Qualitative determination of sweeteners in wine by liquid chromatography coupled with mass spectrometry (LC-MS) (Type-IV)
OIV-MA-AS315-31 Qualitative determination of sweeteners in wine by liquid chromatography coupled with mass spectrometry (LC-MS)
Type IV method
- Scope
This method is suitable for the determination of presence of five artificial sweeteners (aspartame, potassium acesulfame, sodium cyclamate, saccharin and sucralose) as well as the natural sweetener stevioside in white, rosé and red wine.
- Definitions
ESI – Electrospray Ionisation
LC – Liquid chromatography
LC-MS – Liquid chromatography coupled with mass spectrometry
m/z – Mass to charge ratio
MS – Mass spectrometry
MS/MS – Mass spectrometry acquisition mode measuring product ions
QTOF – Quadrupole time-of-flight mass spectrometry
RP – Reverse phase
RT – Retention time
UHPLC – Ultra-high-performance liquid chromatography
- Principle
Wine is analysed directly using a liquid chromatography coupled with mass spectrometry system (LC-MS). In liquid chromatography (LC), separation is performed using a reverse phase (RP) column and detection is accomplished by mass spectrometry (MS) according to the compounds’ mass to charge ratio (m/z). The MS data combined with the retention time (RT) are used for the identification and quantitation of sweeteners.
- Reagents and materials
4.1. Reagents:
4.1.1. Acetonitrile, purity 
99.95 % (CAS Number 75-05-8)
4.1.2. Purified water: 18 MΩ.cm, TOC 
5 μg/L
4.1.3. Formic Acid, purity 98 % (CAS Number 64-18-6)
4.1.4. Aspartame, purity 99.0 % (CAS Number 22839-47-0)
4.1.5. Acesulfame K, purity 99.9 % (CAS Number 55589-62-3)
4.1.6. Cyclamate, Sodium, purity 99.8 % (CAS Number 139-05-9)
4.1.7. Saccharin, purity 99 % (CAS Number 81-07-2)
4.1.8. Sucralose, purity 98.0 % (CAS Number 56038-13-2)
4.1.9. Stevioside, purity 95.0 % (CAS Number 57817-89-7)
4.1.10. Wines representative of the working matrices and previously verified to be absent of any sweeteners in order to be used for the preparation of calibration solutions and standards.
4.2. Solution preparation (as an example)
Standards and calibration solutions are kept in the fridge at approximately 6 °C. Aspartame solutions are unstable in acid media. Therefore, they must be prepared fresh each time the standard is analysed.
4.2.1. Standard solutions
Individual standard solutions at 1 g/L are prepared, e.g., by dissolving 10.0 mg of each sweetener in 10 mL volumetric flasks and filling up to the mark with water (4.1.2) or with ethanol solution at 12% V/V.
4.2.2. Calibration standards
Calibration standards are prepared and analysed by LC-MS as any other sample (see 6).
The calibration standards are prepared in wine (4.1.10) by diluting the appropriate amount of standard solution (4.2.1) to obtain the concentrations 50 μg/L, 100 μg/L, 500 μg/L and 1000 μg/L of each sweetener.
If better method performance is needed it is recommended to perform calibration with the same matrix being evaluated.
-
Apparatus
- Syringe filters: 0.2 μm polypropylene membrane, 25 mm diameter.
- Laboratory glassware, namely class A volumetric flasks.
-
Analytical balance with a resolution of
0.0001 g
- Micropipettes for volumes from 5 μL to 1000 μL.
-
High Performance Liquid Chromatography instrument coupled with mass spectrometer.
- Standard HPLC and UPLC systems are possible given that the chromatographic separation is adjusted accordingly.
- Several MS system configurations are possible such as quadrupole, ion trap, time-of-flight and also hybrid systems.
Each wine sample is prepared by filtration with a syringe filter (5.1) prior to injection.
If necessary, samples are degassed beforehand using, for example, an ultrasound bath or nitrogen bubbling. If concentrations fall outside the calibration range, samples should be diluted.
Better performance may also be achieved with additional sample preparation steps such as dilution (relying on the instrument sensitivity), sample cleanup and extraction.
- Procedure
The following description, given as an example, refers to a UHPLC-QTOF instrument equipped with an ESI source. Modifications may occur according to the type of equipment or manufacturer’s instructions.
