AbstractThe aim of this work was to develop and validate a pesticide multiresidue LC-MS/MS method for the analysis of about 450 analytes eluting in less than 10 min. The MS/MS acquisition method employs triggered multiple reaction monitoring (tMRM), which provides increased confidence in analyte identification through triggered acquisition of additional MRMs when one of the primary MRMs exceeds a set abundance threshold (> 2000 MRMs included in the method). The mobile phase gradient was optimized to spread the analytes evenly throughout the elution window, with special attention paid to the separation of critical pairs. The LC system employs an on-line dilution set-up, ensuring excellent peak shapes of early eluting (more polar) analytes. As a result, acetonitrile extracts (prepared using a QuEChERS-based extraction) are injected directly without a need for a dilution with an aqueous buffer/solution prior to the injection. The method was validated in three different routine laboratories in multiple food commodity types/matrices, with 0.01 mg/kg method validated LOQ achieved for the majority analyte-matrix combinations.

IntroductionLC Problems for Early Eluting AnalytesRetention/peak focusing of early eluting compounds injected in strong solvents, such as relatively polar analytes injected in acetonitrile in reverse-phase separations (including pesticide multiresidue analysis).

Analyte Identification SANTE/11945/2015 guidelines “Analytical quality control and method validation procedures for pesticide residue analysis in food and feed”

Identification using LC-MS/MS:▶ retention time ± 0.1 min▶ ≥ 2 product ions▶ ± 30% max. relative tolerance for ion ratios

Triggered MRM (tMRM)▶ Data-dependent function in Agilent LC-QQQ instruments

▶ Triggered acquisition of additional (secondary) MRMs when one of the primary MRMs exceeds set threshold

▶ Up to 10 MRMs per analytes combined into a “spectrum”

ExperimentalPesticide StandardsAgilent LC-MS standard mixes 1-8 at 100 µg/mL in acetonitrile (Agilent Part # 5190-0551) were combined with several custom mixes to create a composite with >450 analytes.

Sample Preparation▶ Sample size: – 10 g: high-moisture samples – 5 g: low-moisture/low-fat samples – 1 g: low-moisture/high-fat and complex samples

▶ QuEChERS extraction/partition (AOAC 2007.01) – 10 mL acetonitrile with 1% acetic acid – Addition of 10 mL water for low-moisture samples – 4 g MgSO4 and 1 g NaOAc for phase separation/

buffering

▶ No SPE clean-up and no dilution for LC-MS/MS analysis

Development and Validation of a Large Multiresidue LC-MS/MS Method Using On-Line Dilution and Other Features Useful for Routine AnalysisKaterina Mastovska,1* John Zulkoski,1 Erika Deal,2 Lukas Vaclavik3 and Urairat Koesukwiwat4 Covance Food Solutions; 1Madison, WI, USA; 2Greenfield, IN, USA; 3Harrogate, UK; and 4Singapore; *Corresponding author: katerina.mastovska@covance.com

Presented at HPLC Symposium 2017

UHPLC Conditions

On-Line DilutionOn-line dilution set-up employed in the LC-MS/MS method during (A) mixing of the injected sample with mobile phase A (initial 0.2 min) and (B) post-mixing when the binary pump gradient starts.

Components:A – 2 µm in-line direct connect SS filter (Analytical Sales & Services, PN 48812); B – 25 µL high-pressure static mixer (Resolution Systems, PN 402-0025HP); C – Valco SS mixing tee 1/16 inch 0.25 mm bore (Resolution Systems, PN ZT1C or Sigma-Aldrich, PN 58626); D – 0.3 µm in-line filter (Agilent, PN 5067-4638); E – Heat exchanger (PN G1316-80002); F – Guard column; and G – Analytical column

MS/MS ConditionsInstrument: Agilent 6490Ionization mode: Positive ESI with Agilent Jet Stream (AJS)Scan type: Triggered MRM (with 3 repeats)Cycle time: 650 msPrimary MRM: 2-3 per analytes

Results and DiscussionOptimization of MS/MS ConditionsOptimization and selection of MS/MS transitions (typically 10 MRMs per analyte) using MassHunter Optimizer software, followed by a detailed review of the collected information.1

Optimization of UHPLC Conditions and On-Line Dilution▶ Optimum analyte separation and detection within a relatively short

separation time

▶ On-line dilution and mixing using a serial combination of two high-pressure mixers to improve chromatography of early eluting compounds – an alternative setup without the second (quaternary) pump possible with the use a 6-port high-pressure valve2

