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ISSN: 1579-4377
QUALITY CONTROL OF ALOE VERA BEVERAGES
Katrin Lachenmeier1,*, Uta Kuepper1, Frank Musshoff1, Burkhard Madea1, Helmut Reusch2
and Dirk W. Lachenmeier2
1

Institute of Legal Medicine, Rheinische Friedrich-Wilhelms-University of Bonn, Stiftsplatz 12, D-53111 Bonn, Germany
2
Chemisches und Veterinäruntersuchungsamt (CVUA) Karlsruhe, Weißenburger Str. 3, D-76187 Karlsruhe, Germany

KEYWORDS
Aloe vera juice, Aloe barbadensis MILLER, solid-phase microextraction, gas chromatography,
mass spectrometry, thin-layer chromatography.
ABSTRACT
Aloe vera beverages have to be produced exclusively using material of the plant species Aloe barbadensis
MILLER. Commercial material was reported to be frequently adulterated by artificial preservatives or to
lack significant amounts of Aloe ingredients. HPTLC and HS-SPME/GC/MS methods to assess the
authenticity of Aloe vera beverages were developed in this study, allowing to differentiate between
authentic and adulterated products. In one case a commercially available Aloe vera juice could be proven
to be exceedingly watered down. Parallel to the authenticity control, the HS-SPME method employed in
this work allowed to detect the preservatives benzoic acid, sorbic acid and pHB-esters. In 17 of 24 (71%)
currently available Aloe-food products an illegal addition of preservatives of up to 1000 mg/l could be
ascertained. The presented analyses of Aloe vera beverages lead to the conclusion, that this product line
does not give any cause for hygienic but rather legal concerns: controls have to be intensified to ensure
sufficient product quality with regard to preservatives

INTRODUCTION
By law Aloe vera beverages have to be produced using material of the plant species Aloe barbadensis
MILLER, which is a tropical or subtropical plant characterized by lance-shaped leaves with jagged edges
and sharp points [1-3]. In the production process of Aloe vera juices and gels the manufactures often
employ artificial preservatives (e.g. benzoic or sorbic acid) to stabilize the plant extract and conserve the
valued ingredients. Apart from this illegal addition of preservatives to Aloe-beverages, adulterated
products containing no significant amount of Aloe ingredients are also marketed. Aloe juices and gels are
generally high-priced, for 1l Aloe vera juice for example the consumer is charged up to 30 EUR. It cannot
be excluded that manufacturers try to enlarge their profit margins by watering down the original Aloe
juice. Studies from the USA showed that a considerable part of commercial material labelled as Aloe vera
material was not consistent with the label claim [4,5].
Aloe vera products are only suitable for human consumption if they are free from aloin, a native Aloe
vera constituent that acts as a laxative and is supposed to be a DNA-damaging and carcinogenic agent.
Aloin can be prevented from entering the production process if the outer layers of the Aloe plant,
containing the highest quantities of aloin, are discarded before extracting the juice. Aloe vera products
*

Web-address: http://www.cvua-karlsruhe.de, E-Mail: Lachenmeier@web.de

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may be considered aloin-free if the maximum limit of 0.1 mg/l is not exceeded [6]. Therefore, for a
thorough quality control of Aloe beverages, the following analyses have to be performed:


Investigation of authenticity (identity, adulteration, dilution)



Test for inadmissible preservatives



Determination of aloin content

For the detection of preservatives as well as aloin, standard methods are available [7,8]. This paper,
however, describes for the first time HPTLC and HS-SPME methods to assess the authenticity of Aloe
vera beverages.
Solid-phase microextraction (SPME), discovered and developed by Pawliszyn and co-workers [9], has
recently emerged as a versatile solvent-free alternative to conventional liquid-liquid extraction
procedures. Headspace solid-phase microextraction (HS-SPME) is based on the distribution of analytes
between the sample, the headspace above the sample and a coated fused-silica fibre. Analytes are
absorbed by the coating of the fibre where they adhere until equilibrium concentrations between the
phases are reached. Subsequently, the fibre can be introduced directly into a GC injection port for thermal
desorption. In HS-SPME no matrix interferences can result in a diminished chromatographic background,
solvent consumption is markedly reduced and its overall technical performance is fast and simple. The
use of SPME in food analysis was recently reviewed by Kataoka [10].

MATERIALS AND METHODS
Reagents and materials
Cyclodecanone, which was used as internal standard, was purchased as a solid from Fluka (Buchs,
Switzerland). It was stored at 8 °C, and used after dilution to the required concentrations. Further
chemicals were purchased from Merck (Darmstadt, Germany). An SPME device for the autosampler with
a replaceable 100 µm polydimethylsiloxane (PDMS) fibre was obtained from Supelco (Deisenhofen,
Germany). The fibre was conditioned at 250°C for 1 h in the injection port of the GC according to the
supplier's instructions.
Aloe vera beverages (n=17) were sampled in the context of the official food control in the German
Federal State of Baden-Württemberg. Further samples (n=7) were obtained from chemists and
pharmacies.