7.1. LC analysis:
- Mobile phase A: purified water (4.1.2) with 0.1 % formic acid (4.1.3
- Mobile phase B: acetonitrile (4.1.1) with 0.1 % formic acid (4.1.3)
- Injection volume: 2 μL
- Sampler temperature: 10 °C Column: RP C8 2.1 mm x 100 mm, 1.9 μm
- Column Oven: 30 °C
Gradient:
|
Time Min |
Flow mL/min |
% A |
% B |
|
0 |
0.4 |
90 |
10 |
|
3 |
0.4 |
60 |
40 |
|
3 |
0.4 |
1 |
99 |
|
4 |
0.4 |
1 |
99 |
|
4 |
0.8 |
1 |
99 |
|
5.5 |
0.8 |
1 |
99 |
|
5.5 |
0.5 |
90 |
10 |
|
9.5 |
0.5 |
90 |
10 |
|
9.5 |
0.4 |
90 |
10 |
7.2. Mass Spectrometer parameters:
- ESI: negative ionisation
- Source Temp: 200 °C
- Capillary Voltage: 3000 V
- Acquisition Mode: broadband collision-induced dissociation (bbCID)
- Consists of alternating acquisition of spectra of Full Scan and MS/MS modes (acquisition of precursor and product ions respectively)
- Collision Energy: 30 eV
- Acquisition spectra rate: 2.0 Hz
- Dry Gas Flow: 8 L/min;
-
Nebuliser pressure: 2.0x
Pa (2.0 bar)
- Identification
Sweetener identification is confirmed using a standard for each compound (4.1.4, 4.1.5, 4.1.6, 4.1.7, 4.1.8 and 4.1.9). The data gathered for peak confirmation is the RT for guidance (these may vary depending on the chromatographic parameters) and m/z of the precursor and product ions (Table 1).
Table 1 – Sweeteners identification data: RT, precursor m/z and product m/z
|
Sweetener |
RT min |
Ion |
Precursor m/z |
Product m/z |
|
Acesulfame K |
1.24 |
[M]- |
161.9867 |
77.9655 |
|
Aspartame |
2.30 |
[M-H]- |
293.1143 |
261.0881 |
|
Cyclamate Na |
1.66 |
[M]- |
178.0543 |
79.9574 |
|
Saccharin |
1.55 |
[M-H]- |
181.9917 |
41.9985 |
|
Sucralose |
2.14 |
[M-H]- |
395.0073 |
359.0306 |
|
Stevioside |
3.63 |
[M-H]- |
803.3707 |
641.3026 |
Note: The ions used for quantitation are underlined in Table 1.
Ion signals are monitored with extracted ion chromatograms with 
3 mDa tolerance (Figure 1).
|
Aspartame [M-H]- |
Acesulfame K [M]- |
Cyclamate Na [M]- |
|||||||||
|
|
|
|
|||||||||
|
|||||||||||
Note: An example of low standard sensitivity and additional transitions are given in appendix
- Calculus
Results are calculated from the calibration curve which is obtained with the amount (μg/L) vs the peak area of each sweetener:
|
|
Where
is the sweetener concentration (μg/L), 
is the sample peak area, 
is the calibration curve Y-axis interception point and 
is the calibration curve slope.
- Results
Concentrations are expressed in μg/L without decimals.
-
Internal validation
- Matrices
Validation was performed using a total of 43 different wines: 20 red wines, 10 rosé wines and 13 white wines. These wines were selected from several regions with the aim of obtaining great variability of characteristics in order to make a comprehensive approach. Bellow there is a table summarizing the major characteristics of the wines.
Table 2 – Matrices main characteristics
|
Red wine (R) |
Rosé wine (Ro) |
White wine (W) |
||||
|
Regions |
Alentejo |
4 |
Douro |
3 |
Açores |
1 |
|
Bairrada |
1 |
Vinho Verde |
1 |
Alentejo |
2 |
|
|
Dão |
3 |
Other(1) |
6 |
Dão |
1 |
|
|
Douro |
4 |
Douro |
1 |
|||
|
Lisboa |
1 |
Lisboa |
1 |
|||
|
Valladolid |
1 |
Vinho Verde |
4 |
|||
|
Other(1) |
6 |
Other(1) |
3 |
|||
|
Alcoholic Strength by Volume % v/v |
12.1 – 17.2 |
9.8 – 12.6 |
8.7 – 13.6 |
|
Sugar content g/L (glucose + fructose) |
0.5 – 108.0 |
0.7 – 28.8 |
0.2 – 17.1 |
|
Total Acidity g/L (tartaric acid) |
4.6 – 6.4 |
4.7 – 6.0 |
5.2 – 7.1 |
|
pH |
3.5 – 3.8 |
3.2 – 3.5 |
3.2 – 3.4 |
|
Intensity |
2.4 – 16.2 |
0.1 – 0.5 |
0.03 – 0.29(2) |
(1) Without geographical indication
(2) Absorbance at 420 nm instead of intensity
11.2. Linearity
The method proved to be linear within a range of concentrations between 50 μg/L and 1000 μg/L
11.3. Calibration
A total of 14 independent calibrations were made counting 6 red wines, 4 rosé wines and 4 white wines. Then, for each compound, calibrations were made considering 3 different approaches:
- One unified calibration for all the matrices
- 2 groups of matrices consisting in one group for white wines and another group with the remaining wines (red wines and rosé wines)
- 3 groups of matrices consisting of white wines, rosé wines and red wines
Herein presented are the optimized results of the validation study. According to the selected calibration conditions, for acesulfame, saccharin and sucralose calibration functions and subsequent calculations were preformed considering one group for white wines and a second group with the remaining matrices, red wines
and rosé wines. For aspartame, cyclamate and stevioside three groups of matrices were considered: red wines, rosé wines and white wines.
