Analysis of Amino Acids in Animal Feed Matrices Using the Ultivo … · Animal feed was obtained...

Application Note

Food Testing & Agriculture

AuthorsJennifer Hitchcock1, Yanan Yang1, Gaëlle Bridon2, Hélène Lachance3, and Mathieu D’Amours3 1 Agilent Technologies,Inc.

Santa Clara, CA, USA2 Agilent Technologies

Saint‑Laurent, Quebec, Canada

3 Trouw Nutrition, Saint‑Hyacinthe, Quebec, Canada

AbstractThisApplicationNotedemonstratesafastandsimpleanalyticalmethoddevelopedforthequantitationofunderivatizedaminoacidsusinganAgilentUltivotriplequadrupoleLC/MSsystem.

Ultivowasdesignedtoaddressmanychallengesfacedbylaboratories,andthisstudywasconductedtoassesshowthisnovelLC/MScouldperformwithtypicalendogenousanalytesofinterest.InnovativetechnologieswithinUltivoallowedforareducedphysicalfootprintwhilegeneratingananalyticalperformancesimilartothatofphysicallylargerMSsystems.

ThisstudyoutlinesatypicalconfirmationperformanceoffreeaminoacidsusingtheUltivoLC/MSincombinationwithHILICchromatography.Lowerlimitsofquantitation(LLOQ),chromatographicprecision,lineardynamicrange,andaccuracyarediscussed.

AnalysisofAminoAcidsinAnimalFeedMatricesUsingtheUltivoTripleQuadrupoleLC/MSSystem

Figure 1. UltivointegratedintoLCstack.

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The flasks were placed under nitrogen flow for 23 hours, thenremovedandallowedtocooltoroomtemperature.Thesamplesweretransferredto50‑mLvolumetricflasksthathadbeenrinsedwith0.1NHClpriortosampleintroduction.Theflaskswerefilledtothelinewith0.1NHCl,andmixed.AliquotsweretransferredtosamplevialsforintroductionintotheLC/TQsystem.

Data analysisSystemcontrolanddataacquisitionwereperformedbyAgilent MassHunterAcquisitionSoftware(C.01.00).Todetermineoptimalprecursorandproductions,fragmentorvoltages,andcollisionenergiesuponinjectionofaneatsolutionofthecompounds,MRMtransitionswereobtainedusingMassHunterAcquisitionOptimizersoftware.DatawereanalyzedusingAgilent MassHunterQuantitativeAnalysisSoftware(B.09.00)andQualitativeAnalysisSoftware(B.08.00).

InstrumentationAgilent 1260InfinityIIBio‑InertHPLC

• 1260InfinityIIBio‑Inertpump(G5654A)

• 1260InfinityIIBiomultisampler(G5668A)

• 1260InfinityIImulticolumnthermostat(G7116A)

Agilent UltivotriplequadrupoleLC/MSsystem

• Agilent JetStreamelectrosprayionizationsource

MethodTable1summarizesthe1260Infinity IIBio‑InertHPLCconditions.Table2summarizestheUltivotriplequadrupoleparametersandAgilentJetStreamESIsourceparameters.Analysiswascarriedoutwithpositiveionizationanddynamicmultiplereactionmonitoring(dMRM).DatawereevaluatedusingMassHunterQuantitativeAnalysisSoftwareB.09withtheQuant‑My‑Waytool.

Results and discussion

ChromatographyWiththePoroshellHILIC‑Zcolumnandinertchromatographysystem,the17 aminoacidswerewellseparatedwithinaseven‑minutewindow(Figures 2and3).Abaselineseparationof0.3 minuteswasalsoachievedforleucineandisoleucine(Figure4),andtheretentiontimesdemonstratedexcellentstability,with %RSDsoflessthan0.5%foreachcompound.

IntroductionAminoacidsarethebuildingblocksofproteins,andarenecessarycomponentsofabalanceddiet.Forpetsandfarmanimals,thedietmustsupplytheessentialaminoacids,which includes cysteine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine,threonine,tryptophan,tyrosine,andvaline.Toensurethequalityoftheirfoodinprovidingabalanceddiet,petfoodandanimalfeednutritioncompaniesmonitortheirproductsforthepresenceoftheseaminoacids,alongwiththeremainingnonessentialones.

