Method Development for Trace Analysis in Foods

©2012 Waters Corporation 1

Method Development forMethod Development forTrace Analysis in FoodsTrace Analysis in Foods

Esa [email protected]: +358-9-5659 6288Fax: +358-9-5659 6282

Waters FinlandKutomotie 1600380 Helsinki


Scientific Challenges Trace Analysis

� Sensitivity is required to accurately identify contaminants in a wide variety sample matrices– Regulated methods– Emerging contaminants

� High throughput is a necessity– Hundreds of samples – Fast turnaround time

� Method ruggedness and reliability is essential – Co-eluting endogenous materials can result in reduced assay accuracy

� Data quality must be maintained – Better, more informed decisions


ChallengesConventional Method Development

� LC/MS/MS is the analytical platform of choice– MRM provides a high degree of selectivity– Interference from endogenous materials is minimized – High level technology requiring specialized expertise

� Chromatography– Simple gradients using C18 columns– Low pH MS compatible (Formic Acid) modifiers

� Sample preparation– Minimal use of sample preparation due to cost and method development time


Critical Components of Method Development

Define method objectives

Understand analyte properties and intended use of method

Understand analyte properties and intended use of method

Devise initial LC Conditions

Develop adequate separation by systematic screening or computer assisted development

Develop adequate separation by systematic screening or computer assisted development

Sample preparation procedure

Suitable sample clean-up procedure based on physical and chemical properties of matrix

Suitable sample clean-up procedure based on physical and chemical properties of matrix

Standardization [data processing] Determine method linearity, accuracy and precisionDetermine method linearity, accuracy and precision

Final method optimization/

robustness testing

Challenge method and identify weak spotsChallenge method and identify weak spots

Method validation Prove assay meets intended useProve assay meets intended use


Outline� Introduction– Approaches Toward Method Development– UPLC Technology– Success Criteria� Controlling Selectivity and Retention– Stationary Phase and Particle Substrate Design– Organic Modifier– Mobile Phase pH� Method Development Strategy– Systematic Screening Protocol� Implementing the Approach– Case Study� Conclusion


Approaches Toward Method Development:Deriving Initial LC Conditions

� Match LC conditions to the chemical properties of the analyte[s]– Educated guess based on past experience [speculation]– Usually supplemented with a literature search– Ask a colleague

� Stepwise iterative approach– Next step experimental design based on results from previous experiment

� Systematic screening protocol– Evaluate combinations of mobile phase pH, organic modifier and stationary phaseo Select best combination of these parameters

– Method optimizationo Gradient slope/Temperature


UPLC Technology Can Streamline Method Development

Develop methods in a single work day

� Systematic screening protocol involving pH, organic modifier and column chemistry

� High resolution sub 2 µm column technology creates high resolution separations, faster

� Automated column and mobile phase selection

� Quaternary solvent mixing[ACQUITY UPLC H-Class]

UPLC Technology enables faster method development

ACQUITY UPLC H-Class


Before You Start:Information Gathering

� Chemical properties [functional groups]– Ionizable species, polarity, pKa, molecular weight� Sample solubility� Number of compounds present– How many components are you trying to separate?� Sample matrix� Detection technique [UV, ELS, RI, FL, MS…etc.]– Based on available equipment or sensitivity requirements of assay

� Criteria for success– Concentration range and quantitative requirements– System suitability


Chemical Factors that Impact Selectivity

Selectivity [α]

Stationary Phase

Organic Modifier

Mobile Phase pH


Improving Resolution with Complementary Selectivity

1

1

+

−

k

k

α

α

4

N=Rs

Maximized in UPLC Separations by:

�Range of column chemistries�Multiple particle substrates�Wide usable pH range [BEH]�High retentivity [HSS]�Wide range in selectivity [CSH]

Maximized in UPLC Separations by:

�Ultra-low dispersion system�Small [< 2 µm] particles�Higher pressure capability�Well-designed columns

Impact on Resolution

Double NDouble k Double α

% Improvement

20 – 40 %15 – 20 %> 400 %


Stationary Phase Selectivity:Bonded-Phase [Ligand] and Particle Substrate

� Silanol activity and surface charge– Influences secondary interactions [ion-exchange], peak shape and sample loadability

� Hydrophobicity– Longer alkyl chain lengths will provide increased retention– Shorter, ionizable ligands will increase polarity� Hydrolytic stability– Column lifetime will be impacted by the number of attachment points to the particle surface