Table 3 – Calibration scheme for each compound
|
Calibrations |
Individual |
Combined |
||
|
Matrices |
White wine |
Rosé wine |
Red wine |
Red wines + Rosé wines |
|
Acesulfame |
X |
X |
||
|
Aspartame |
X |
X |
X |
|
|
Cyclamate |
X |
X |
X |
|
|
Saccharin |
X |
X |
||
|
Stevioside |
X |
X |
X |
|
|
Sucralose |
X |
X |
||
Given the heteroskedasticity and normal distribution of the residuals, the regression model employed was the weighted least square regression.
As an example, sucralose for the group of red and rosé wines at a concentration range 50 μg/L to 1000 μg/L is presented below.
|
Figure 2 – Calibration curve, standardized residuals and Q-Q plot for the combined red and rosé wines calibration for sucralose |
|
|
|
|
|
|
|
|
Yellow |
2x standard deviation |
|
red |
3x standard deviation |
|
|
|
11.4. Limits of detection and limits of quantitation
The limits of quantitation were obtained through calculation from the calibration curves
Table 4 – LOD and LOQ values obtained for each compound
|
LOD (mg/L) |
LOQ (mg/L) |
|||||
|
White wine |
Rosé wine |
Red wine |
White wine |
Rosé wine |
Red wine |
|
|
Acesulfame K |
0.003 |
0.003 |
0.011 |
0.011 |
||
|
Aspartame |
0.004 |
0.006 |
0.004 |
0.014 |
0.019 |
0.014 |
|
Cyclamate Na |
0.002 |
0.005 |
0.004 |
0.006 |
0.015 |
0.014 |
|
Saccharin |
0.002 |
0.005 |
0.006 |
0.016 |
||
|
Stevioside |
0.002 |
0.002 |
0.005 |
0.005 |
0.005 |
0.016 |
|
Sucralose |
0.014 |
0.007 |
0.048 |
0.022 |
||
11.5. Repeatability
Repeatability was assessed at three spiking levels: 50 µg/L corresponding to the reporting limit, 250 μg/L and 1000 μg/L. This evaluation is based on 8 replicate injections at each spiking level and for each matrix.
In the following tables the repeatability values obtained for each sweetener are presented including the mean concentration measured in each sample, the standard deviation (Std. Dev.), the percentual relative standard deviation for repeatability (RSDr %) and the Horwitz Ratio for repeatability (HorRat (r)).
Table 5 – Repeatability values for potassium acesulfame at 3 spiking levels
|
Acesulfame |
White wine (W) |
||||||||||
|
Sample |
W1 |
W2 |
W3 |
W4 |
W5 |
W6 |
W7 |
W8 |
W9 |
||
|
Mean μg/L |
45 |
42 |
49 |
233 |
207 |
240 |
1036 |
926 |
1060 |
||
|
Std. Dev. |
1.4 |
2.1 |
0.8 |
3.3 |
5.2 |
2.6 |
13.2 |
13.7 |
15.8 |
||
|
Recovery % |
89 % |
84 % |
98 % |
93 % |
83 % |
96 % |
104 % |
93 % |
106 % |
||
|
RSDr % |
3.2 % |
5.0 % |
1.6 % |
1.4 % |
2.5 % |
1.1 % |
1.3 % |
1.5 % |
1.5 % |
||
|
HorRat (r) |
0.13 |
0.20 |
0.06 |
0.07 |
0.13 |
0.05 |
0.08 |
0.09 |
0.09 |
||
|
Acesulfame |
Rosé wine (Ro) |
||||||||||
|
Sample |
Ro1 |
Ro2 |
Ro3 |
Ro4 |
Ro5 |
Ro6 |
Ro7 |
Ro8 |
Ro9 |
||
|
Mean μg/L |
49 |
52 |
53 |
248 |
248 |
247 |
1063 |
1091 |
1097 |
||
|
Std. Dev. |
2.0 |
1.2 |
1.4 |
2.9 |
3.5 |
3.9 |
14.1 |
13.2 |
15.5 |
||
|
Recovery % |
98 % |
104 % |
107 % |
99 % |
99 % |
99 % |
106 % |
109 % |
110 % |
||
|
RSDr % |
4.1 % |
2.3 % |
2.6 % |
1.2 % |
1.4 % |
1.6 % |
1.3 % |
1.2 % |
1.4 % |
||
|
HorRat (r) |
0.17 |
0.09 |
0.10 |
0.06 |
