Vanilla – Natural or Out of the Reaction Flask?

Anita Piper, Scientist Instrumental Analysis R&D; Stephan Altmaier, Principal Scientist Advanced Analytical R&D;
Matthias Nold, Product Manager Reference Materials

HPLC fngerprint method and reference materials help to distinguish natural from synthetic or adulterated vanilla

Ice creamVanilla is one of the most popular flavors in food and beverage products. The demand far exceeds the global supply of naturally grown vanilla; therefore, in addition to natural vanilla, artificial vanilla flavors are used in the food industry. Natural vanilla is commonly substituted for synthetically produced vanillin or by other compounds with a similar flavor such as ethyl vanillin. And because of the large price difference between natural and synthetic vanilla, this is a very attractive target for food criminals and frauds.

Analysis of the chromatographic fingerprint of a vanilla flavor represents an efficient method to detect these types of adulteration and mislabeling.1 Characteristic markers for natural vanilla are vanillic acid, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde and vanillin (Figure 1).

For artificially produced vanilla, cheap chemicals such as guaiacol or eugenol are typically used as starting materials. The presence of traces of these compounds are indicators of synthetically produced vanilla. Ethyl vanilin or coumarin are also often added to enhance the flavor (Figure 2).

 

Figure 1. Compounds found in natural vanilla.



4-Hydroxybenzoic acid (Cat.No. 92596)


4-Hydroxybenzaldehyde (Cat.No. 91554)


Vanillic acid (Cat.No. 68654)


Vanillin (Cat.No. 30304)

Figure 2. Markers for artificial vanilla.







Guaiacol (Cat.No. PHR1136)


Ethyl vanillin (Cat.No. 75042)


Coumarin (Cat.No. 72609)


Eugenol (Cat.No. 79891)

We recently launched a set of two reference materials for natural and synthetic vanilla extracts for the testing of vanilla authenticity by chromatographical fingerprint. These two reference extracts are also available individually (Table 1).

 

Table 1. Vanilla Extract Reference Materials (Natural and Synthetic)

Description Quantified components Qualitatively confirmed components Package size Cat. no.
Vanilla extract, natural Vanillin, Vanillic acid Vanillin, Vanillic acid, Ethyl Vanillin (absence) 1 mL 06261501
Vanilla extract, synthetic Vanillin, Vanillic acid Vanillin, Vanillic acid, Ethyl Vanillin 1 mL 06271501
Vanilla extract set, natural and synthetic Vanillin, Vanillic acid Vanillin, Vanillic acid, Ethyl Vanillin (absence / presence) 2x1 mL 06281501

The products are developed and manufactured by HWI pharma services GmbH in Rülzheim, Germany, and are qualified as secondary standards, traceable to HWI primary reference standards quantified by qNMR. These products add to a range of plant extract reference materials designed for rapid identification and quantification of typical constituents of plants used as food additives or as herbal medicinal products.

HPLC fingerprint method

In the following we present an HPLC method to detect natural and synthetic vanilla markers using a Chromolith® Performance RP-18 endcapped 100x2 mm column (Table 2). Results for both the synthetic and the natural vanilla extract are shown. In addition, samples of food and beverage products containing vanilla flavor, such as ice cream, rooibos tea and Bourbon vanilla, were tested.

For all the standards, extensive studies were made to determine LOD, LOQ, linearity, repeatability and standard deviation (see below under Method Validation Data).