� Ligand density– Influences retention and sample loadability


EverEver--Expanding ACQUITY UPLCExpanding ACQUITY UPLC®®Column OfferingColumn Offering

� Five particle substrates • 130Å, 200Å and 300Å BEH [Bridged Ethylene Hybrid],

HSS [High Strength Silica] and CSH [Charged Surface Hybrid]• All are available in HPLC and UPLC particle sizes

� Wide and growing selection of column chemistries• BEH 130Å C18, C8, Shield RP18, Phenyl, HILIC & Amide• BEH 300Å C18 & C4

• BEH 125Å & 200Å SEC• HSS C18, T3, C18 SB, PFP & Cyano• CSH C18, Fluoro-Phenyl & Phenyl-Hexyl

� Proven application-based solutions • AAA, OST, PST, PrST and Glycan

� Transferability between HPLC and UPLC� XBridge HPLC and ACQUITY UPLC BEH columns� XSelect HSS HPLC and ACQUITY UPLC HSS columns� XSelect CSH HPLC and ACQUITY UPLC CSH columns

� VanGuard Pre-columns� eCord Technology


CSH [Charged Surface Hybrid] CSH [Charged Surface Hybrid] Chemistries of UPLC TechnologyChemistries of UPLC Technology

� CSH C18– Trifunctionally bonded C18– Wide pH range for maximum selectivity [pH 1-11]– Superior peak shape and efficiency in buffered and low

ionic strength mobile phases

� CSH Phenyl-Hexyl– Trifunctionally bonded C6-Phenyl– Wide pH range [1-11]– Complementary selectivity for aromatic species

� CSH Fluoro-Phenyl– Trifunctionally bonded, non-endcapped,

pentafluorophenyl [pH 1-8]– Unique selectivity compared to alkyl columns– Stable and reproducible manufacturing process



Selectivity [α]

Stationary Phase

Organic Modifier

Mobile Phase pH


Veterinary Drug StructuresVeterinary Drug Structures


Stationary Phase Selectivity:Basic and Neutral Compounds at Low pH

ObservationsCSH Fluoro-Phenyl gives the largest difference in selectivity, followed by CSH C18

Large differences in stationary phase selectivity for overall mixture

Acetonitrile,pH 2,7

Test Probes:C: Ciprofloxacin [B]E: Enrofloxacin [B]Er: Erythromycin [B]O: Oxytetracycline [B]T: Tetracycline [B]S1: Sulfamerazine [N]S2: Sulfamethazine [N]


Stationary Phase Selectivity:Acidic Compounds at Low pH

ObservationsNo selectivity change

Acetonitrile,pH 2,7

Test Probes:P: Penicillin [A]Oc: Oxacillin [A]



Selectivity [α]

Stationary Phase

Organic Modifier

Mobile Phase pH


Organic Solvent PropertiesOrganic Solvent Properties

� Methanol– Protic solvent [hydrogen bond donor] – Weak elution solvent [compared to acetonitrile]

� Acetonitrile– Aprotic solvent [hydrogen bond acceptor] – Strong elution solvent [compared to methanol]– Low viscosity


Solvent Selectivity:Solvent Selectivity:Basic and Neutral Compounds at High pHBasic and Neutral Compounds at High pH

ObservationsMethanol is a weaker elution solvent than acetonitrile resulting in greater retention for all analytesSelectivity difference between bases relative to neutral test probes are larger



Reversed-Phase Retention Map:The Impact of pH on Ionizable Compounds


Mobile Phase pH Selectivity:Mobile Phase pH Selectivity:Basic and Neutral CompoundsBasic and Neutral Compounds

ObservationsSelectivity change with mobile phase pH change for all the compounds



Mobile Phase pH Selectivity:Mobile Phase pH Selectivity:Acidic and Neutral CompoundsAcidic and Neutral Compounds

ObservationsAt low pH, better resolution and peak shape for most compounds

Test Probes:P: Penicillin [A]Oc: Oxacillin [A]S1: Sulfamerazine [N]S2: Sulfamethazine [N]



Selectivity [α]

Stationary Phase

Organic Modifier

Mobile Phase pH


Maximizing Selectivity Differences:Maximizing Selectivity Differences:Combining Stationary Phase, Organic Modifier and Combining Stationary Phase, Organic Modifier and Mobile Phase pHMobile Phase pH

Test Probes:C: Ciprofloxacin [B]E: Enrofloxacin [B]Er: Erythromycin [B]O: Oxytetracyline [B]T: Tetracyline [B]S1: Sulfamerazine [N]S2: Sulfamethazine [N]

