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anillin content is an important quality parameter of vanilla extract. Vanillin and ethyl vanillin contents are important quality parameters of imitation and fortified va-
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
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
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
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
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
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
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
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 2. Repeatability and reproducibility of ethyl vanillin determination as a function of ethyl vanillin concentration.