 

Table 2. Experimental conditions & sample preparation

Chromatographic conditions
Column: Chromolith® Performance RP-18 endcapped 100x2 mm (1.52006)
Detection: Dionex Ultimate 3000 VWD-3400 @ UV = 280 nm (micro flow cell; 1.4 µL/7 mm)
Mobile phases: [A] 0.05% TFA in water, [B] Acetonitrile
Injection volume: 0.5 µL
Flow Rate: 0.8 mL/min
Temperature: 40 °C
Pressure drop: 75-118 bar (1088-1711 psi)
Gradient : Time %A %B
  0 90   10
  0.5 65   35
  1.3 25   75
  1.5   0 100
  2   0 100
Samples
Standard solution: The standards 4-hydroxybenzoic acid (c = 0.1 mg/mL), vanillic acid (0.1 mg/mL), 4‑hydroxybenzaldehyde (0.2 mg/mL), vanillin (0.1 mg/mL), guaiacol (0.2 mg/mL), ethyl vanillin (0.1 mg/mL), coumarin (0.1 mg/mL), and eugenol (0.2 mg/mL) were dissolved in mobile phases A/B 90/10 (v/v), Figure 3.
Matrix standard solution natural: Transfer approximately 100 mg of vanilla extract natural into a 5 mL volumetric flask, dissolve in mobile phases A/B 95/5 (v/v) and fill up to mark with mobile phases A/B 95/5 (v/v), Figure 4.
Matrix standard solution synthetic: Transfer approximately 25 mg of vanilla extract synthetic into a 25 mL volumetric flask, dissolve in mobile phases A/B 95/5 (v/v) and fill up to mark with mobile phases A/B 95/5 (v/v), Figure 6.
Sample solution
Bourbon vanilla:
One piece of Bourbon vanilla was cut into small pieces and placed in a 50 mL volumetric flask. The flask was filled to the mark with ethanol and after ultrasonic extraction at room temperature for 30 min the sample mixture was filtered through a 0.2 µm membrane filter.
Sample solution
ice cream:
500 mg vanilla ice cream was placed in a 50 mL volumetric flask and filled up with ethanol to the mark. After ultrasonic extraction at room temperature for 30 min the sample mixture was filtered through a 0.45 µm syringe filter.
Sample solution
Rooibos tea:
One pad of vanilla flavored Rooibos tea was placed in a 50 mL volumetric flask and filled up with ethanol to the mark. After ultrasonic extraction at room temperature for 30 min the sample mixture was filtered through a 0.45 µm membrane filter.

Blank run:

Results & discussion

The prepared standard solution was used for method development and validation. The chromatogram and retention data is shown in Figure 3. Vanillin is the main component of the natural vanilla extract reference material (cat. no. 06261501), in addition traces of 4‑hydroxybenzoic acid, vanillic acid and 4‑hydroxybenzaldehyde could be detected. No ethyl vanillin, guaiacol, coumarin or eugenol were present (Figure 4a). For verification purposes also a spiked extract was injected (Figure 4b). For comparison, the chromatogram of the commercial Bourbon vanilla sample (Figure 5) is very similar to the natural vanilla extract reference material.

In contrast to natural vanilla, the synthetic vanilla extract reference material (cat.no. 06271501) shows, besides vanillin as the major peak, ethyl vanillin and coumarin as well as traces of eugenol (Figure 6a). For verification purposes also a spiked extract was injected (Figure 6b). In the ice cream sample, guaiacol instead of vanillin is the major peak (Figure 7). In addition, traces of ethyl vanillin, coumarin and eugenol were detected, indicating the synthetic nature of the used material. In the Rooibos tea sample the coumarin peak is pronounced (Figure 8)

Chromatographic data

Figure 3. Standard solution.

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 t0 void volume 0.37      
2 4-Hydroxybenzoic acid 0.59 0.64   1.537 1.5
3 Vanillic acid 0.68 0.74   2.256 1.28
4 4-Hydroxybenzaldehyde 0.81 0.88 18.34 1.03
5 Vanillin 0.92 0.00   5.06 1.05
6 Guaiacol 1.09 1.18   2.391 1.16
7 Ethyl vanillin 1.15 1.25   4.243 1.02
8 Coumarin 1.21 1.32   4.384 1.05
9 Eugenol 1.63 1.77   1.416 1.32

Figure 4a. Matrix standard solution natural (vanilla extract, natural, cat. no. 06261501).