Thin-layer chromatography (HPTLC)
Classic thin-layer chromatographic methods are suitable to investigate the authenticity of Aloe beverages.
Separation was performed on pre-coated 10x10cm HPTLC glass plates (sorbent: silica gel; pore size:
60Ǻ; fluorescence indicator: F254; Merck, Darmstadt, Germany). Sample volumes of 20 µl were applied
to the plates as bands with a width of 10 mm using a TLC applicator (Automatic TLC Sampler III,
Camag, Berlin, Germany). The plates were developed using a freshly prepared mobile phase of n-Butanol
: n-Propanol : glacial acetic acid : water (30 : 10 : 10 : 10, v/v/v/v).
After drying at room temperature, the spots were stained with a solution of anisaldehyde reagent R (0.5
ml of anisaldehyde, 10 ml acetic acid, 85 ml methanol, and 5 ml sulphuric acid) by dipping, followed by
heating for 5 min at 105-110°C.

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Following chromatographic separation bands can be compared to Aloe-typical bands of an authentic Aloe
vera-standard and authenticity of the sample can be evaluated.

Gas chromatography – mass spectrometry (GC/MS) method
For further confirmation, a detection method combining solid-phase microextraction (SPME) and GC/MS
was developed. The applicability of this novel method could be assured by the successful identification of
volatile analytes, which have already been recommended for the characterization of Aloe, e.g. limonene,
eucalyptol and β-pinene [11,12].
The analyses were performed on a model 6890 Series Plus gas chromatograph, in combination with an
Agilent 5973 N MSD mass spectrometer (Chromtech, Idstein, Germany). Substances were separated on a
fused silica capillary column (HP-5MS, 30 m x 0.25 mm I.D., film thickness 0.25 µm). The temperature
program was applied as follows: start temperature 35°C, 10 °C/min increase up to 300 °C. The
temperatures for the injection port, ion source, quadrupole and interface were set at 260 °C, 230 °C,
150 °C and 280 °C, respectively. Injection was carried out in splitless injection mode, and helium at a
flow rate of 1.0 ml/min was used as carrier gas. Electron impact (EI) mass spectra for the analytes were
recorded in Full Scan mode.

Headspace-SPME method
500 µl aliquots of each sample were deposited into two 10 ml headspace vials with 50 µl cyclodecanone
(50 ng/ml) each. No further sample preparation was necessary. The vials were sealed using silicone
septums and magnetic caps. One vial was shaken for 5 min at 40° C, the other one at 80° C in the agitator
of the autosampler (650 rpm, agitator on time: 0:05 min, agitator off time: 0:02 min). For absorption the
needle of the SPME device containing the extraction fibre was exposed to the vapours in the headspace of
the vials for 11 min. The absorbed compounds were desorbed from the fibre by incubating it in the
injection port for 5 min.
In order to develop optimal conditions in the sample preparation step, the influence of extraction,
incubation temperature, incubation time and desorption on recovery was determined. Samples (500 µl
plus 50 µl ISTD) were either diluted with buffer solutions (500 µl phosphate buffer pH 4-11) to solubilize
the analytes or hydrolysed using acid or base (500 µl 0.1 M HCl and 0.1 M NaOH). The following
preparation and analysis was as described above. Furthermore, samples were incubated at different
temperatures (30-100° C). The incubation time was evaluated between 5 and 15 min. Optimal conditions
were defined as those yielding the highest recoveries, i.e. the highest peak areas, of the internal standard
cyclodecanone, which was chosen to represent all volatile compounds.

Multivariate data analysis
The quantitative data of HS-SPME/GC/MS analyses were exported to the software Unscrambler v9.0
(CAMO Process AS, Oslo, Norway). The data set was pre-processed by standardization to give all
variables the same variance. Then Principal Component Analysis (PCA) was used to transform the
original measurement variables into new variables called principal components (PC). The technique of
cross-validation was applied to determine the number of principal components (PCs) needed. During
cross-validation, one sample at a time (out of n samples) is left out, and the prediction ability is tested on
the omitted sample. This procedure is repeated n times resulting in n models and giving an estimate on the
average prediction ability for the n models. This result is used to select the number of PCs needed. By
plotting the data in a coordinate system defined by the two largest principal components, it is possible to
identify key relationships in the data as well as to find similarities and differences.