Historically,aminoacidshavebeenanalyzedusingalabor‑intensivehydrolysisandderivatizationforsamplepreparation,whichcanlimitthespeedofanalysis.However,ithasbeenfoundthatunderivatizedaminoacidscanbeanalyzedwithexcellentsensitivityandaccuracyusinghydrophilicinteractionchromatography(HILIC)withlowpHsolventsandpositiveionmodeMSdetection.

TheUltivoisdesignedtoaddressmanyofthechallengesfacedbylabsperformingroutineanalyses.InnovativetechnologieshousedwithinUltivocreatedareducedoverallfootprintwhileconservingtheperformancefoundintraditionalsystems(Figure 1).InnovationssuchastheCycloneIonGuide,VortexCollisionCell,andtheHyperbolicQuadsmaximizequantitativeperformanceinasmallpackage,enhancinginstrumentreliability,robustness,anduptime.

Inthisapplication,wedemonstratethesensitiveandprecisequantitationof17 underivatizedaminoacidsinanimalfeedusingthenovelUltivotriplequadrupoleLC/MS(Figure1).

Experimental

Reagents and chemicalsAllreagentsusedwereHPLCorLC/MSgrade.AcetonitrilewaspurchasedfromHoneywell(Morristown,NJ,USA),andultrapurewaterwassourcedfromaMilli‑QIntegralsystemwithanLC‑PakPolisheranda0.22‑µmpoint‑of‑usemembranefiltercartridge(EMDMillipore,Billerica,MA,USA).FormicacidandammoniumacetatewerepurchasedfromFluka(Sigma‑AldrichCorp.,St. Louis,MO,USA).ChemicalstandardswerepurchasedfromPierce(ThermoScientfic,Waltham,MA,USA).

Sample preparationStandardswerepreparedthroughserialdilutionintowater.Animalfeedwasobtainedfromlocalsuppliers,andgroundup.Asampleof0.1gwasweighedintoa250‑mLErlenmeyerflask,andhydrolyzedusing25mLof6NHClwithphenol.

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Table 1. 1260InfinityIIBio‑InertHPLCparameters.

Parameter Value

Column Agilent Poroshell 120 HILIC-Z, 2.1 × 150 mm, 2.7 µm (p/n 6x3775-924)

Column temperature

25 °C

Injection volume 2 µL

Mobile phase A) 20 mM ammonium acetate + 0.1 % formic acid in water, pH 3B) 20 mM ammonium acetate in 90 % acetonitrile, pH 3

Flow rate 0.50 mL/min

Gradient

Time %B 0 100 11.5 70 12.0 100 15.0 100 stop time 19.0 100 post time

Table 2. AJSsourceandUltivotriplequadrupoleparameters.

Parameter Value

Drying gas temperature 150 °C

Drying gas flow 15 L/min

Sheath gas temperature 400 °C

Sheath gas flow 12 L/min

Nebulizer pressure 20 psi

Capillary voltage 2,000 V(+)

Nozzle voltage 0 V(+)

Cycle time 500 ms

Table 3. TransitionsforaminoaciddetectionindMRMmode.

CompoundPrecursor

(m/z)Product

(m/z)RT

(min)

RT Window

(min)Frag. (V)

CE (V) Polarity

Alanine 90.1 62.1 6.83 2 75 0 Positive

Alanine 90.1 44.1 6.83 2 75 0 Positive

Arginine 175.1 116.1 10.43 2 105 2 Positive



Aspartic acid 134.1 88.0 9.03 2 75 0 Positive



Cystine 241.0 241.0 11.16 2 105 0 Positive




Glutamic acid 148.1 148.1 8.27 2 85 0 Positive




Glycine 76.0 48.0 7.36 2 65 0 Positive

Glycine 76.0 30.0 7.36 2 65 0 Positive

Histidine 156.1 110.1 9.81 2 95 4 Positive

Histidine 156.1 95.1 9.81 2 95 6 Positive

Isoleucine 132.1 86.1 4.90 2 85 0 Positive




Leucine 132.1 86.1 4.62 2 85 0 Positive

CompoundPrecursor

(m/z)Product

(m/z)RT

(min)

