lc-ms/ms analysis of chlorates in milk and whey … ms ms...res product ms 2 res dwell fv ce cav...

LC-MS/MS analysis of Chlorates in Milk and Whey Powder using the Agilent 6470 QQQ

Anthony Sullivan, LC/MS Product Specialist

Melanie Mülek and Christoph MüllerLC-MS Applications SpecialistsHewlett-Packard-Str. 876337 WaldbronnGermany

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Use/Occurence of Chlorate

Non-selectiveherbicide

(banned since2010)

Disinfection by-product

(Chlorine dioxide, hypochlorite

disinfectants, but also in caustic

soda)

By-product ofchlorination ofdrinking water

(wash andirrigation water)

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Why are Chlorate Levels Regulated?

Negative health effects• Competes with iodide for transport to the thyroid,

reversibly inhibits the absorption of iodide

At high doses• Cause health hazards in sensitive groups such as

children, pregnant women or people with thyroid disfunction

Can cause damage to red blood cells• Irreversible formation of methemoglobin from

hemoglobin after cell lysis

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Where is Chlorate Regulated?

Drinking Water• No maximum levels for chlorate in drinking

water have been set in the European Union • WHO has established a guideline level for

chlorate in drinking water of 0.7 mg/L

Food• No specific maximum residue levels established

for chlorate under Regulation (EC) No 396/2005• Therefore, a default MRL of 0.01 mg/kg is

applicable

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Relevance to Dairy Processing Industry

Chlorinated process water

Cleaning of processing equipment

Amount of chlorate depending on type of chlorination(chlorine, chlorine dioxide, hypochlorite)

Chlorination often required to kill microorganisms, that could cause greater negative health effects than chlorate!

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Aim of method development for chlorateanalysis in milk and whey powder

To balance presence of chlorate by (inevitable) cleaning/disinfection and remain within regulated MRL, frequent analysis of chlorate levels is required

Improve sensitivity and speed compared to establishedmethods (QuPPe porous graphite column, mixedmode column)

Evaluate sample preparation options

Determine performance of Agilent 6470 QQQ in chlorate analysis

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Method development steps

Optimize mass spectrometer forchlorate detection

Explore alternative to establishedmethods good retention but faster, good peak shape and reproducibility

Test different sample preparationtechniques for successful clean-up,

simplicity

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LC Development and Optimization

Columns

• Porous Graphite• Mixed mode (RP + ion

exchange)• Poroshell PFP 2.1 x

100 mm; 2.7 µm

PoroshellPFP

• Better peak intensity• Best retention• excellent RT

reproducibility

Eluents

• Formic vs acetic acid• Increasing acetic acid

concentration: 0/0.01/0.1/1 %

• Methanol/Acetonitrile

0

200000

400000

600000

800000

1000000

0% 0.01% 0.1% 1%C

hlor

ate

83>6

7 [P

eak

area

]

Influence of the amount of acetic acid in water as mobile phase (A)

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Optimized Method 1290 Infinity II UHPLC

Stationary Phase: Poroshell PFP, 2.1 x 100 mm, 2.7 µmTemperature: 40 ˚C

Mobile Phase A: 1 % HAC in WaterMobile Phase B: MeOHFlow: 0.4 mL/minGradient: 0 min 0.1 % B

2.50 min 60 % B2.51 min 100 % B4 min 100 % B4.01 min 5 % B6 min 5 % B

Stop Time: 6 min

Injection: 1 µL Needle Wash: 10 s Flush Port;

Methanol:Water:Formic acid (50:50:0.1; v/v)

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Electrospray Ionization with Agilent Jet Stream Ion SourceDrying Gas: 150° C, 8 L/minSheath Gas: 400° C, 11 L/minNebulizer: 45 psiCapillary: 2000 V Nozzle: 0 V

MS-Parameter ESI Polarity: negative Scan Type: MRM Transitions: 2Cycle Time: 207 msΔEMV: +200 V

