 mL, and
22.0 mg/100 mL over the same range. AOAC Official Method 990.25 has been modified to include determination of ethyl vanillin in vanilla extract and artificial vanilla flavor.

nilla flavors. The presence of ethyl vanillin is often an indicator


of adulteration in products fraudulently represented as pure vanilla extract. For these reasons, a reliable method for analysis
of the vanillin and ethyl vanillin contents of natural and imitation vanilla extracts is needed.
The current validated methods for analysis of vanillin and
ethyl vanillin in vanilla products involve chromatographic
separation, by either column or paper chromatography, followed by UV spectrophotometry. This approach is typified by
AOAC Official Methods 966.13 and 955.31 for vanillin and
ethyl vanillin (1). These methods are time-consuming and are
considered obsolete in the modern analytical laboratory. An approximate determination of vanillin may be conveniently obtained by UV spectrophotometry without prior separation, according to AOAC Official Methods 964.10 and 966.12, but
these methods can be inaccurate and are ineffective at distinguishing vanillin from ethyl vanillin.
Recently, methods have been developed for analysis of vanillin and ethyl vanillin in vanilla products by reversed-phase
liquid chromatography (LC). Kahan (2) recently reported a collaborative study of such a method for analysis of vanillin and
related flavor compounds in vanilla extract (2). This method
has been approved as AOAC Official Method 990.25 (1). The
present study extends the applicability of this method to analysis of vanillin and ethyl vanillin in fortified and imitation vanilla flavors.
Collaborative Study

anillin content is an important quality parameter of vanilla extract. Vanillin and ethyl vanillin contents are important quality parameters of imitation and fortified va-

Submitted for publication October 26, 1995.


The recommendation was approved by the Methods Committee on
Additives, Beverages, and Food Process Related Analytes, and was adopted
by the Official Methods Board of the Association. See Official Methods
Board Actions (1996) J. AOAC Int. 79, 42A, and Official Methods Board
Actions (1996) The Referee, March issue.

The collaborative study samples consisted of pure vanilla


extract fortified with various levels of vanillin and ethyl vanillin, as indicated in Tables 1 and 2. Sample pairs 1, 4 and 3, 5
were duplicates. A separate preliminary method comparison
and recovery study used 3 other samples of vanilla extract fortified with 3 levels of ethyl vanillin as shown in Table 3. Collaborators were instructed to perform each analysis in duplicate
and to submit both calculated results and chromatograms to the
Associate Referee.

990.25 Vanillin, Vanillic Acid, p-Hydroxybenzaldehyde, and p-Hydroxybenzoic Acid and


Ethyl Vanillin in Vanilla Extract and Artificial Vanilla
Flavor, Liquid Chromatographic Method
First Action 1990
Final Action 1992
Revised First Action 1996
Caution: See Appendix: Laboratory Safety for Safe Handling
of Special Chemical Hazardsmethanol and Safe Handling of
Acidsacetic acid. Dispose of waste solvents in an appropriate
manner compatible with applicable environmental rules and regulations.
Method Performance:
See Tables 990.25A, B, and C for method performance data.

A. Principle
Flavor compounds in test sample are resolved by liquid
chromatography (LC) on C8 column using external standard
solution and are detected at 254 nm.

B. Apparatus
(a) Liquid chromatograph.Capable of flow rate of
2.5 mL/min and UV detection at 254 nm.
(b) LC column.Stainless steel, 25 cm 4.6 mm id,
packed with C8 on 10 m particles, capable of complete separation of the standard substances under the test conditions. (See
Figure 990.25 for chromatograms of vanillin and ethyl vanillin
standard solution and typical sample.)
(c) Filters.0.45 m, alcohol-compatible.

C. Reagents
(a) Solvents.LC grade methanol and H2O, 95% nondenatured ethanol, and reagent grade glacial acetic acid.
(b) Mobile phase.Methanolacidified H2O (10 + 90).
Acidify 800 mL H2O with 10 mL acetic acid.
(c) Standard solutions for determination of vanillin, vanillic acid, p-hydroxybenzaldehyde, and p-hydroxybenzoic
acid.Weigh 1.2000 g vanillin, 0.0800 g vanillic acid,
0.0600 g p-hydroxybenzaldehyde, and 0.0200 g p-hydroxybenzoic acid into 100 mL volumetric flask and dilute to volume
with 95% ethanol. Dilute 10 mL of this solution to 100 mL with
40% ethanol. Make all standard and sample dilutions at same
temperature and as close to 20C as possible. Each 100 mL
standard solution contains 120 mg vanillin, 8.0 mg vanillic
acid, 6.0 mg p-hydroxybenzaldehyde, and 2.0 mg p-hydroxybenzoic acid.
(d) Standard solutions for determination of vanillin and
ethyl vanillin.(1) Standard stock solution.120 mg/100 mL
vanillin and 40 mg/100 mL ethyl vanillin. Weigh 1.2000 g vanillin and 0.400 g ethyl vanillin into 100 mL volumetric flask
and dilute to volume with 95% ethanol. Standard stock solution
is stable indefinitely when stored in refrigerator. (2) Working
standard solution.Dilute 10 mL standard stock solution to
100 mL with 40% ethanol and use for analysis. Prepare working standard solution daily.