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 t0 void volume 0.35      
2 4-Hydroxybenzoic acid 0.72 0.72 0.0317 1.13
3 Vanillic acid 0.84 0.84 0.0621 1.12
4 4-Hydroxybenzaldehyde 0.92 0.92 0.2579 1.03
5 Vanillin 1.00 0.00 1.5233 1.16
6 Guaiacol        
7 Ethyl vanillin        
8 Coumarin        
9 Eugenol        

Figure 4b. Matrix standard solution natural (vanilla extract, natural, cat. no. 06261501) spiked with 100 µL standard solution.

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 t0 void volume 0.35      
2 4-Hydroxybenzoic acid 0.68 0.69 0.1949 1.15
3 Vanillic acid 0.79 0.81 0.2715 1.04
4 4-Hydroxybenzaldehyde 0.89 0.91 1.9767 0.99
5 Vanillin 0.98 0.00 0.5564 1.10
6 Guaiacol 1.13 1.15 0.1910 1.00
7 Ethyl vanillin 1.17 1.19 0.4899 1.12
8 Coumarin 1.23 1.26 0.5541 1.25
9 Eugenol 1.64 1.67 0.4369 1.23

Figure 5. Commercial bourbon vanilla sample.

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 t0 void volume 0.37      
2 4-Hydroxybenzoic acid 0.58 0.64 0.127 1.05
3 Vanillic acid 0.67 0.74 0.153 1.15
4 4-Hydroxybenzaldehyde 0.80 0.87 0.340 1.08
5 Vanillin 0.91 0.00 3.094 0.89
6 Guaiacole        
7 Ethyl vanillin        
8 Coumarin        
9 Eugenol        

Figure 6a. Matrix standard solution synthetic (vanilla extract, synthetic, cat. no. 06271501).

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 t0 void volume 0.37      
2 4-Hydroxybenzoic acid        
3 Vanillic acid        
4 4-Hydroxybenzaldehyde        
5 Vanillin 1.00 0.00 2.2404  1.16
6 Guaiacol        
7 Ethyl vanillin 1.19 1.19 0.7156  1.22
8 Coumarin 1.24 1.24 0.9187  1.33
9 Eugenol 1.66 1.66 0.0216  1.27

Figure 6b. Matrix standard solution synthetic (vanilla extract, synthetic, cat. no. 06271501) spiked with 100 µL standard solution.

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 t0 void volume 0.37      
2 4-Hydroxybenzoic acid 0.68 0.69 0.1823 1.10
3 Vanillic acid 0.80 0.82 0.2164 1.04
4 4-Hydroxybenzaldehyde 0.89 0.91 1.9434 1.06
5 Vanillin 0.98 0.00 2.7772 1.08
6 Guaiacol 1.13 1.15 0.1532 1.04
7 Ethyl vanillin 1.17 1.19 1.1713 1.11
8 Coumarin 1.22 1.24 1.2770 1.28
9 Eugenol 1.64 1.67 0.0760 1.27

Figure 7. Ice cream sample

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 t0 void volume 0.37      
2 4-Hydroxybenzoic acid 0.58 0.64 0.001 1.29
3 Vanillic acid 0.67 0.74 0.004 1.22
4 4-Hydroxybenzaldehyde 0.81 0.89 0.008 1.06
5 Vanillin 0.91 0.00 0.099 0.96
6 Guaiacol 1.09 1.20 0.244 0.85
7 Ethyl vanillin 1.14 1.25 0.001  
8 Coumarin 1.19 1.31 0.009 1.25
9 Eugenol 1.63 1.79 0.008 0.88