CompoundName

Precursor MS1Res

Product MS 2 Res

Dwell FV CE CAV Polarity

Chlorate 85 Unit 69 Wide 100 130 26 4 Negative

Chlorate 83 Unit 67 Wide 100 140 26 5 Negative

Optimized Method 6470 QQQ

Optimized transitions

Optimized source parameters in solvent, milk and whey powder samples

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6470 Performance: Serial Dilution in Solvent- Sensitivity / Linearity / Repeatability -

Sensitivity: LLOQ 0.05 ppb

∅S/N (peak-to-peak; noise region: 1.6 – 1.8 min; n= 3): 9.2

Quantifier: Qualifier:

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Serial dilution of chlorate in water (11 levels):

0.05 – 1000 ng/mL

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Sample preparation- Method Development Approach -

Post-extraction method(1,2): set of three samples per technique

Standard solution(contains analytes)

Standards spiked into

blank matrix

Extracted SPIKED samples

Sample matrix (with

analytes)

Standards spiked into extracted

matrix

POST-extracted SPIKED samples

Blank sample matrix

(no analytes)

(1) B.K. Matuszewski, M.L. Constanzer, and C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. AnalyticalChemistry 75 (2003) 3019-3030. (2) E. Chambers et al., Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. J Chromatogr B Analyt TechnolBiomed Life Sci 852 (2007) 22-34.

Recovery:

Matrix effects:

Process efficiency:

ME [%] = Response post−extracted spiked sampleResponse standard solution

−1 x 100

RE [%] = Response extracted spiked sampleResponse post−extracted spiked sample

x 100

PE [%] = Response extracted spiked sampleResponse standard solution

x 100

Sample preparation

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Chlorate in milk- Sample preparation -

Sample volume:0.5 mL milk (homogenized pasteurized low-fatmilk; 1.5% fat)+ 10 ppb chlorate spiked

Sample Preparation Process

PPT 1 Addition of MeOHcold (1:2)

PPT 2 Addition of MeOHcold (1:3)

PPT 3 Addition of 1% HAC in MeOHcold (1:3) PPT 4 Addition of 1% HAC in MeOHcold (1:1)

PPT 5 Addition of 1% HAC in MeOHcold (1:2)

PPT 6 Addition of ACNcold (1:2)

PPT 7 Addition of ACNcold (1:1)

PPT 8 Addition of 1% HAC in ACNcold (1:3)

PPT 9 Addition of 1% FA in ACNcold (1:3)

PPT 10 Addition of 1% HAC in ACNcold (1:2) SPE Bond Elut Plexa

PPT: protein precipitation; SPE: solid phase extraction

Set 1: Extracted spiked samples containing chlorate (10 ppb)Set 2: Post-extracted spiked samples; chlorate (10 ppb) was added to the extracted matrix blankSet 3: Standard solution; chlorate (10 ppb) in 100% water

0

5

10

15

20

25

30

35

40

45

PPT

1

PPT

2

PPT

3

PPT

4

PPT

5

PPT

6

PPT

7

PPT

8

PPT

9

PPT

10

SPE

1Pl

exa

Rec

over

y C

hlor

ate

[%]

0

5

10

15

20

25

30

35

40

PPT

1

PPT

2

PPT

3

PPT

4

PPT

5

PPT

6

PPT

7

PPT

8

PPT

9

PPT

10

SPE

1Pl

exa

Proc

ess

effic

ienc

y C

hlor

ate

[%]

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Chlorate in milk- Sensitivity

2x10

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

Blank [Chlorate]

1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=0.012 S/N=1.1

85.0 -> 69.0 S/N=0.9

Blank Milk [Chlorate]

1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=1.138 S/N=139.1

85.0 -> 69.0 S/N=78.7

1 ppb [Chlorate]

1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=2.015 S/N=223.6

85.0 -> 69.0 S/N=112.3

2.5 ppb [Chlorate]

1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=3.695 S/N=431.6

85.0 -> 69.0 S/N=152.1

Water Milk (unspiked)S/N* 139

Milk (1 ppb spike)S/N* 223

Milk (2.5 ppb spike)S/N* 431

(about 1 ppb chlorate in blank milk prevents direct determination of LLOQ, estimated LLOQ based on S/N at 1 ppb spike level with consideration of contribution from chlorate in blank milk is 0.1 ppb)