RT Window

(min)Frag. (V)

CE (V) Polarity




Lysine 147.1 130.1 11.12 2 85 0 Positive

Lysine 147.1 84.1 11.12 2 85 6 Positive

Methionine 150.1 104.1 5.16 2 75 0 Positive




Phenylalanine 166.1 120.1 4.23 2 85 4 Positive




Proline 116.1 70.1 6.01 2 85 6 Positive

Proline 116.1 43.1 6.01 2 85 25 Positive

Serine 103.1 88.1 7.63 2 65 0 Positive

Serine 103.1 60.1 7.63 2 65 0 Positive

Threonine 120.0 74.1 6.98 2 75 0 Positive

Threonine 120.0 56.1 6.98 2 75 6 Positive

Tyrosine 182.1 136.1 5.53 2 95 0 Positive




Valine 118.1 72.1 5.84 2 75 0 Positive

Valine 118.1 55.1 5.84 2 75 14 Positive

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Figure 2. CompositedMRMchromatogramofaminoacids,10ppbspike.

3.40

×101

Acquisition time (min)

PheLeu

Ile

Met

Tyr

Val

Ala

Thr

GlySer Glu

Asp

HisArg

Cys

Lys

Pro

Co

un

ts (

%)

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

4.4

4.8

5.2

5.6

6.0

6.4

6.8

7.2

7.6

8.0

8.4

8.8

9.2

9.6

3.8 4.2 4.6 5.0 5.4 5.8 6.2 6.6 7.0 7.4 7.8 8.2 8.6 9.0 9.4 9.8 10.2 10.6 11.0 11.4 11.8

Figure 3. CompositedMRMchromatogramofaminoacidsinananimalfeedsample.

3.40

×105


Phe

Leu

Ile

Met

Tyr

Val

Ala

Thr

Gly Ser

Glu

Asp

His

Arg

Lys

Pro

Co

un

ts (

%)

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

4.4

4.8

5.2

5.6

6.0

6.4

6.8

7.2

7.6

8.0

8.4

8.8

9.2

9.6

10.0

3.8 4.2 4.6 5.0 5.4 5.8 6.2 6.6 7.0 7.4 7.8 8.2 8.6 9.0 9.4 9.8 10.2 10.6 11.0 11.4 11.8

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Figure 4. Separationofisobarsleucineandisoleucine.

4.4

0

×106


Leucine

Isoleucine

Co

un

ts

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3

Linearity, accuracy, and reproducibilityThecalibrationconcentrationsrangedfrom1.12to18,844 ppbforthevariousanalytes.Table4givesthelimitsofquantitation(LOQs),andcurvefitparameters.EachcurvehadanR2valuegreaterthan0.992,andresponsesshowedexcellentreproducibilityfromruntorun.Calibrationcurveaccuracieswerewithin11.5%oftheexpectedconcentrationatthelowestlevel,anddemonstratedRSDswithin20 %attheLOQs,andwithin5 %atthehigherlevels.Figure5showsexamplesofcalibrationcurvesforsixselectedcompounds,whilesixreplicateinjectionsofthreeselectedcompoundsinmatrixareshowninFigure6,demonstratingexcellentprecision.

Table 4. Calibrationcurvefit,LOQs,andS/N.

Compound Curve fit R2 LOQ (ppb) S/N at LOQ

Alanine Linear 0.9997 46.33 4.35

Arginine Quadratic 0.9920 8.71 10.75

Aspartic acid Linear 0.9971 13.31 5.76

Cystine Linear 0.9969 61.04 13.51

Glutamic acid Quadratic 0.9998 7.65 5.37

Glycine Linear 0.9986 75.07 3.51

Histidine Quadratic 0.9998 7.76 15.92

Isoleucine Linear 0.9961 1.36 5.88

Leucine Linear 0.9978 1.36 4.03

Lysine Quadratic 0.9934 7.60 18.94

Methionine Linear 0.9977 1.55 14.57

Phenylalanine Linear 0.9974 1.72 4.82

Proline Linear 0.9954 1.15 6.03

Serine Quadratic 0.9987 10.51 5.43

Threonine Linear 0.9995 11.91 4.35

Tyrosine Linear 0.9943 1.88 3.86

Valine Linear 0.9972 1.12 6.19

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Figure 5. Calibrationcurvesforselectedcompounds.