ObservationsLarge differences in selectivity are observed when evaluating combinations of stationary phase, organic modifier and mobile phase pH

BASIC AND NEUTRAL TEST PROBES


Maximizing Selectivity Differences:Maximizing Selectivity Differences:Combining Stationary Phase, Organic Modifier and Combining Stationary Phase, Organic Modifier and Mobile Phase pHMobile Phase pH

ObservationsSome differences in selectivity are observed when evaluating combinations of stationary phase, organic modifier and mobile phase pH

Test Probes:P: Penicillin [A]Oc: Oxacillin [A]S1: Sulfamerazine [N]S2: Sulfamethazine [N]

ACIDIC TEST PROBES


Automated Method DevelopmentAutomated Method DevelopmentSystematic Screening Protocol

� ACQUITY UPLC H-Class Quaternary Solvent manager [QSM] with solvent select valve– Mix up to 4 solvents– Optional solvent select valve enables

an additional 5 solvent lines� ACQUITY UPLC H-Class Column Manager– Flexible modules to select between

2 and 6 columns– Utilize 2,1 x 100 mm, 1,7/1,8 µm

columns� Fast, 5 minute gradient from 5 to 95 % organic at 0,3 ml/min

SSV

2 % H

COOH

2 % N

H 4OH

1 2 3 4 5 6

200 m

MNH

4COO

H, pH

3

200 m

MCH

3COO

NH4,

pH 5

200 m

M NH

4(HCO

3), pH

10

50:5

0 ACN

:H2O

H 2O

ACN

MeO

H

A B C

DA B C

PUMPSAMPLE MANAGER

COLUMN MANAGER DETECTOR


ACQUITY UPLC Column Selection:ACQUITY UPLC Column Selection:Systematic ScreeningSystematic Screening

� CSH C18– Wide pH range for maximum selectivity [pH 1 - 11]– Superior peak shape and efficiency in buffered and low ionic strength mobile phases

� CSH Phenyl-Hexyl– Trifunctionally - bonded C6-Phenyl [pH 1-11]– Complementary selectivity for aromatic species

� CSH Fluoro-Phenyl– Trifunctionally bonded, non-endcapped, pentafluorophenyl[pH 1 – 8]

– Unique selectivity compared to alkyl columns

� HSS T3– Low ligand density, trifunctionally - bonded C18 [pH 2 – 8]– Aqueous - compatible C18 chemistry with long column lifetimes

– Recommended for maximum polar compound retention by RPLC


Systematic Screening ProtocolSystematic Screening Protocol


Case Study:Multiresidue Veterinary Drug Method Development for Milk


Veterinary Drug StructuresVeterinary Drug Structures


Systematic Screening Protocol:Systematic Screening Protocol:Instrument ParametersInstrument Parameters

System ACQUITY UPLC H-Classwith TQD MS

Mobile phase A) water,B) acetonitrile, C) methanol,D) 2,0 % HCOOH @ pH 2,7 or200 mM NH4(HCO3) @ pH 10

Temperature 35 °CInjection 5 µlFlow rate 0,3 ml/minGradient 5 % to 95 % organic in 5 min

Capillary (kV) 3,00Extractor (V) 3,00RF (V) 0,10Source Temperature (°C) 150Desolvation Temperature (°C) 350Cone Gas Flow (l/h) 30Desolvation Gas Flow (l/h) 800Collision Gas Flow ( ml/min) 0,10

ACQUITY UPLC H-Class Conditions: TQD MS Conditions:

Electrospray ionization (ESI)Multiple reaction monitoring (MRM)


pH 10 in Methanol

C: Ciprofloxacin [B]E: Enrofloxacin [B]Er: Erythromycin [B]O: Oxytetracycline [B]T: Tetracycline [B]S1: Sulfamerazine [N]S2: Sulfamethazine [N]P: Penicillin [A]Oc: Oxacillin [A]


pH 10 in Acetonitrile



0,1 % Formic Acid in Methanol0,1 % Formic Acid in MethanolC: Ciprofloxacin [B]E: Enrofloxacin [B]Er: Erythromycin [B]O: Oxytetracycline [B]T: Tetracycline [B]S1: Sulfamerazine [N]S2: Sulfamethazine [N]P: Penicillin [A]Oc: Oxacillin [A]


0,1 % Formic Acid in Acetonitrile0,1 % Formic Acid in Acetonitrile

Final ChoiceBetter resolution and peak symmetry



OptimizationOptimization� Gradient Optimization

– Shallower gradient slope to increase resolution– Start at higher %-organic to elute peaks earlier

