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 Müller LC-MS Applications Specialists Hewlett-Packard-Str. 8 76337 Waldbronn Germany
Use/Occurence of Chlorate Non-selective herbicide (banned since 2010) Disinfection byproduct (Chlorine dioxide, hypochlorite disinfectants, but also in caustic soda) By-product of chlorination of drinking water (wash and irrigation water) Page 2
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 Page 3
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 Page 4
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! Page 5
Aim of method development for chlorate analysis 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 established methods (QuPPe porous graphite column, mixed mode column) Evaluate sample preparation options Determine performance of Agilent 6470 QQQ in chlorate analysis Page 6
Method development steps Optimize mass spectrometer for chlorate detection Explore alternative to established methods good retention but faster, good peak shape and reproducibility Test different sample preparation techniques for successful clean-up, simplicity Page 7
LC Development and Optimization Columns Porous Graphite Mixed mode (RP + ion exchange) Poroshell PFP 2.1 x 100 mm; 2.7 µm Poroshell PFP 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 1000000 Influence of the amount of acetic acid in water as mobile phase (A) Chlorate 83>67 [Peak area] 800000 600000 400000 200000 0 0% 0.01% 0.1% 1% Page 8
Optimized Method 1290 Infinity II UHPLC Stationary Phase: Poroshell PFP, 2.1 x 100 mm, 2.7 µm Temperature: 40 C Mobile Phase A: 1 % HAC in Water Mobile Phase B: MeOH Flow: 0.4 ml/min Gradient: 0 min 0.1 % B 2.50 min 60 % B 2.51 min 100 % B 4 min 100 % B 4.01 min 5 % B 6 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) Page 9
Optimized Method 6470 QQQ Electrospray Ionization with Agilent Jet Stream Ion Source Drying Gas: 150 C, 8 L/min Sheath Gas: 400 C, 11 L/min Nebulizer: 45 psi Capillary: 2000 V Nozzle: 0 V MS-Parameter ESI Polarity: negative Scan Type: MRM Transitions: 2 Cycle Time: 207 ms ΔEMV: +200 V Optimized source parameters in solvent, milk and whey powder samples Optimized transitions Compound Name Precursor MS1 Res 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 Page 10
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: Page 11
6470 Performance: Serial Dilution in Solvent - Sensitivity / Linearity / Repeatability - Serial dilution of chlorate in water (11 levels): 0.05 1000 ng/ml Page 12
6470 Performance: Serial Dilution in Solvent - Sensitivity / Linearity / Repeatability - Page 13
Sample preparation - Method Development Approach - Post-extraction method (1,2) : set of three samples per technique Standards spiked into blank matrix Blank sample matrix (no analytes) Recovery: Sample preparation RE [%] = Response extracted spiked sample Response post extracted spiked sample x 100 Sample matrix (with analytes) Standards spiked into extracted matrix Matrix effects: ME [%] = Response post extracted spiked sample 1 x 100 Response standard solution Process efficiency: PE [%] = Response extracted spiked sample Response standard solution x 100 Extracted SPIKED samples POSTextracted SPIKED samples Standard solution (contains 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. Analytical Chemistry 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 Technol Biomed Life Sci 852 (2007) 22-34. Page 14
Chlorate in milk - Sample preparation - Sample volume: 0.5 ml milk (homogenized pasteurized low-fat milk; 1.5% fat) + 10 ppb chlorate spiked PPT: protein precipitation; SPE: solid phase extraction Sample Preparation Process PPT 1 Addition of MeOH cold (1:2) PPT 2 Addition of MeOH cold (1:3) PPT 3 Addition of 1% HAC in MeOH cold (1:3) PPT 4 Addition of 1% HAC in MeOH cold (1:1) PPT 5 Addition of 1% HAC in MeOH cold (1:2) PPT 6 Addition of ACN cold (1:2) PPT 7 Addition of ACN cold (1:1) PPT 8 Addition of 1% HAC in ACN cold (1:3) PPT 9 Addition of 1% FA in ACN cold (1:3) PPT 10 Addition of 1% HAC in ACN cold (1:2) SPE Bond Elut Plexa 45 40 35 30 Process efficiency Chlorate [%] Recovery Chlorate 0[%] 25 20 15 10 5 0 40 35 30 25 20 15 10 5 PPT 1 PPT 2 PPT 3 PPT 4 PPT 5 PPT 6 PPT 7 PPT 8 PPT 9 PPT 10 SPE 1 Plexa Set 1: Extracted spiked samples containing chlorate (10 ppb) Set 2: Post-extracted spiked samples; chlorate (10 ppb) was added to the extracted matrix blank Set 3: Standard solution; chlorate (10 ppb) in 100% water PPT 1 PPT 2 PPT 3 PPT 4 PPT 5 PPT 6 PPT 7 PPT 8 PPT 9 PPT 10 SPE 1 Plexa Page 15