0.07 |
0.08 |
0.08 |
0.08 |
0.09 |
||
|
Acesulfame |
Red wine (R) |
||||||||
|
Sample |
R1 |
R2 |
R3 |
R4 |
R5 |
R6 |
R7 |
R8 |
R9 |
|
Mean μg/L |
56 |
50 |
57 |
275 |
241 |
260 |
1195 |
1064 |
1160 |
|
Std. Dev. |
1.2 |
2.0 |
1.4 |
3.2 |
5.1 |
4.3 |
13.8 |
14.5 |
10.0 |
|
Recovery % |
112 % |
101 % |
115 % |
110 % |
96 % |
104 % |
120 % |
106 % |
116 % |
|
RSDr % |
2.1 % |
3.9 % |
2.4 % |
1.2 % |
2.1 % |
1.6 % |
1.2 % |
1.4 % |
0.9 % |
|
HorRat (r) |
0.08 |
0.16 |
0.10 |
0.06 |
0.11 |
0.08 |
0.07 |
0.09 |
0.05 |
Table 6 - Repeatability values for aspartame at 3 spiking levels
|
Aspartame |
White wine (W) |
||||||||
|
Sample |
W1 |
W2 |
W3 |
W4 |
W5 |
W6 |
W7 |
W8 |
W9 |
|
Mean μg/L |
34 |
51 |
45 |
237 |
231 |
235 |
981 |
973 |
982 |
|
Std. Dev. |
7.3 |
4.2 |
6.7 |
27.5 |
7.6 |
10.9 |
29.0 |
18.0 |
23.2 |
|
Recovery % |
68 % |
101 % |
91 % |
95 % |
92 % |
94 % |
98 % |
97 % |
98 % |
|
RSDr % |
21.6 % |
8.3 % |
14.7 % |
11.6 % |
3.3 % |
4.6 % |
3.0 % |
1.8 % |
2.4 % |
|
HorRat (r) |
0.87 |
0.33 |
0.59 |
0.59 |
0.17 |
0.24 |
0.19 |
0.12 |
0.15 |
|
Aspartame |
Rosé wine (Ro) |
||||||||
|
Sample |
Ro1 |
Ro2 |
Ro3 |
Ro4 |
Ro5 |
Ro6 |
Ro7 |
Ro8 |
Ro9 |
|
Mean μg/L |
38 |
42 |
41 |
200 |
211 |
210 |
833 |
905 |
916 |
|
Std. Dev. |
3.0 |
2.9 |
4.3 |
6.8 |
5.2 |
5.9 |
20.9 |
34.0 |
22.5 |
|
Recovery % |
75 % |
85 % |
82 % |
80 % |
84 % |
84 % |
83 % |
90 % |
92 % |
|
RSDr % |
8.0 % |
6.9 % |
10.6 % |
3.4 % |
2.5 % |
2.8 % |
2.5 % |
3.8 % |
2.5 % |
|
HorRat (r) |
0.32 |
0.28 |
0.43 |
0.17 |
0.13 |
0.14 |
0.16 |
0.24 |
0.15 |
|
Aspartame |
Red wine (R) |
||||||||
|
Sample |
R1 |
R2 |
R3 |
R4 |
R5 |
R6 |
R7 |
R8 |
R9 |
|
Mean μg/L |
46 |
51 |
50 |
227 |
254 |
230 |
956 |
1099 |
1013 |
|
Std. Dev. |
8.6 |
3.2 |
8.1 |
16.9 |
10.4 |
7.3 |
21.8 |
39.0 |
20.2 |
|
Recovery % |
92 % |
103 % |
100 % |
91 % |
102 % |
92 % |
96 % |
110 % |
101 % |
|
RSDr % |
18.5 % |
6.3 % |
16.2 % |
7.4 % |
4.1 % |
3.2 % |
2.3 % |
3.5 % |
2.0 % |
|
HorRat (r) |
0.74 |
0.25 |
0.65 |
0.38 |
0.21 |
0.16 |
0.14 |
0.22 |
0.13 |
Table 7 - Repeatability values for sodium cyclamate at 3 spiking levels
|
Cyclamate |
White wine (W) |
||||||||
|
Sample |
W1 |
W2 |
W3 |
W4 |
W5 |
W6 |
W7 |
W8 |
W9 |
|
Mean μ g/L |
51 |
50 |
50 |
261 |
247 |
246 |
1092 |
1040 |
1045 |
|
Std. Dev. |
1.0 |
1.4 |
1.4 |
2.8 |
4.2 |
3.5 |
12.2 |
17.7 |
14.4 |
|
Recovery % |
103 % |
100 % |
101 % |
104 % |
99 % |
99 % |
109 % |
104 % |
105 % |
|
RSDr % |
1.9 % |
2.9 % |
2.9 % |
1.1 % |
1.7 % |
1.4 % |
1.1 % |
1.7 % |
1.4 % |
|
HorRat (r) |
0.08 |
0.12 |
0.11 |
0.05 |
0.09 |
0.07 |
0.07 |
0.11 |
0.09 |
|
Cyclamate |
Rosé wine (Ro) |
||||||||
|
Sample |
Ro1 |
Ro2 |
Ro3 |
Ro4 |
Ro5 |
Ro6 |
Ro7 |
Ro8 |
Ro9 |
|
Mean μg/L |
42 |
42 |
44 |
232 |
228 |
233 |
982 |
992 |
1002 |
|
Std. Dev. |
1.6 |
1.3 |
0.8 |
2.8 |
4.4 |
4.5 |
14.9 |
6.0 |
12.9 |
|
Recovery % |
84 % |
85 % |
88 % |
93 % |
91 % |
93 % |
98 % |
99 % |
100 % |
|
RSDr % |
3.9 % |
3.0 % |
1.7 % |
1.2 % |
2.0 % |
1.9 % |
1.5 % |
0.6 % |
1.3 % |
|
HorRat (r) |
0.16 |
0.12 |
0.07 |
0.06 |
0.10 |
0.10 |
0.10 |
0.04 |