� Column Flow Optimization– Shorten the analysis time while balancing resolution

� Column Temperature– Shorten the analysis time while balancing resolution– May improve peak shape


Gradient Optimization:Gradient Optimization:CSH C18, Low pH, ACNCSH C18, Low pH, ACN

Initial Gradient

Final Gradient

Time

(min)

Flow

(mL/min)

%A

H2O

%B

ACN

%D

2% FAInitial 0.3 90 5 55.00 0.3 0 95 55.01 0.4 0 95 56.00 0.4 0 95 56.10 0.4 90 5 59.00 0.3 90 5 510.00 0.3 90 5 5

Time (min)

Flow (mL/min)

%A H2O

%B ACN

%D 2% FA

Initial 0.3 80 15 52.50 0.3 55 40 53.90 0.3 0 95 54.90 0.3 0 95 55.00 0.4 80 15 56.00 0.4 80 15 56.10 0.3 80 15 57.00 0.3 80 15 5



Flow Rate OptimizationFlow Rate Optimization

Flow Rate = 0,4 ml/min

Flow Rate = 0,3 ml/min

Final Choice



Temperature OptimizationTemperature Optimization

Final Choice



Final UPLC ConditionsFinal UPLC Conditions

System ACQUITY UPLC H-Classwith TQD MS

Mobile phase A) water,B) acetonitrile, D) 2,0 % HCOOH @ pH 2,7

Temperature 30 °CInjection 5 µlFlow rate 0,3 ml/min

UPLC H-Class Conditions:

Time (min)

Flow (mL/min)

%A H2O

%B ACN

%D 2% FA

Initial 0.3 80 15 52.50 0.3 55 40 53.90 0.3 0 95 54.90 0.3 0 95 55.00 0.4 80 15 56.00 0.4 80 15 56.10 0.3 80 15 57.00 0.3 80 15 5


Sample PreparationSample Preparation-- Reagent PreparationReagent Preparation• Succinic buffer:5,9 g succinic acid in 1 l water adjusted with NH4OH to pH 5,0

• Extraction solution:37,4 g EDTA in 1 l of succinate buffer pH 5


Sample PretreatmentSample Pretreatment

� Spike standards into 1 ml of milk� Extract 1:

Add 3 ml ACN to milk.Vortex for 1 min. Centrifuge and collect supernatant.Evaporate supernatant to just under 1 ml using nitrogen at 45 °C. (Extracts penicillin/some sulfonamides)

� Extract 2:Add 3 ml of succinate/EDTA buffer at pH 5 to the solid milk pellet. (Extracts tetracycline/remaining sulfonamides) Centrifuge and collect supernatant.

� Combine Extracts 1 and 2 and dilute to 10 ml with water.

Sample Pretreatment


Solid Phase Extraction (SPE) Solid Phase Extraction (SPE) ProtocolProtocol

The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.1 cc 30 mg Cartridge Condition/Equilibrate1 ml MeOH, 1 ml water

Load Samplefrom pretreatment

Wash 0,5 ml 5 % Methanol/water

Elute 0,5 ml 60:40 methanol/

Water 50 mM NH4CH3COO 0,5 ml 0,2 % FA in 60:40 methanol

/water 50 mM NH4CH3COO

Oasis® HLBSPE protocol

&

Add 1 ml of ACN, evaporate to dryness,Reconstituted in 250 µl 20 % methanol/water

Filtered by 2 µm filter


Chromatogram of Milk Sample Chromatogram of Milk Sample Spiked at 100 Spiked at 100 µµg/lg/l



OutlineOutline� Introduction– Approaches Toward Method Development– UPLC Technology– Success Criteria� Controlling Selectivity and Retention– Stationary Phase and Particle Substrate Design– Organic Modifier– Mobile Phase pH� Method Development Strategy– Systematic Screening Protocol� Implementing the Approach– Case Study� Conclusion


Conclusion� The most challenge for food analysis is detect trace level of analytes under complex matrix which gives rise to interference during analysis. Frequently the removal of matrix interference is necessary in order to reach the required detection limits.� Food analysis tends to be multi-residue analysis. Mass spectrometric (MS) determination usually is the choice of detection due to its high sensitivity and selectivity.� The combination of UPLC technology with MS detection is a powerful tool to achieve the strong demand of speed, efficiency and sensitivity for food analysis.� The unique of CSH column chemistry provides a better choice for the food analysis.

Method Development for Trace Analysis in Foods

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