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RESULTS AND DISCUSSION
HS-SPME method optimization
The SPME-method parameters extraction temperature, extraction time and desorption conditions were
successively optimized. The desorption velocity of analytes from the fibre can be regarded as uncritical at
and above 260° C. A desorption time of 5 min was proven to eliminate all residual analytes from the
PDMS-phase. The fully automated extraction of the samples either in buffer solution (phosphate buffer
pH 4-11) or after acidic or alkaline hydrolysis did not significantly affect recovery as compared to
untreated samples. The influence of different pH-values was shown to be negligible (Fig. 1). Extraction
time and extraction temperature were most critical for the optimization of the method. The optimal
extraction temperature of cyclodecanone was found to be 60° C (Fig. 2). However, it was shown that peak
areas of the volatile substances were greater at lower temperatures (30-50° C), while peak areas of fatty
acids where significantly greater in the upper temperature ranges (80-100°C). For this reason samples
were analysed in duplicate (40° C and 80°C), to cover the whole spectrum of analytes. For HS-SPME it is
necessary, that a 3-phase equilibrium between the liquid sample, the gas phase and the solid fibre is
formed. The optimal extraction time for sample analysis was 11 min and it can be presumed that at this
time equilibrium prevails. Longer extraction times lead to a decrease in recovery (Fig. 3). A typical HSSPME/GC/MS chromatogram of an Aloe vera juice is shown in Fig. 4.

160000000

150000000

Peakarea

140000000

130000000

120000000

110000000

Cyclodecanone (ISTD)

100000000
pH4 pH5

pH7

pH9

pH11

0.1M

0.1M

Sample without

NaOH

HCl

additives

Different pH-values

Fig. 1. Influence of different pH-values on the recovery of cyclodecanone.

The new HS-SPME procedure proved to be suitable for the determination of volatile compounds of Aloe
vera in food products in an automated and therefore convenient procedure. All steps (e.g. heating and
shaking of the sample, absorption, pre-concentration and desorption into the injector of the GC) are
programmable and automatically executed, thereby reducing the possible sources of error and distinctly
improving reproducibility.

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1.60E+008

1.40E+008

Peakarea

1.20E+008

1.00E+008

8.00E+007

6.00E+007

Cyclodecanone (ISTD)
4.00E+007
20

30

40

50

60

70

80

90

100

110

Extraction temperature (°C)

Fig. 2. Optimization of SPME extraction temperature.

195000000

190000000

Peakarea

185000000

180000000

175000000

Cyclodecanone (ISTD)
170000000

4

6

8

10

12

14

16

Extraction time (min)

Fig. 3. Optimization of SPME extraction time.

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7.43
7.38

1000000

800000

5.43

600000

400000

5.85

4.52 5.11
5.31
4.71
4.76
4.64
5.47
4.60
4.46
5.66

2.80 3.34

200000

Time-->

α-Pinen

4.24

6.82

6.70
6.61

3.00

4.00

5.00

6.00

7.00

Caryophyllene

13.06

14.25

10.34

8.40

9.30

14.75

13.85

9.83
10.28

14.15

14.97
14.50
14.54
14.64
10.42
14.66
12.84
15.53
9.40 10.12
14.69
7.79 8.34
14.57
15.56
10.20
7.48
14.01
13.30 13.90
14.40
8.57 9.10
10.05
11.53
7.85
16.54
13.24
9.23
12.81
12.07
14.93
13.5814.0514.4614.88
10.38
15.10
11.36
12.69 13.43
11.60
15.16
11.45
11.68
10.57
9.6110.01
10.84
10.99
11.26
8.68
15.29 15.79 16.32
11.88 12.56
11.03
13.54
7.657.99
7.728.04

8.88

8.21

6.36

13.12
13.16 13.71

α-Farnesen

7.28
1200000

Linalylpropanoat

1400000

11.95
12.32

10.47

Borneol

β-Myrcen

1600000

Eucalyptol
Limonen
α-Phellandren
1-Octanol

7.55

1800000

9.04

Camphene

7.93

2.92
2000000

Cyclodecanon (ISTD)

Abundance

8.00

9.00

9.77

10.00

10.63

11.99

11.00

12.00

13.00

14.00

15.00

16.00

17.00

18.2818.77
18.00

19.83

19.00

Fig. 4. HS-SPME/GC/MS chromatogram of an Aloe vera-juice (100%).

