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Noora Perkola

Finnish Environment InstituteNordic MS Symposium

November 9, 2011, Båstad, Sweden

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Artificial Sweeteners Used as additives in various food products to replace

sugar

4 non-caloric sweeteners

Acesulfame Potassium (ACS-K)

Saccharin (SAC)

Cyclamate (cyclamic acid) (CYC)

Sucralose (SCL)

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Table 1. Analytes; Properties and origin of the standards

1 TRC = Toronto Research Chemicals Inc. (North York, Ontario, Canada)2 Ehrenstorfer = Dr. Ehrenstorfer-Schäfers (Augsburg, Germany)3 ADI = Acceptable Daily Intake

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Artificial Sweeteners in Environment ACS-K, SAC, CYC and SCL detected in environmental

water samples

Levels up to over 2 µg/L of ACS-K

SAC and CYC 10-150 ng/L

SCL 10-1000 ng/L

Loos et al, Journal of Chromatography A (2009) 1216: 1126-1131

Scheurer et al, Anal Bioanal Chem (2009) 394: 1585-1594

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Artificial Sweeteners in Environment Pass through human body and WWTPs

SAC and CYC eliminated over 90 %

ACS-K up to 41 %, SCL 20 %

ACS-K and SCL – tracers of domestic waste water

Effects to aquatic ecosystems unknown

Scheurer et al, Anal Bioanal Chem (2009) 394: 1585-1594

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Artificial Sweeteners in Environment Pass through human body and WWTPs

SAC and CYC eliminated over 90 %

ACS-K up to 41 %, SCL 20 %

ACS-K and SCL – tracers of domestic waste water

Effects to aquatic ecosystems unknown

Scheurer et al, Anal Bioanal Chem (2009) 394: 1585-1594

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Sample pretreatment Reversed phase SPE (Waters Oasis HLB and J.T. Baker

Bakerbond SDB 1) and ion exchange SPE (Waters Oasis WAX and Oasis MAX) were tested

Oasis WAX chosen – possibility to have SCL in different extract (different concentration factor)

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Sample pretreatment SPE procedure (Oasis WAX cartridges):

Conditioning 10 ml MeOH + 5 ml H2O

Load sample (pH 3.0)

Wash with 2 ml 2 % HCOOH (aq)

1st elution 6 ml MeOH

2nd elution 6 ml 1 % NH3 in MeOH

Recoveries between 70 and 120 % with extractionvolumes 50 – 200 ml

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Sample

• 50-200 mL

• pH 3.0

• Add ISTD (d-ACS-K, d-SCL, d-CYC, 13C-SAC)

SPE (WAX)

• Condition: 10 mL MeOH + 5 mL H2O

• Load Sample: 1 drop sec-1

• Elute: 6 mLMeOH + 6 mL 1 % NH3 (MeOH)

Evaporation

• To dryness

• Redissolve to H2O

LC-MS/MS analysis

Figure 1. Sample pretreatment procedure

LC separation Acquity UPLC

Flow rate 0.400 mL/min, Column T 40 °C

• LC eluents A: 2 mM NH4OAc (aq); B: 2 mM NH4OAc (MeOH)

Gradient:

Initial: 99.9 % A

0.10 min: 99.9 % A

2.00-2.30 min: 0.1 % A

2.40-3.00 min: 99.9 % A

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

LC separation• Comparison of HSS T3 1.8 µm 2.1 x 50 mm

and BEH C18 1.7 µm 2.1 x 50 mm columns

• HSS: better retention for polar analytes –longer retention times but less matrixinteractions?

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Figure 2. Chromatogram of standard solution of ACS-K (1), SAC (2), CYC (3) and SCL (4) with BEH C18 column (1a-4a) and HSS column (1b-4b).

14-Oct-2011 2 WAX, HSS

Time0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20

%

0

100

chromtest_111014_21 Sm (Mn, 2x1) 1: MRM of 6 Channels ES- 162 > 82 (acesulfame)

2.47e50.96

14-Oct-2011 2 WAX, HSS

Time1.12 1.14 1.16 1.18 1.20 1.22 1.24 1.26 1.28 1.30 1.32 1.34 1.36 1.38 1.40 1.42 1.44 1.46 1.48 1.50 1.52 1.54 1.56 1.58

%

0

100

chromtest_111014_21 Sm (Mn, 2x1) 2: MRM of 3 Channels ES- 178 > 80 (cyclamate)

7.70e31.29

1.10

1.15 1.39

Figure 3. Example: Chromatogram of river water sample

ACS-K

CYC

MS/MS analysis Xevo TQ MS

ESI-

MRM

Table 2. Optimization of MS parameters

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

ESI- MS/MS LOQs for ACS-K, SAC, CYC and SCL 25 ng/L, 25 ng/L,

6.5 ng/L and 210 ng/L, respectively

Sample volume 100 mL

Extract volume 1.0 mL

Matrix suppression

70-80 % for ACS-K and SAC

60-90 % for SCL

Less than 20 % for CYC

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Enhancing ionization MS optimization with matrix?

Lower matrix effects in MeOH than in 1 % NH3

(MeOH)

More efficient wash step in SPE or clean-up for extract?

Modificator to enhance ionization in ES?

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

IDMS Isotope Dilution Mass Spectrometry

Mass labelled standards for all analytes

d4-ACS-K

d11-CYC

d6-SCL

13C6-SAC

Quantification using labelled standards

Reliable results despite low recoveries

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

AcknowledgementsI wish to thank

Waters

Association of Maa- ja Vesitekniikan Tuki ry for financial support

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011

Thank You!

Noora Perkola - 2nd Nordic MS Symposium 9.11.2011