D. Preparation of Sample
If vanillin content is <1.0 g/100 mL extract, filter extract and
use undiluted for analysis. If vanillin content is >1.0 g/100 mL
extract, dilute extract with 40% ethanol to below that level and
filter. If vanillin or ethyl vanillin content is <0.05 g/100 mL extract, further dilute standard solution C(c) or C(d)(2) (depending on compound that is analyzed) or use larger sample volume
for analysis.

E. Liquid Chromatography
Inject 10 L standard solution onto LC column. Establish
retention times, measure peak heights (or areas), and check reproducibility. Repeat analysis for sample solution.
Calculate concentration of compound as follows:
Concentration, mg/100 mL =

S F Pt
Ps

where S = concentration of compound in standard solution C(c)


or C(d)(2), mg/mL; F = dilution factor for sample, defined as
ratio of final sample volume to initial aliquot volume (F = 1 for
undiluted sample); Pt = peak height (or area) of compound in
test sample; and Ps = peak height (or area) of compound in
standard solution C(c) or C(d)(2).
Ref.: J. Assoc. Off. Anal. Chem. 72, 614(1989); J. AOAC Int.
80, 564570(1997)
CAS-121-33-5 (vanillin)
CAS-121-34-6 (vanillic acid)
CAS-123-08-0 (p-hydroxybenzaldehyde)
CAS-99-96-7 (p-hydroxybenzoic acid)
Results and Discussion
Eight laboratories, all familiar with examination of vanilla
extracts, agreed to collaborate on this study. Because most of
them were familiar with AOAC Official Method 990.25, they
found the current method relatively simple and straightforward. Typical chromatograms of standard and sample injections are shown in Figure 990.25. The collaborative study data
are summarized in Tables 1 and 2. All entries in the tables are
single determinations, and pairs of duplicate analyses were
used as cells in a uniform-level experiment with n = 2 for statistical analysis. The cell differences were tested for outliers by
Cochrans test, and the cell averages were tested for outliers by
Dixons test (3). No outliers were detected by either of these
methods.
For each set of duplicate results, the values for repeatability
(r) and reproducibility (R) were calculated by standard methods
for a uniform-level experiment (3). Results are listed in Tables
1 and 2.
The vanillin contents of the samples ranged from about 155
to 737 mg/L (the true values are not known exactly because of
uncertainty in the vanillin content of the starting extract). This
extends the range of vanillin contents tested in the original
study of the AOAC Official Method 990.25, which previously
was only up to 200 mg/L. Recoveries of vanillin from 3 fortified samples ranged from 96 to 100%. The r values were

8 mg/100 mL at a level of 155 mg/100 mL (mean of 5.7, 9.5,


and 9.4), 12 mg/100 mL at a level of 484 mg/100 mL, and
31 mg/100 mL at a level of 723 mg/100 mL (mean of 22.6 and
38.8). The R values were 20 mg/100 mL (mean of 12.2, 26.1,
and 20.7), 55 mg/100 mL, and 137 mg/100 mL (mean of 154.8
and 118.8) over the same range. The R values for the low-value
samples in this study (samples 1, 4, and 6) are similar to those
determined for the high-value samples in the original study of
the Method 990.25 (samples F1, F2, G1, G2, H1, and H2). The
r values in this study are somewhat lower. The r and R values
are proportional to the concentration (C), as shown in Figure 1.
Best-fit regression lines passing through the origin yield the
relationships r = 0.040C and R = 0.172C
None of the collaborators experienced difficulties with the
vanillin determination. Collaborator 7 had to dilute the samples
to significantly less than the 1 g/100 mL level specified in the
method; samples 2, 3, and 5 exceeded the linear range of the
detector until diluted to below 0.2 g/100 mL.
The ethyl vanillin contents of the samples ranged from 0 to
180 mg/L. None of the collaborators found any ethyl vanillin
in the blank sample. Recoveries ranged from 90 to 104%. The
r values were 2.4 mg/100 mL at a level of 37 mg/100 mL,
3.2 mg/100 mL at a level of 74 mg/100 mL (mean of 2.7 and
3.6), and 8.6 mg/100 mL at a level of 180 mg/100 mL (mean of
6.9 and 10.4). The R values were 6.4 mg/100 mL,
5.4 mg/100 mL (mean of 4.3 and 6.4), and 22.0 mg/100 mL
(mean of 26.9 and 17.1) over the same range. The r and R values are proportional to the concentration, as shown in Figure 2.
Best-fit regression lines passing through the origin yield the
relationships r = 0.047C and R = 0.114C
None of the collaborators experienced difficulties with the
ethyl vanillin determination. Collaborator 7 detected a small
peak, corresponding to ca 10 mg/100 mL ethyl vanillin, in the
blank sample eluting near the ethyl vanillin retention time. Coinjection with a small quantity of ethyl vanillin yielded 2 components.
Reproducibility results were statistically acceptable, as
judged by comparison with the CVR results predicted from the
Horwitz equation (4). Actual CVR (%) values are all very close
to, and generally slightly below, the predicted results. This
demonstrates that the method has a precision typical of analytical methods useful at these concentration levels. For both analytes, r values were significantly lower than R values. Therefore, as is typically the case, interlaboratory bias is a significant
component of the overall analytical uncertainty.
Prior to the main collaborative study, a smaller preliminary
study was performed to evaluate the recovery of the method for
ethyl vanillin. It was also intended to compare results obtained
from the method with those from the existing validated methods for ethyl vanillin determination. Table 3 lists values obtained for ethyl vanillin, determined both by the proposed LC