Authenticity control using HPTLC and HS-SPME/GC/MS
Thin-layer-chromatography is a simple and rapid method to detect adulterations of Aloe vera products
(Fig. 5). In one case a commercially available Aloe vera juice could be proven to be exceedingly watered
down. In comparison to HPTLC, HS-SPME/GC/MS-chromatograms contain considerably more data,
which can be made accessible using multivariate data analysis (Fig. 6).
Table 1. Analysis results of 24 Aloe vera products (+ positive, - negative)

No.
Sample
HPTLC identity SPME identity Sorbic acid Benzoic acid pHB-Esters
Bio
Juice
+
+
+
1
+
+
+
2 Juice from concentrate
Juice
+
+
3
Juice
+
+
+
+
4
Juice
+
+
+
+
5
Juice
+
6
Juice
+
+
+
+
7
Juice
+
+
+
+
8
Juice
+
+
+
9
Gel
+
+
+
+
10
Juice
+
+
+
+
11
Juice
+
+
+
+
12
Juice
+
+
+
+
13
Premium juice
+
+
+
+
+
14
Juice
+
+
+
+
15
Juice
+
+
+
16
Juice
+
+
17
Juice
+
+
18
Juice
+
+
19
Gel
+
+
20
Fresh juice
+
+
21
Gel
+
+
22
Tea
+
23
Gel
+
+
+
+
24

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This technique was used successfully to differentiate between products containing just a minimal amount
of Aloe ingredients and material made exclusively from Aloe (Table 1). However, substantial differences
in the HS-SPME/GC/MS-fingerprints of different products were detected, thus it was impossible to
identify a single compound as a characteristic marker for Aloe in drinks. Parallel to the authenticity
control, the HS-SPME method employed in this work allowed to detect the preservatives benzoic acid,
sorbic acid and pHB-ester, which was confirmed using standard HPLC.

Fig. 5. HPTLC chromatogram of Aloe vera products and authentic samples for comparison (Aloe vera gel in
pharmaceutical quality.

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100
0
-100

PC2

Lachenmeier et al. EJEAFChe, 4 (4), 2005. [1033-1042]

Adulterated sample
(watered-down juice)

-200

Typical authentic
100% Aloe vera samples

-300
-400
-500
-600

Sample with low quantity
of Aloe vera (Aloe tea)

-700

PC1

-800
-20000

-15000

-10000

-5000

0

5000

Fig. 6. PCA scores plot of 24 Aloe vera products

Problem of artificial preservatives
In 17 of 24 (71%) currently available Aloe-food products an illegal addition of preservatives was
ascertained (Table 1). Many Aloe vera juices are preserved by addition of sorbic and/or benzoic acid in
concentrations of up to 1000 mg/l. As preservation of Aloe juices is prohibited in the European Union
(EU) [13], several manufacturers add ascorbic acid to their products and declare their Aloe-juices to be
“dietary supplements”. This is done to legally bypass the above mentioned EU-directive, as dietary
supplements may contain preservatives. It has to be considered though that Aloe juices or gels do not
contain concentrated nutrients or other ingredients of dietary or physiological value, and even with the
addition of ascorbic acid Aloe juices do not qualify as dietary supplements. Otherwise bottling plants
could vitaminize all their fruit- or vegetable-juices, declare them to be “liquid dietary supplements” and
add preservatives up to a concentration of 2000 mg/l, thereby circumnavigating the current ban on food
preservatives. The classification of vitamin C-enriched Aloe vera juice/gel as a dietary supplement is
therefore not admissible.

CONCLUSIONS
The presented analyses of Aloe vera beverages lead to the conclusion, that this product line does not give
any cause for hygienic but rather legal concerns.
Using standard HPLC only one of the tested products had to be reprehended due to an Aloin
concentration above the legal limit. In earlier quality control experiments such an excess of Aloin was
more frequent, leading to the interpretation that manufacturers have improved their production methods
and/or the quality assurance. Aloe vera beverages are extensively controlled by the official food control
and nowadays most products abide by the legal aloin limit. The considerable percentage of illegally
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preserved products we found during this study makes us postulate that controls have to be intensified to
ensure sufficient product quality with regard to preservatives.

ACKNOWLEDGMENTS
The authors thank A. Felgner, C. Pickert, M. Brossart and S. Gonzalez for excellent technical assistance.
Presented at the Regional Meeting North Rhine-Westphalia 2005 of the German Food-Chemical Society
(Wuppertal, Germany).

REFERENCES
1.

D. Grindlay, T. Reynolds. The Aloe vera phenomenon: a review of the properties and modern
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2.

T. Reynolds, A. C. Dweck. Aloe vera leaf gel: a review update. J. Ethnopharmacol. 68, 3-37
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3.

K. Eshun, Q. He. Aloe vera: a valuable ingredient for the food, pharmaceutical and cosmetic
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P. A. G. M. De Smet. Health risks of herbal remedies: an update. Clin. Pharmacol. Ther. 76, 117 (2004). DOI: 10.1016/j.clpt.2004.03.005

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7.

M. Yamamoto, M. Ishikawa, T. Masui, H. Nakazawa, Y. Kabasawa. Liquid chromatographic
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PMID: 4019374

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F. Zonta, P. Bogoni, P. Masotti, G. Micali. High-performance liquid chromatographic profiles of
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9.

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13. European Parliament and European Council. European Parliament and Council Directive No
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