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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

 

  1. 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.

  1. 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

  1. 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.

  1. 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.

  1. Apparatus
    1.                         Syringe filters: 0.2 μm polypropylene membrane, 25 mm diameter.
    2.                         Laboratory glassware, namely class A volumetric flasks.
    3.                         Analytical balance with a resolution of 0.0001 g
    4.                         Micropipettes for volumes from 5 μL to 1000 μL.
    5.                         High Performance Liquid Chromatography instrument coupled with mass spectrometer.
      1.                 Standard HPLC and UPLC systems are possible given that the chromatographic separation is adjusted accordingly.
      2.                 Several MS system configurations are possible such as quadrupole, ion trap, time-of-flight and also hybrid systems.
  1. Sampling

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.

  1. 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)
  1. 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]-

Saccharin [M-H]-

Stevioside [M-H]-

Sucralose [M-H]-

Figure 1 – PFigure 1-Precursor and product peak confirmation for 250 μg/L standard

Note: An example of low standard sensitivity and additional transitions are given in appendix

  1. 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.

  1. Results

Concentrations are expressed in μg/L without decimals.

  1. Internal validation
     
    1.                         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

  1.            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