method and by the paper chromatographic method (AOAC Official Method 966.13). The ethyl vanillin content by LC compares well with the existing paper chromatographic method.
LC results averaged 106% of the paper chromatographic results. Both methods yielded good recovery of ethyl vanillin.
This method is satisfactory for determination of ethyl vanillin in vanilla flavors. It is also satisfactory for determination of
vanillin at the high concentration levels commonly found in
artificial and fortified vanilla flavors. In addition to good repeatability and reproducibility, the method yields good recovery of ethyl vanillin, and results correlate well with existing
validated methods. Recently published work suggests that the
method may also have applicability to the determination of
other flavor additives in vanilla, such as piperonal (2, 5) and
coumarin (5).
Recommendation
On the basis of the results of this study, it is recommended
that the AOAC Official Method 990.25 be extended to determination of ethyl vanillin in vanilla and artificial vanilla flavor.
We also recommend that the method be further evaluated for its
suitability in the determination of other artificial vanilla flavoring substances.
Acknowledgments
We thank the following for their participation:
R. Berger, Beck Flavors, St. Louis, MO
A. Filandro, Virginia Dare Extract Co., Brooklyn, NY
L.R. Hageman, Nanolab, Inc., Dublin, OH
T. Korpinski, Certified Laboratories, Inc., Corona, NY
S. Lin, Krueger Food Laboratories, Inc., Cambridge, MA
N.E. Lally, Bush Boake Allen, Chicago, IL
M. Parrish, McCormick and Co., Hunt Valley, MD
K.L. Schoen, David Michael and Co., Philadelphia, PA
We also thank Certified Laboratories for permitting use of
their facilities in preparation of the extracts.
References
(1) Official Methods of Analysis (1995) 16th Ed., AOAC INTERNATIONAL, Arlington, VA, secs 955.31, 964.10, 966.12,
966.13, 990.25
(2) Kahan, S. (1989) J. Assoc. Off. Anal. Chem. 72, 614618
(3) Statistical Methods, ISO Standards Handbook 3 (1989) 3rd
Ed., International Organization for Standardization, Geneva,
Switherland, ISO 57251986 (E)
(4) Horwitz, W. (1982) Anal. Chem. 54, 67A76A
(5) Thompson, R.D., & Hoffmann, T.J. (1988) J. Chromatogr.
438, 369382

Table 990.25A. Method performance for determination of vanillin, vanillic acid, p-hydroxybenzaldehyde, and
p-hydroxybenzoic acid in vanilla extract by liquid chromatography
Compound

p-Hydroxybenzoic acid
p-hydroxybenzaldehyde
Vanillic acid
Vanillin
a
b

r = 2.8 sr.
R = 2.8 sR.

sr

sR

RSDr, %

RSDR, %

ra

Rb

0.29
0.92
0.85
7.45

0.39
1.20
1.12
7.50

8.98
6.16
7.47
3.43

11.68
6.95
9.97
3.48

0.81
2.68
2.38
20.86

1.09
3.36
3.13
21.00

Table 990.25B. Method performance for determination of vanillin in artificial vanilla flavor by liquid chromatography

Sample
1
c
2
d
3
4
e
5
6
a
b
c
d
e

Mean
Vanillin
concn,
added,
Recovery,
mg/100 mL mg/100 mL mg/100 mL Recovery, %
155
484
712
157
734
155

0.00
328
581
0.00
581
0.00

b
328
556

578

Samples 1, 4 and 3, 5 are duplicates.