Chlorate in milk - Sensitivity x10 Blank [Chlorate] 2 83.0 -> 67.0 Final Conc.=0.012 S/N=1.1 85.0 -> 69.0 S/N=0.9 5.2 5 Water 4.8 4.6 4.4 Blank Milk [Chlorate] 83.0 -> 67.0 Final Conc.=1.138 S/N=139.1 85.0 -> 69.0 S/N=78.7 Milk (unspiked) S/N* 139 1 ppb [Chlorate] 83.0 -> 67.0 Final Conc.=2.015 S/N=223.6 85.0 -> 69.0 S/N=112.3 Milk (1 ppb spike) S/N* 223 2.5 ppb [Chlorate] 83.0 -> 67.0 Final Conc.=3.695 S/N=431.6 85.0 -> 69.0 S/N=152.1 Milk (2.5 ppb spike) S/N* 431 4.2 4 3.8 3.6 3.4 3.2 3 Quantification of chlorate in milk by standard addition after precipitation 2.8 2.6 2.4 0.99 ng/ml 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 (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): Page 16
Sample Dilution The excellent instrument sensitivity allows dilution of the sample, resulting in reduced matrix effect, which means more accurate quantification Dilution factor considered in calculated concentration (Expected concentration 10 ng/ml) x10 3 1.5 1.45 1.4 1.35 1.3 PPT 3 spiked [Chlorate] 83.0 -> 67.0 Final Conc.=3.01 S/N=1651.1 85.0 -> 69.0 S/N=494.8 1.55 Undiluted Conc. 3.01 ng/ml PPT 3 spiked Dil 1_10 [Chlorate] 83.0 -> 67.0 Final Conc.=5.04 S/N=220.8 85.0 -> 69.0 S/N=72.6 10x diluted Conc. 5.04 ng/ml PPT 3 spiked Dil 1_20 [Chlorate] 83.0 -> 67.0 Final Conc.=5.52 S/N=72.9 85.0 -> 69.0 S/N=23.2 20x diluted Conc. 5.52 ng/ml PPT 3 spiked Dil 1_50 [Chlorate] 83.0 -> 67.0 Final Conc.=5.58 S/N=37.8 85.0 -> 69.0 S/N=10.5 50x diluted Conc. 5.58 ng/ml PPT 3 spiked Dil 1_100 [Chlorate] 83.0 -> 67.0 Final Conc.=5.26 S/N=18.1 85.0 -> 69.0 S/N=5.8 100x diluted Conc. 5.26 ng/ml 1.25 1.2 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 Lower concentration found in undiluted sample due to ion suppression Already 10x dilution sufficient to remove matrix effects 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 Page 17
Chlorate in milk - Repeatability Page 18
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 Page 19
Chlorate in whey powder Sample Preparation by Precipitation Sensitivity: LLOQ could not be established directly in matrix, due to high chlorate concentration in available whey powder x10 4 2.3 2.2 2.1 Whey Blank [Chlorate] 83.0 -> 67.0 Final Conc.=332.003 S/N=219.9 2.4 85.0 -> 69.0 S/N=157.7 Whey Powder S/N* 220 75 ppb [Chlorate] 83.0 -> 67.0 Final Conc.=69.092 S/N=255.2 85.0 -> 69.0 S/N=196.2 75 ppb spike S/N* 255 100 ppb [Chlorate] 83.0 -> 67.0 Final Conc.=105.459 S/N=233.7 85.0 -> 69.0 S/N=172.0 100 ppb spike S/N* 233 250 ppb [Chlorate] 83.0 -> 67.0 Final Conc.=275.833 S/N=355.0 85.0 -> 69.0 S/N=276.7 250 ppb spike S/N* 355 500 ppb [Chlorate] 83.0 -> 67.0 Final Conc.=487.999 S/N=480.7 85.0 -> 69.0 S/N=359.1 500 ppb spike S/N* 480 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 Quantification of chlorate in whey powder by standard addition after precipitation 1.1 1 0.9 332 ng/g 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 *(peak-to-peak; noise region: 1.6 1.8 min): Page 20
Chlorate in whey powder Sample 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 and Bond Elut Nexus SPE, chlorate in whey powder could be quantified 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 Page 21
18 O 3 -Chlorate as Internal Standard No isotopically labeled internal standard was used in this study in order to understand the true 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 18 O 3 -Chlorate Page 22
Summary Excellent sensitivity of new UHPLC-QQQ method Allows for significant sample dilution to reduce matrix effects Short runtime (6 min) Facilitates accurate quantification through standard addition instead of use of expensive 18 O 3 -labeled internal standard. Compatible with different sample preparation techniques for both milk and whey powder Page 23