0.08 |
|
Cyclamate |
Red wine (R) |
|||||||||||||||||
|
Sample |
R1 |
R2 |
R3 |
R4 |
R5 |
R6 |
R7 |
R8 |
R9 |
|||||||||
|
Mean µg/L |
51 |
55 |
54 |
250 |
265 |
243 |
1069 |
1160 |
1086 |
|||||||||
|
Std. Dev. |
1.2 |
1.3 |
1.4 |
5.5 |
5.2 |
4.2 |
27.4 |
13.9 |
18.4 |
|||||||||
|
Recovery % |
103 % |
110 % |
108 % |
100 % |
106 % |
97 % |
107 % |
116 % |
109 % |
|||||||||
|
RSDr % |
2.4 % |
2.4 % |
2.6 % |
2.2 % |
2.0 % |
1.7 % |
2.6 % |
1.2 % |
1.7 % |
|||||||||
|
HorRat (r) |
0.10 |
0.10 |
0.10 |
0.11 |
0.10 |
0.09 |
0.16 |
0.08 |
0.11 |
|||||||||
Table 8 - Repeatability values for saccharin at 3 spiking levels
|
Saccharin |
White wine (W) |
||||||||
|
Sample |
W1 |
W2 |
W3 |
W4 |
W5 |
W6 |
W7 |
W8 |
W9 |
|
Mean μg/L |
45 |
45 |
59 |
216 |
214 |
252 |
920 |
909 |
1055 |
|
Std. Dev. |
1.5 |
1.4 |
1.4 |
5.1 |
5.1 |
3.7 |
21.3 |
23.7 |
21.5 |
|
Recovery % |
89 % |
91 % |
119 % |
86 % |
86 % |
101 % |
92 % |
91 % |
105 % |
|
RSDr % |
3.3 % |
3.0 % |
2.4 % |
2.4 % |
2.4 % |
1.5 % |
2.3 % |
2.6 % |
2.0 % |
|
HorRat (r) |
0.13 |
0.12 |
0.10 |
0.12 |
0.12 |
0.08 |
0.15 |
0.16 |
0.13 |
|
Saccharin |
Rosé wine (Ro) |
|||||||||||
|
Sample |
Ro1 |
Ro2 |
Ro3 |
Ro4 |
Ro5 |
Ro6 |
Ro7 |
Ro8 |
Ro9 |
|||
|
Mean µg/L |
58 |
56 |
56 |
303 |
276 |
278 |
1263 |
1190 |
1204 |
|||
|
Std. Dev. |
1.4 |
2.0 |
0.6 |
5.5 |
3.5 |
4.8 |
28.8 |
24.8 |
25.2 |
|||
|
Recovery % |
116 % |
112 % |
112 % |
121 % |
110 % |
111 % |
126 % |
119 % |
120 % |
|||
|
RSDr % |
2.4 % |
3.5 % |
1.1 % |
1.8 % |
1.3 % |
1.7 % |
2.3 % |
2.1 % |
2.1 % |
|||
|
HorRat (r) |
0.10 |
0.14 |
0.04 |
0.09 |
0.07 |
0.09 |
0.14 |
0.13 |
0.13 |
|||
|
Saccharin |
Red wine (R) |
|||||||||||
|
Sample |
R1 |
R2 |
R3 |
R4 |
R5 |
R6 |
R7 |
R8 |
R9 |
|||
|
Mean μg/L |
47 |
44 |
46 |
224 |
203 |
199 |
955 |
906 |
885 |
|||
|
Std. Dev. |
1.4 |
0.5 |
1.5 |
4.4 |
2.2 |
2.9 |
20.6 |
20.1 |
25.8 |
|||
|
Recovery % |
94 % |
88 % |
92 % |
89 % |
81 % |
80 % |
95 % |
91 % |
88 % |
|||
|
RSDr % |
3.0 % |
1.1 % |
3.2 % |
2.0 % |
1.1 % |
1.5 % |
2.2 % |
2.2 % |
2.9 % |
|||
|
HorRat (r) |
0.12 |
0.04 |
0.13 |
0.10 |
0.06 |
0.07 |
0.14 |
0.14 |
0.18 |
|||
Table 9 - Repeatability values for stevioside at 3 spiking levels
|
Stevioside |
White wine (W) |
||||||||
|
Sample |
W1 |
W2 |
W3 |
W4 |
W5 |
W6 |
W7 |
W8 |
W9 |
|
Mean μg/L |
41 |
43 |
30 |
262 |
265 |
204 |
1094 |
1116 |
860 |
|
Std. Dev. |
0.4 |
0.4 |
0.7 |
2.0 |
31.2 |
1.9 |
13.6 |
12.9 |
6.6 |
|
Recovery % |
83 % |
86 % |
60 % |
105 % |
106 % |
81 % |
109 % |
112 % |
86 % |
|
RSDr % |
1.0 % |
1.0 % |
2.2 % |
0.8 % |
11.8 % |
0.9 % |
1.2 % |
1.2 % |
0.8 % |
|
HorRat (r) |
0.04 |
0.04 |
0.09 |
0.04 |
0.60 |
0.05 |
0.08 |
0.07 |
0.05 |
|
Stevioside |
Rosé wine (Ro) |
||||||||
|
Sample |
Ro1 |
Ro2 |
Ro3 |
Ro4 |
Ro5 |
Ro6 |
Ro7 |
Ro8 |
Ro9 |
|
Mean μg/L |
50 |
39 |
41 |
237 |
254 |
286 |
935 |
1104 |
1109 |
|
Std. Dev. |
0.8 |
1.3 |
0.9 |
2.6 |
5.3 |
7.1 |
10.5 |
10.2 |
18.3 |
|
Recovery % |
99 % |
77 % |
81 % |
95 % |
102 % |
114 % |
93 % |
110 % |
111 % |
|
RSDr % |
1.7 % |
3.4 % |
2.2 % |
1.1 % |
2.1 % |
2.5 % |
1.1 % |