Figure 8. Rooibos tea sample

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 t0 void volume 0.37      
2 4-Hydroxybenzoic acid 0.62 0.69 0.526  
3 Vanillic acid 0.68 0.76 0.406  
4 4-Hydroxybenzaldehyde 0.84 0.93 0.295  
5 Vanillin 0.90 0.00 6.804 1.37
6 Guaiacole 1.11 1.23 0.018 1.25
7 Ethyl vanillin 1.14 1.28 0.021 1.27
8 Coumarin 1.20 1.36 0.347 0.80
9 Eugenol 1.63 1.81 0.012 0.76

Method validation data

1. Specificity: inject standard solution and determine the retention time and content of desired analyte @280 nm

Peak no. Compound Retention time (min) RRT Area (mAU*min) Tailing factor
1 4-Hydroxybenzoic acid 0.59 0.64 1.537 1.50
2 Vanillic acid 0.68 0.74 2.256 1.28
3 4-Hydroxybenzaldehyde 0.81 0.88 18.34 1.03
4 Vanillin 0.92 0.00 5.060 1.05
5 Guaiacol 1.09 1.18 2.391 1.16
6 Ethyl vanillin 1.15 1.25 4.243 1.02
7 Coumarin 1.21 1.32 4.384 1.05
8 Eugenol 1.63 1.77 1.416 1.32

2. Standard repeatability sample solution of Rooibos tea in mAU*min

Sample 4-Hydroxy-benzoic acid Vanillic acid 4-Hydroxy-benzaldehyde Vanillin Guaiacol Ethyl vanillin Coumarin Eugenol
STD 1 0.5880 0.6730 0.2980 6.5040 0.0180 0.0200 0.3710 0.0110
STD 2 0.5810 0.6730 0.2970 6.4980 0.0190 0.0200 0.3790 0.0110
STD 3 0.5900 0.6670 0.3090 6.5070 0.0190 0.0210 0.3800 0.0120
STD 4 0.5970 0.6730 0.2990 6.5060 0.0190 0.0210 0.3810 0.0110
STD 5 0.5870 0.6730 0.3060 6.5010 0.0180 0.0210 0.3790 0.0120
                 
Mean 0.5886 0.6718 0.3018 6.5032 0.0186 0.0206 0.3780 0.0114
Standard Deviation 0.0058 0.0027 0.0054 0.0037 0.0005 0.0005 0.0040 0.0005
(%) RSD 1.0 0.4 1.8 0.1 2.9 2.7 1.1 4.8

3. LOD &LOQ

4-Hydroxybenzoic acid
Conc. (µg/mL) Mean area (mAU*min)
0.910 0.014
5.000 0.079
11.375 0.214
22.750 0.376
45.500 0.763
68.250 1.141
91.000 1.534
136.500 2.240
   
STEYEX 0.016049632
Slope 0.016517569
LOD (µg/mL) 3.351
LOQ (µg/mL) 10.156

Vanillic acid
Conc. (µg/mL) Mean area (mAU*min)
    1.250 0.023
    6.250 0.118
  15.625 0.272
  31.250 0.544
  62.500 1.144
  93.750 1.608
125.000 2.230
187.500 3.344
   
STEYEX 0.026315012
Slope 0.017775088
LOD (µg/mL) 5.255
LOQ (µg/mL) 15.926

4-Hydroxybenzaldehyde
Conc. (µg/mL) Mean area (mAU*min)
    2.4   0.181
  12.0   0.920
  30.0   2.579
  60.0   4.681
120.0   9.046
180.0 14.448
240.0 18.723
360.0 28.476
   
STEYEX 0.218213451
Slope 0.078874069
LOD (µg/mL) 9.860
LOQ (µg/mL) 29.879

Vanillin
Conc. (µg/mL) Mean area (mAU*min)
    1.2 0.053
    5.90 0.284
  14.75 0.624
  29.50 1.300
  59.00 2.451
  88.50 3.672
118.00 5.017
177.00 7.480
   