*(peak-to-peak; noise region: 1.6 – 1.8 min):

Quantification ofchlorate in milk bystandard additionafter precipitation

0.99 ng/mL

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Sample DilutionThe excellent instrument sensitivity allows dilution of the sample, resulting in reduced matrixeffect, which means more accurate quantification

Dilution factor considered in calculated concentration (Expected concentration 10 ng/mL)3x10

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

1.4

1.45

1.5

1.55

PPT 3 spiked [Chlorate]

1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=3.01 S/N=1651.1

85.0 -> 69.0 S/N=494.8

PPT 3 spiked Dil 1_10 [Chlorate]

1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=5.04 S/N=220.8

85.0 -> 69.0 S/N=72.6


1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=5.52 S/N=72.9

85.0 -> 69.0 S/N=23.2


1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=5.58 S/N=37.8

85.0 -> 69.0 S/N=10.5


1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4

83.0 -> 67.0 Final Conc.=5.26 S/N=18.1

85.0 -> 69.0 S/N=5.8

UndilutedConc. 3.01 ng/mL

10x dilutedConc. 5.04 ng/mL




Lower concentration foundin undiluted sample due toion suppression

Already 10x dilution sufficient to remove matrix effects

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Chlorate in milk- Repeatability

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Chlorate in whey powder- Sample preparation -Sample volume: 0.5 g whey powder – 100 ng/g chlorate spiked

(0% casein, 1% fat, 12% protein, 1% ash, high lactose)

Approach:0.5 g whey powder + 4.4 ml water + 0.1 mL spike solution chlorate in water

0.5 mL for sample clean-up

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4x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

Whey Blank [Chlorate]

1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

83.0 -> 67.0 Final Conc.=332.003 S/N=219.9

85.0 -> 69.0 S/N=157.7

75 ppb [Chlorate]

1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

83.0 -> 67.0 Final Conc.=69.092 S/N=255.2

85.0 -> 69.0 S/N=196.2

100 ppb [Chlorate]

1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

83.0 -> 67.0 Final Conc.=105.459 S/N=233.7

85.0 -> 69.0 S/N=172.0

250 ppb [Chlorate]

1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

83.0 -> 67.0 Final Conc.=275.833 S/N=355.0

85.0 -> 69.0 S/N=276.7

500 ppb [Chlorate]

1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

83.0 -> 67.0 Final Conc.=487.999 S/N=480.7

85.0 -> 69.0 S/N=359.1

Chlorate in whey powderSample Preparation by PrecipitationSensitivity: LLOQ could not be established directly in matrix, due to high chlorate concentration in available whey powder

Whey PowderS/N* 220

75 ppb spikeS/N* 255




*(peak-to-peak; noise region: 1.6 – 1.8 min):

Quantification ofchlorate in whey

powder bystandard additionafter precipitation

332 ng/g

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Chlorate in whey powderSample Preparation by Solid Phase Extraction

In contrast to milk, whey powder could also be cleaned up by solid phase extraction, retaining matrix on the cartridge and leaving chlorate in the load/wash liquid

Whey powder: cleared by SPE Milk: passes through milky

Several SPE cartridges tested

Best recovery of chlorate was found with Bond Elut Nexus

Polymeric sorbent with no pre-conditioning required

Improved simplicity!

Using standard addition andBond Elut Nexus SPE, chlorate

in whey powder could bequantified as 349 ng/g

* Quant. by external calibration delivers significantly lower results at 130 ng/g with PPT and 253 ng/g with standard addition

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18O3-Chlorate as Internal StandardNo isotopically labeled internal standard was used in this study in order to understand thetrue performance of the UHPLC-QQQ method, and it was shown that with the more rapid LC method, standard addition could be a more economical alternative to 18O3-Chlorate

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Summary

• Allows for significant sample dilution to reduce matrix effects

Excellent sensitivity of new UHPLC-QQQ method

• Facilitates accurate quantification through standard addition instead of use of expensive 18O3-labeled internal standard.

Short runtime (6 min)

Compatible with different sample preparation techniques for both milk and whey powder

lc-ms/ms analysis of chlorates in milk and whey … ms ms...res product ms 2 res dwell fv ce cav...

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