Chromatography

Sweetener Analysis (Part 3 of 3)

To do:

The first attempts with CHROMABOND® HR-XAW show that enrichment by ionic interaction works much better for Acesulfame than for cyclamate, than the hydrophobic interaction using CHROMABOND® HR-X. The results of these experiments will be summarized in an application note and will be published soon.

Subsequent analysis with HPLC-MS/MS

Core-shell phases are suited for residue analysis in the HPLC. Core-shell silica particles exist of a non-porous solid core and a porous shelf which allow the highest column efficiency with short analysis times and low back pressure. With conventional fully porous particles the diffusion within the particle usually leads to broad peaks. The dwell time of the sweetener molecules in the stationary phase is reduced by the short paths in the core-shell particles, so that even at high flow velocities of the mobile phase, optimal separation results can be obtained.

Mass spectrometry is most suitable for the respective analytes due to the low detection limits as well as the missing UV-active molecule structure elements of some sweeteners. A method proposal using a NUCLEOSHELL® RP 18plus, 2.7 µm column is introduced that allows a very good and quick separation and sure quantification of the residues of sweeteners.

Eluent exchange:

Eluent exchange is performed manually. Eluates from the SPE are evaporated to dryness at 40 °C under a stream of nitrogen and then dissolved in water – acetonitrile (95:5, v/v).

Chromatographic conditions:

Column: EC 100/2 NUCLEOSHELL® RP 18plus, 2.7 µm

Eluent: A) H2O (Millipore) + 0.1 % formic acid, B) acetonitrile + 0.1 % formic acid

Gradient: 5–55 % in 5 min, 55–95 % in 1 min, 95 % for 2 min, 95–5 % in 0.1 min, 5 % for 4.9 min

Flow rate: 0.3 mL/min

Temperature: 30 °C

Injection volume: 10 µL

MS conditions for Neotame and Advantame:

API 3200, ion source ESI, positive ionization mode, curtain gas 15 psig, ion spray voltage 4500 V, temperature 650 °C, nebulizer gas 40 psig, turbo gas 50 psig, CAD 3.0 psig

MS conditions for the other analytes:

API 3200, ion source ESI, negative ionization mode, curtain gas 15 psig, ion spray voltage -4500 V, temperature 650 °C, nebulizer gas 40 psig, turbo gas 50 psig, CAD 3.0 psig

Table 2: MRM transitions

 

Analyte [M+H]+ [M-H] Q1 (quantifier) Q2 (qualifier)
Acesulfame 162.0 82.1 77.9
Aspartame 293.1 200.1 261.1
Cyclamate 178.0 80.0 79.0
NHDC 611.2 303.1 125.0
Saccharin 182.0 42.1 105.9
Sucralose 395.0 35.2 359.1
Advantame 459.2 102.1 252.2
Neotame 379.3 172.2 85.2

 

Figure 6: MRM transitions Q1 (quantifier)

Sweetener Analyis 7

Sweetener Graph 2

Sweetener Graph 3

Sweetener Graph 4

Sweetener Graph 5

Sweetener Graph 6

Sweetener Graph 7

Sweetener Graph 8

Figure 7: Calibration curves, linear range 5 ng/mL to 100 ng/mL

Sweetener Analysis 8

Sweetener Analysis 9

Table 3: Limits of detection and limits of quantification
(estimation of signal to noise ratio)

Substance LOD [µg/L] LOQ [µg/L]
Acesulfame 1.2 1.3
Aspartame 0.8 1.0
Cyclamate 0.8 0.9
NHDC 0.6 0.7
Saccharin 0.6 1.0
Sucralose 1.6 6.7
Advantame 0.7 1.0
Neotame 0.7 0.7

 

Conclusion

It could be shown that an enrichment of the most investigated sweeteners in fresh water with CHROMABOND® HR-X is well possible.

The subsequent analysis with the core-shell phase NUCLEOSHELL® RP 18plus, 2.7 µm features a very good and fast separation and allows a reliable quantification of residues of sweeteners.

Image credits

Candy: © #48654890, www.fotolia.com

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Sweetener Analysis (Part 2 of 3)

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Modern TLC (Part 1 of 2)

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