, not applicable.
Sample 2 = sample 1 (mean 155 mg/mL) + spike.
Sample 3 = sample 1 + spike.
Sample 5 = sample 4 (mean 157 mg/mL) + spike.

100
96

100

RSDr, %

RSDR, %

sr

sR

1.3
0.9
1.1
2.2
1.9
2.2

2.8
4.0
7.8
5.9
5.8
4.8

2.0
4.5
7.9
3.4
13.9
3.4

4.4
19.5
55.3
9.3
42.4
7.4

5.7
12.5
22.6
9.5
38.8
9.4

12.2
54.6
154.8
26.1
118.8
20.7

Table 990.25C. Method performance for determination of ethyl vanillin in artificial vanilla flavor by liquid
chromatography

Samplea
1
2
3
4
5
6
a
b

Mean
concn,
mg/100 mL

Ethyl vanillin added,


mg/100 mL

Recovery, %

RSDr, %

RSDR, %

sr

sR

75.4
33.2
181.4
75.4
185.9
0.00

74.0
37.0
179.6
74.0
179.6
0.00

102
90
101
102
104
b b

1.3
2.6
1.4
1.7
2.0

2.0
6.9
5.3
3.0
3.3

1.0
0.9
2.5
1.3
3.7

1.5
2.3
9.6
2.3
6.1

2.7
2.4
6.9
3.6
10.4

4.3
6.3
26.9
6.4
17.1

Samples 1, 4 and 3, 5 are duplicates.


, Not applicable.

Table 1. Collaborative study results for vanillin (mg/100 mL)


Sample a
Laboratory
1
2
3
4
5
6
7
8

Mean
r
RSDr, %
R
RSDR, %
Vanillin added
Recovery
Recovery, %
a

163
163
155
159
155
156
154
156
159
154
151
152
148
150
153
157

519
514
501
505
466
465
485
489
463
459
465
477
490
491
470
480

763
775
733
729
665
683
769
777
722
712
714
728
728
726
595
609

162
162
159
165
155
159
169
177
157
151
152
157
151
149
142
144

769
772
755
772
670
678
790
810
688
710
743
701
730
731
710
718

159
158
158
167
151
155
165
169
147
148
154
159
146
150
148
153

155
5.7
1.3
12.2
2.8
0

484
12.5
0.9
54.6
4.0
328
328
100

712
22.6
1.1
154.8
7.8
581
556
96

157
9.5
2.2
26.1
5.9
0

734
38.8
1.9
118.8
5.8
581
578
100

155
9.4
2.2
20.7
4.8
0

Sample pairs 1, 4 and 3, 5 are duplicates.

Table 2. Collaborative study results for ethyl vanillin (mg/100 mL)


Sample a
Laboratory
1
2
3
4
5
6
7
8
Mean
r
RSDr, %
R
RSDR, %
Ethyl vanillin added
Recovery, %
a

1
75
75
76
76
75
76
74
74
75
75
76
74
74
75
77
80
75.4
2.7
1.3
4.3
2.0
74.0
102

Sample pairs 1, 4 and 3, 5 are duplicates.


n/a, not applicable.

2
29
30
33
33
34
35
32
30
34
32
33
34
35
35
37
36
33.2
2.4
2.6
6.4
6.9
37.0
90

3
179
183
178
177
185
190
184
187
193
189
188
191
176
178
164
160
181.4
6.9
1.4
26.9
5.3
179.6
101

4
74
74
76
79
78
76
79
78
75
76
74
71
75
74
73
74
75.4
3.6
1.7
6.4
3.0
74.0
1021

5
180
178
185
185
190
185
191
187
188
184
196
185
176
178
196
190
185.9
10.4
2.0
17.1
3.3
179.6
104

6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
b
n/ab
n/a
n/a
n/a
0
n/a

Table 3. Preliminary study of the recovery of ethyl vanillin by LC method and by AOAC Official Method 966.13
Sample

Added ethyl vanillin,


mg/100 mL

Ethyl vanillin by LCa

Ethyl vanillin by AOAC


966.13b

Recovery by LC method,
%

Recovery by Method
966.13, %

222
50
400

216
57
366

202
51
366

97
114
92

91
102
92

A
B
C
a
b

Mean of 7 analysts
Mean of 3 analysts

Figure 990.25. Chromatograms of (a) vanillin and ethyl vanillin working standard solution and (b) typical artificial
vanilla flavor with added ethyl vanillin.

Figure 1. Repeatability and reproducibility of vanillin determination as a function of vanillin concentration.

Figure 2. Repeatability and reproducibility of ethyl vanillin determination as a function of ethyl vanillin concentration.