0

0

×105

Concentration (ppb)

Phenylalanine1.72–17,180 ppbR2 = 0.997

Re

sp

on

se

0.40.81.21.62.02.42.83.23.64.0

3,000 6,000 9,000 12,000 15,000 17,000

0

0

×105

Concentration (ppb)

Valine1.12–11,246 ppbR2 = 0.997

Re

sp

on

se

0.20.40.60.81.01.21.41.6

3,000 6,000 9,000 0

0

×105

Concentration (ppb)

Proline1.15–5,757 ppbR2 = 0.995

Re

sp

on

se

0.4

0.8

1.2

1.6

1,000 2,000 3,000 4,000 5,000

0

0

×105

Concentration (ppb)

Methionine1.55–15,518 ppbR2 = 0.998

Re

sp

on

se

1

2

3

4

5

6

7

3,000 6,000 9,000 12,000 15,0000

0

×105

Concentration (ppb)

Leucine1.36–13,642 ppbR2 = 0.998

Re

sp

on

se

1

2

3

4

5

3,000 6,000 9,000 12,000

3,000 6,000 12,000 15,0009,0000

0

×105

Concentration (ppb)

Histidine1.76–15,515 ppbR2 = 0.999

Re

sp

on

se

0.3

0.6

0.9

1.2

1.5

1.8

Figure 6. Excellentprecisiondemonstratedforsixreplicateinjectionsofthreeaminoacidsinsamplematrix.

4.90

×104


Methionine

RSD% = 1.04Avg. S/N = 10,819

Co

un

ts

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

4.4

4.8

5.2

5.6

6.0

6.4

6.8

7.2

7.6

8.0

8.4

8.8

5.0 5.1 5.2 5.3 5.4 5.5 5.6 3.90

×105


Phenylalanine

RSD% = 1.06Avg. S/N = 35,506

Co

un

ts

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

4.4

4.8

5.2

5.6

6.0

6.4

4.0 4.1 4.2 4.3 4.4 4.5 4.6 10.90

×105


Lysine

RSD% = 0.42Avg. S/N = 48,697

Co

un

ts

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3.0

3.3

3.6

3.9

4.2

4.5

4.8

5.1

5.4

11.0 11.1 11.2 11.3 11.4 11.5 11.6

7

ConclusionsUltivoisaninnovativetriplequadrupolemassspectrometerthatcanminimizelaboratoryworkspacerequirementsandreducemaintenancechallenges.TheLC/MSsystemdemonstratedtheaccurateandsensitivedetectionofcommonlymonitoredaminoacidsinananimalfeedmatrix,whiletheuseofthePoroshell120HILIC‑Zcolumnenabledanalytestoremainunderivatized,resultinginasimplifiedworkflow.

www.agilent.com/chem

This information is subject to change without notice.

© Agilent Technologies, Inc. 2019 Printed in the USA, January 8, 2019 5994-0586EN

References1. MethodsfortheAnalysisofUnderivatizedAmino

Acids by LC/MS, Agilent Technologies Application Note, publicationnumber5991‑8582EN.

2. QuantitativeAnalysisofUnderivatizedAminoAcidsinPlantMatrixbyHydrophilicInteractionChromatography(HILIC)withLC/MSDetection,Agilent Technologies Application Note,publicationnumber5991‑8922EN.

3. AnalysisofUnderivatizedAminoAcidsbyLC/MSforBioreactor Cell Culture Monitoring, Agilent Technologies Application Note,publicationnumber5991‑8816EN.

4. Otter,D.E.Standardisedmethodsforaminoacidanalysisoffood.British Journal of Nutrition 2012, 108,S230‑S237.

5. Wu,G.Dietaryrequirementsofsynthesizableaminoacidsbyanimals:aparadigmshiftinproteinnutrition.Journal of Animal Science and Biotechnology 2014, 5,34‑45.

Analysis of Amino Acids in Animal Feed Matrices Using the Ultivo … · Animal feed was obtained...

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