STEYEX 0.039267368
Slope 0.042121875
LOD (µg/mL) 3.322
LOQ (µg/mL) 10.067

Guaiacol
Conc. (µg/mL) Mean area (mAU*min)
    2.430 0.025
  12.150 0.117
  30.375 0.250
  60.750 0.565
121.500 1.143
182.250 1.696
243.000 2.263
345.000 3.328
   
STEYEX 0.030186278
Slope 0.009559148
LOD (µg/mL) 11.048
LOQ (µg/mL) 33.479

Guaiacol
Conc. (µg/mL) Mean area (mAU*min)
    2.430 0.025
  12.150 0.117
  30.375 0.250
  60.750 0.565
121.500 1.143
182.250 1.696
243.000 2.263
345.000 3.328
   
STEYEX 0.030186278
Slope 0.009559148
LOD (µg/mL) 11.048
LOQ (µg/mL) 33.479

Coumarin
Conc. (µg/mL) Mean area (mAU*min)
    1.0 0.037
    5.2 0.193
  13.0 0.485
  26.0 0.965
  52.0 1.949
  78.0 2.920
104.0 3.871
156.0 5.830
   
STEYEX 0.006761945
Slope 0.037365165
LOD (µg/mL) 0.635
LOQ (µg/mL) 1.924

Coumarin
Conc. (µg/mL) Mean area (mAU*min)
    1.0 0.037
    5.2 0.193
  13.0 0.485
  26.0 0.965
  52.0 1.949
  78.0 2.920
104.0 3.871
156.0 5.830
   
STEYEX 0.006761945
Slope 0.037365165
LOD (µg/mL) 0.635
LOQ (µg/mL) 1.924

4. Linearity (area mAU*min)

4-Hydroxy-benzoic acid
Conc. (µg/mL) Mean area
    0.910 0.014
    5.000 0.079
  11.375 0.214
  22.750 0.376
  45.500 0.763
  68.250 1.141
  91.000 1.534
136.500 2.240
Vanillic acid
Conc. (µg/mL) Mean area
    1.250 0.023
    6.250 0.118
  15.625 0.272
  31.250 0.544
  62.500 1.144
  93.750 1.608
125.000 2.230
187.500 3.344
4-Hydroxy-benzaldehyde
Conc. (µg/mL) Mean area
    2.4   0.181
  12.0   0.920
  30.0   2.579
  60.0   4.681
120.0   9.046
180.0 14.448
240.0 18.723
360.0 28.476
Vanillin
Conc. (µg/mL) Mean area
    1.2 0.053
    5.90 0.284
  14.75 0.624
  29.50 1.300
  59.00 2.451
  88.50 3.672
118.00 5.017
177.00 7.480
Guaiacol
Conc. (µg/mL) Mean area
    2.430 0.025
  12.150 0.117
  30.375 0.250
  60.750 0.565
121.500 1.143
182.250 1.696
243.000 2.263
345.000 3.328
Guaiacol
Conc. (µg/mL) Mean area
    2.430 0.025
  12.150 0.117
  30.375 0.250
  60.750 0.565
121.500 1.143
182.250 1.696
243.000 2.263
345.000 3.328
Coumarin
Conc. (µg/mL) Mean area
    1.0 0.037
    5.2 0.193
  13.0 0.485
  26.0 0.965
  52.0 1.949
  78.0 2.920
104.0 3.871
156.0 5.830
Coumarin
Conc. (µg/mL) Mean area
    1.0 0.037
    5.2 0.193
  13.0 0.485
  26.0 0.965
  52.0 1.949
  78.0 2.920
104.0 3.871
156.0 5.830

Conclusion

The examples shown demonstrate the applicability and value of matrix reference materials to help detect food adulterations and mislabeling. The developed and validated method in conjunction with the presented reference materials allow an efficient and reliable analysis of vanilla flavored samples.

 

Reference

  1. Cicchetti, Chaintreau J. Sep. Sci. 2009, 32, 3043 – 3052.

 

Materials