0.9 % |
1.6 % |
|
HorRat (r) |
0.07 |
0.14 |
0.09 |
0.06 |
0.11 |
0.13 |
0.07 |
0.06 |
0.10 |
|
Stevioside |
Red wine (R) |
||||||||
|
Sample |
R1 |
R2 |
R3 |
R4 |
R5 |
R6 |
R7 |
R8 |
R9 |
|
Mean μg/L |
60 |
40 |
43 |
262 |
211 |
210 |
1048 |
904 |
921 |
|
Std. Dev. |
0.9 |
0.7 |
0.4 |
4.0 |
4.0 |
2.6 |
18.0 |
18.4 |
11.9 |
|
Recovery % |
120 % |
80 % |
86 % |
105 % |
85 % |
84 % |
105 % |
90 % |
92 % |
|
RSDr % |
1.5 % |
1.8 % |
1.0 % |
1.5 % |
1.9 % |
1.3 % |
1.7 % |
2.0 % |
1.3 % |
|
HorRat (r) |
0.06 |
0.07 |
0.04 |
0.08 |
0.10 |
0.06 |
0.11 |
0.13 |
0.08 |
Table 10 - Repeatability values for sucralose at 3 spiking levels
|
Sucralose |
White wine (W) |
|||||||||
|
Sample |
W1 |
W2 |
W3 |
W4 |
W5 |
W6 |
W7 |
W8 |
W9 |
|
|
Mean μg/L |
53 |
52 |
53 |
221 |
225 |
223 |
986 |
973 |
1021 |
|
|
Std. Dev. |
5.3 |
7.8 |
8.1 |
10.8 |
27.5 |
6.5 |
29.8 |
43.9 |
31.5 |
|
|
Recovery % |
106 % |
103 % |
105 % |
88 % |
90 % |
89 % |
99 % |
97 % |
102 % |
|
|
RSDr % |
10.0 % |
15.1 % |
15.4 % |
4.9 % |
12.2 % |
2.9 % |
3.0 % |
4.5 % |
3.1 % |
|
|
HorRat (r) |
0.40 |
0.61 |
0.62 |
0.25 |
0.63 |
0.15 |
0.19 |
0.28 |
0.19 |
|
|
Sucralose |
Rosé wine (Ro) |
|||||||||
|
Sample |
Ro1 |
Ro2 |
Ro3 |
Ro4 |
Ro5 |
Ro6 |
Ro7 |
Ro8 |
Ro9 |
|
|
Mean µg/L |
35 |
43 |
36 |
215 |
236 |
194 |
944 |
1075 |
905 |
|
|
Std. Dev. |
4.1 |
2.1 |
2.2 |
7.2 |
7.4 |
7.7 |
21.3 |
27.5 |
19.3 |
|
|
Recovery % |
70 % |
86 % |
71 % |
86 % |
94 % |
78 % |
94 % |
108 % |
91 % |
|
|
RSDr % |
11.7 % |
5.0 % |
6.2 % |
3.3 % |
3.1 % |
4.0 % |
2.3 % |
2.6 % |
2.1 % |
|
|
HorRat (r) |
0.47 |
0.20 |
0.25 |
0.17 |
0.16 |
0.20 |
0.14 |
0.16 |
0.13 |
|
|
Sucralose |
Red wine (R) |
||||||||
|
Sample |
R1 |
R2 |
R3 |
R4 |
R5 |
R6 |
R7 |
R8 |
R9 |
|
Mean μg/L |
50 |
46 |
48 |
236 |
255 |
228 |
1017 |
1194 |
1041 |
|
Std. Dev. |
7.7 |
3.1 |
6.8 |
11.5 |
9.2 |
8.4 |
16.9 |
27.5 |
23.0 |
|
Recovery % |
100 % |
92 % |
96 % |
94 % |
102 % |
91 % |
102 % |
119 % |
104 % |
|
RSDr % |
15.3 % |
6.9 % |
14.1 % |
4.9 % |
3.6 % |
3.7 % |
1.7 % |
2.3 % |
2.2 % |
|
HorRat (r) |
0.61 |
0.28 |
0.57 |
0.25 |
0.18 |
0.19 |
0.10 |
0.15 |
0.14 |
Table 11 – Repeatability summary table
|
Compound |
Recovery |
RSDr % |
HorRat (r) |
|
Acesulfame |
83 % – 120 % |
0.9 % – 5.0 % |
0.05 – 0.20 |
|
Aspartame |
68 % – 110 % |
1.8 % – 21.6 % |
0.12 – 0.87 |
|
Cyclamate |
84 % – 116 % |
0.6 % – 3.9 % |
0.04 – 0.16 |
|
Saccharin |
80 % – 126 % |
1.1 % – 3.5 % |
0.04 – 0.18 |
|
Stevioside |
60 % – 112 % |
0.8 % – 11.8 % |
0.04 – 0.60 |
|
Sucralose |
70 % – 119 % |
1.7 % – 15.4 % |
0.10 – 0.63 |
11.6. Intermediate Precision
Intermediate precision was evaluated by analyzing samples spiked with 50 μg/L, 250 μg/L and 1000 μg/L in different moments spanning throughout several days. The results are presented in the following tables. Count represents the number of points considered for the determination of the mean values and respective standard deviation (Std. Dev.). The recovery percentage, the relative standard deviation (RSD%) and the Horwitz ratio (HorRat) are also displayed for each case.