▶ Improved retention and peak shape of early eluting, more polar analytes:

▶ Carry-over reduction/elimination with in-run solvent injections - injections of acetonitrile during the column- wash period at 100% B (after the elution of analytes) using an autosampler program

Final LC-MS/MS Method OptimizationExtracted ion chromatograms of the analytes included in the method and MRM histograms showing distribution of the analytes and their primary MRMs and all MRMs:

Interlaboratory Method Transfer and Validation▶ Method transferred onto multiple identically configured Agilent 1290/6490

systems in three different pesticide routine testing laboratories in the US, Europe and Asia (verification and/or update of the analyte retention times)

▶ Validated in multiple commodity types/matrices using the SANTE method validation guidelines and criteria3

▶ Very good repeatability (% RSD) obtained in the method validation throughout the chromatographic run:

Analyte Identification Using Triggered MRM▶ Increased identification confidence▶ Tool for elimination of false positive results▶ Fast decision-making for suspect hits – examples:

A suspect fenhexamid result dismissed due to the missing m/z 302.1 >143.1 transition and two additional MRMs with incorrect ion ratio in the sample:

A high identification confidence achieved for imazathepyr in a botanical extract sample due to 5 matching MRMs (2 primary and 3 triggered):

Summary▶ Successful interlaboratory method development and transfer of a globally

harmonized method

▶ Optimized LC and MS/MS conditions for separation of ≈ 450 analytes in <10 min

▶ Robust on-line dilution set-up for improved peak shape and retention of more polar pesticides

▶ In-run solvent injections for carry-over reduction/elimination

▶ Triggered MRM (tMRM) function for improved identification confidence

▶ Global validation in multiple commodity and matrix types with 0.01 mg/kg method validated LOQ for the majority of analyte-matrix combinations

Acknowledgements▶ Jerry Zweigenbaum, Thomas Glauner, John Lee, Steve Royce and Andre

Santos from Agilent Technologies

▶ J.-F. Halbardier, Laura Harrison, Camilla Midtlien and Max Chang from Covance Food Solutions

References1. K. Mastovska, J. Zulkoski, and J. Zweigenbaum: Triggered MRM

LC-MS/MS method development – Practical considerations for MRM optimizations using Agilent MassHunter Optimizer software. Application Note 5991-7195EN, Agilent Technologies, 2017.

2. K. Mastovska, J. Zulkoski, E. Deal, L. Vaclavik, U. Koesukwiwat, J.-F. Halbardier, J. Zweigenbaum, and T. Glauner: Improved LC-MS/MS pesticide residue analysis using triggered MRM and on-line dilution. Application Note 5991-7193EN, Agilent Technologies, 2017.

3. K. Mastovska, J. Zulkoski, E. Deal, L. Vaclavik, U. Koesukwiwat, J.-F. Halbardier, J. Zweigenbaum, and T. Glauner: Validation results for LC-MS/MS pesticide residue analysis using triggered MRM and on-line dilution. Application Note 5991-7194EN, Agilent Technologies, 2017.

Instrument: Agilent 1290

Analytical column: ZORBAX Eclipse Plus C18, Rapid Resolution HD 2.1 x 100 mm, 1.8 µm (Agilent Technologies)

Guard column: ZORBAX Eclipse Plus C18 2.1 x 5 mm, 1.8 µm (Agilent Technologies)

Column temperature: 40°C

Mobile phase A: 10 mM ammonium formate in water-methanol (98:2, v/v) + 0.1% formic acid

Mobile phase B: 10 mM ammonium formate in methanol-water (99:1, v/v) + 0.1% formic acid

Injection volume: 2 μL

Binary pump gradient and flow: Time (min) A% B% Flow (mL/min)0.00 100 0 0.1000.20 100 0 0.1000.21 100 0 0.5000.50 50 50 0.5002.50 45 55 0.5005.50 25 75 0.5007.50 15 85 0.5008.30 0 100 0.50012.00 0 100 0.50012.10 100 0 0.50014.80 100 0 0.50014.90 100 0 0.100

Quaternary pump gradient and flow: Time (min) A% B% C% D% Flow (mL/min)

0.00 100 0 0 0 0.5000.20 100 0 0 0 0.5000.40 100 0 0 0 0.00014.80 100 0 0 0 0.00014.90 100 0 0 0 0.500