Table 12 – Intermediate precision values for potassium acesulfame at 3 spiking levels
|
Acesulfame |
White wine (W) |
Red (R) and Rosé wines (Ro) |
|||||||
|
Sample |
W1 |
W2 |
W3 |
Ro1 |
Ro2 |
Ro3 |
R1 |
R2 |
R3 |
|
Count |
12 |
12 |
11 |
12 |
12 |
12 |
12 |
12 |
12 |
|
Mean µg/L |
46 |
223 |
928 |
54 |
252 |
1089 |
53 |
252 |
1113 |
|
Std. Dev. |
6.1 |
31.4 |
76.6 |
6.1 |
23.6 |
66.3 |
2.4 |
9.1 |
41.3 |
|
Recovery % |
91 % |
89 % |
93 % |
108 % |
101 % |
109 % |
106 % |
101 % |
111 % |
|
RSD% IP |
13.2 % |
14.1 % |
8.2 % |
11.3 % |
9.4 % |
6.1 % |
4.5 % |
3.6 % |
3.7 % |
|
HorRat |
0.53 |
0.72 |
0.52 |
0.46 |
0.48 |
0.38 |
0.18 |
0.18 |
0.23 |
Table 13 - Intermediate precision values for aspartame at 3 spiking levels
|
Aspartame |
White wine (W) |
Rosé wine (Ro) |
Red (R) |
||||||
|
Sample |
W1 |
W2 |
W3 |
Ro1 |
Ro2 |
Ro3 |
R1 |
R2 |
R3 |
|
Count |
11 |
10 |
10 |
11 |
12 |
12 |
11 |
12 |
12 |
|
Mean μg/L |
57 |
281 |
1190 |
41 |
202 |
841 |
41 |
222 |
998 |
|
Std. Dev. |
5.5 |
21.8 |
91.9 |
4.1 |
12.9 |
73.9 |
7.5 |
15.4 |
43.5 |
|
Recovery % |
114 % |
113 % |
119 % |
82 % |
81 % |
84 % |
83 % |
89 % |
100 % |
|
RSD % IP |
9.6 % |
7.7 % |
7.7 % |
10.0 % |
6.4 % |
8.8 % |
18.1 % |
6.9 % |
4.4 % |
|
HorRat |
0.38 |
0.40 |
0.49 |
0.40 |
0.32 |
0.55 |
0.73 |
0.36 |
0.27 |
Table 14 - Intermediate precision values for sodium cyclamate at 3 spiking levels
|
Cyclamate |
White wine (W) |
Rosé wine (Ro) |
Red (R) |
||||||
|
Sample |
W1 |
W2 |
W3 |
Ro1 |
Ro2 |
Ro3 |
R1 |
R2 |
R3 |
|
Count |
10 |
10 |
10 |
11 |
12 |
12 |
12 |
12 |
12 |
|
Mean μg/L |
48 |
237 |
1011 |
40 |
210 |
918 |
49 |
226 |
999 |
|
Std. Dev. |
5.5 |
27.3 |
134.3 |
2.5 |
20.1 |
70.7 |
1.3 |
7.4 |
26.6 |
|
Recovery % |
97% |
95% |
101% |
80% |
84% |
92% |
98% |
91% |
100% |
|
RSD% IP |
11.3% |
11.5% |
13.3% |
6.3% |
9.6% |
7.7% |
2.7% |
3.3% |
2.7% |
|
HorRat |
0.45 |
0.59 |
0.84 |
0.25 |
0.49 |
0.48 |
0.11 |
0.17 |
0.17 |
Table 15 - Intermediate precision values for saccharin at 3 spiking levels
|
Saccharin |
White wine (W) |
Red (R) and Rosé wine (Ro) |
|||||||
|
Sample |
W1 |
W2 |
W3 |
Ro1 |
Ro2 |
Ro3 |
R1 |
R2 |
R3 |
|
Count |
11 |
10 |
10 |
12 |
12 |
12 |
12 |
12 |
12 |
|
Mean μg/L |
51 |
241 |
1010 |
56 |
270 |
1166 |
44 |
195 |
857 |
|
Std. Dev. |
2.6 |
8.4 |
36.8 |
2.8 |
10.7 |
47.6 |
3.0 |
8.3 |
31.7 |
|
Recovery % |
103% |
96% |
101% |
112% |
108% |
117% |
88% |
78% |
86% |
|
RSD % IP |
5.0% |
3.5% |
3.6% |
5.1% |
4.0% |
4.1% |
6.9% |
4.3% |
3.7% |
|
HorRat |
0.20 |
0.18 |
0.23 |
0.20 |
0.20 |
0.26 |
0.28 |
0.22 |
0.23 |
Table 16 - Intermediate precision values for stevioside at 3 spiking levels
|
Stevioside |
White wine (W) |
Rosé wine (Ro) |
Red (R) |
||||||
|
Sample |
W1 |
W2 |
W3 |
Ro1 |
Ro2 |
Ro3 |
R1 |
R2 |
R3 |
|
Count |
11 |
10 |
10 |
12 |
12 |
12 |
12 |
12 |
12 |
|
Mean μg/L |
35 |
232 |
977 |
31 |
210 |
921 |
41 |
208 |
905 |
|
Std. Dev. |
6.5 |
45.8 |
184.1 |
8.1 |
45.4 |
184.4 |
3.1 |
22.2 |
84.5 |
|
Recovery % |
70% |
93% |
98% |
61% |
84% |
92% |
81% |
83% |
91% |
|
RSD% IP |
18.5% |
19.7% |
18.8% |
26.4% |
21.6% |
20.0% |
7.6% |
10.7% |
9.3% |
|
HorRat |
0.74 |
1.01 |
1.19 |
1.06 |
1.10 |
1.26 |
0.31 |
0.55 |
0.59 |
Table 17 - Intermediate precision values for sucralose at 3 spiking levels
|
Sucralose |
White wine (W) |
Red (R) and Rosé wine (Ro) |
|||||||
|
Sample |
W1 |
W2 |
W3 |
Ro1 |
Ro2 |
Ro3 |
R1 |
R2 |
R3 |
|
Count |
10 |
11 |
11 |
10 |
11 |
12 |
12 |
12 |
12 |
|
Mean μg/L |
51 |
197 |
776 |
51 |
295 |
1196 |
42 |
228 |
1069 |
|
Std. Dev. |
10.4 |
41.6 |
137.5 |
11.9 |
48.7 |
184.5 |
5.3 |
18.0 |
51.3 |
|
Recovery % |
101% |
79% |
78% |
101% |
118% |
120% |
85% |
91% |
107% |
|
RSD % IP |
20.5% |
21.1% |
17.7% |
23.5% |
16.5% |
15.4% |
12.5% |
7.9% |
4.8% |
|
HorRat |
0.82 |
1.08 |
1.12 |
0.94 |
0.84 |
0.97 |
0.50 |
0.40 |
0.30 |
Table 18 - Intermediate precision summary table
|
Compound |
Recovery |
RSD% |
HorRat |
|
Acesulfame |
89 % – 111 % |
3.6 % – 14.1 % |
0.18 – 0.72 |
|
Aspartame |
81 % – 119 % |
4.4 % – 18.1 % |
0.27 – 0.73 |
|
Cyclamate |
80 % – 101 % |
2.7 % – 13.3 % |
0.11 – 0.84 |
|
Saccharin |
78 % – 117 % |
3.5 % – 6.9 % |
0.18 – 0.28 |
|
Stevioside |
61 % – 98 % |
7.6 % – 26.4 % |
0.31 – 1.26 |
|
Sucralose |
78 % – 120 % |
4.8 % – 23.5 % |
0.30 – 1.12 |
- Bibliography
- EUROPEAN COMMISSION DIRECTORATE GENERAL FOR HEALTH AND FOOD SAFETY, SANTE/11813/2017, “Analytical quality control and method validation procedures for pesticide residues and analysis in food and feed”. Implemented by 01/01/2018
- Horwitz W., Albert R., 2006. The Horwitz Ratio (HorRat): A Useful Index of Method Performance with Respect to Precision. J AOAC Int, 89, 1095-1109
- OIV, 2021. International Code of Oenological Practices. Issue 2021, OIV, Paris.
Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives (Text with EEA relevance), 2008. OJ, L354, 16–33.
Appendix
A1. Quantitation performance for a wine sample spiked with 50 μg/L of each sweetener
|
Sweetener |
S/N |
|
Acesulfame K |
789.4 |
|
Aspartame |
586.5 |
|
Cyclamate Na |
282.5 |
|
Saccharin |
24.3 |
|
Sucralose |
80.5 |
|
Stevioside |
224.1 |
A2 . Sweeteners identification data - additional transitions given as guidance
|
Sweetener |
Additional transition in ESI negative. |
|
Ace sulfame K |
162 > 82 |
|
Aspartame |
293 > 200 |
|
Cyclamate Na |
178 > 96 |
|
Saccharin |
182 > 106 |
|
Sucralose |
397 > 361 |
|
Stevioside |
641 > 479 641 > 317 |