Introduction Introduction

Determination of Multi Determination of Multi- -Residues of Tetracyclines and Their Metabolites in Milk by High Residues of Tetracyclines and Their Metabolites in Milk by High Performance Liquid Chromatography Performance Liquid Chromatography- - Tandem Mass Spectrometry Tandem Mass Spectrometry
Yanyan, Fang Yanyan, Fang
1 1
, Jerry, Zweigenbaum , Jerry, Zweigenbaum
2 2 1 1
Agilent Technologies, Inc. Agilent Technologies, Inc. Shanghai, China Shanghai, China
2 2
Agilent Technologies, Inc. Agilent Technologies, Inc. US US
Antibiotics areusedworldwidetocontrolbacterialinfectionandpromotehealthy farm
animals for milkprodution. Becausetetracyclines arebroadspectrumantibioticsthey
arewidelyused. However,it is veryundesirabletohavetheminthemilksupply.
FDAs regulationsfor tetracyclines includingoxytetracycline,andchlortetracyclineare
set toprovidefor anacceptable dailyintake(ADI)andfor settinga tolerancefor
residues inmilk. TheADI for totalresidues ofthesecompounds is 25micrograms per
kilogramofbodyweight per day. Sixtypercent (60%)oftheADI is reserved for milkand
40% for edibletissues. BasedontheADI,a toleranceof300ppb is set for thesumof
residues ofthetetracyclines includingchlortetracycline,oxytetracycline,and
tetracyclineinmilk. Withtheestablishmentofa toleranceof300ppbfor thesumof
residues oftetracyclines, a toleranceof300ppbfor eachofthethree tetracyclines is
alsoaccepted.
IntheEU,themaximumresiduelimit (MRL) for antibioticsis establishedccordingto
(EEC)2377/90and for thetetracyclines inmilkis at 100g/kg(100ppb). InChina,the
Government Standard(GB/T 213172007) alsoestablishedthemethodfor
determinationofthesecompounds inmilkandanimaltissues andthis regulationtook
effectiveApr. 1,2008.
Sample Preparations Sample Preparations
Instrument Setting Instrument Setting
ResultsandDiscussions ResultsandDiscussions
Structures Structures
Results and Discussions Results and Discussions
Conclusions Conclusions
Agilent 1 Agilent 12 200 00 RRLC RRLC series series
Column: Agilent Zorbax RXC8,2.1150mm, 5 um;
Flow rate : 0.3 mL/min
Temperature: 30
Mobilephase : A0.1% Formic acidwater; B 100% ACN;
Totalrun: 28minand5minfor post run
Gradient: 010minB from5%30%, 1012min,B from3040%,
12.518min,B 65% and185.525minB 95%; 25.5min,B 5%
MS Source settings MS Source settings
Source: ESI
Ionpolarity: Positive
DryingGas flow rate: 10L/min
DryingGas temp. 350
Nebulizer: 45psi
Vcap.: 4000V
Thepurposeofthis studywas todeterminethepresenceoftetracyclines andtheir
metaboliteresidues inmilk. Arapidandeasytousemethodwas developedonthe
Agilent 6410LC/MS/MS.
Collect theeluent anddryunder nitrogenbelow40. Dissolvetheresiduewithmobilephaseto1.0mL
(LC/MS/MS),filtratedwith0.45mfiltermembraneandinjection.
Extraction: Extraction: Weigh5gmilksample(accurateto0.01g)intoa 50mL colorimetric tube,
dissolvedwith0.1mol/L Na2EDTAMcllvainebuffer solutionandbring volumeto50mL.
Vortex for 1minandultrasonic extract inicewater bathfor 10 min,thentransfer to50mL polypropylene
centrifugaltube,cooltobetween 0 and4,centrifugeat a speedof5000rpmfor 10min(<15),
filtratedwithfast filterpaper.
Purification: Purification: AgilentSampliQ OPT cartridges werepreconditionedwith5mL of
methanol,then5mL ofa 10mmol/L TFAsolution.A10mL extract (equivalenttoa 1g
sample)was passedthroughtheSampliQ OPT cartridgeat a speedof 1mL/min. After
thesampleeffusedcompletely,thecartridgewas washedwith3mL ofwater (pH
adjustedto4.0withTFA). Theentireeffluent was discarded.Thecartridgewas dried
under negativepressurebelow2.0 kPa for 3minutes. Finally,thecartridgewas eluted
with8mL of10mmol/L oxalic acid inmethanol. Theeluent was collectedanddried
under nitrogenbelow40C. Theresultingresiduewas dissolvedandmadetoa
constant volumeof1.0mL using mobilephase. Thentheresiduewas filteredthrougha
0.45mfiltermembrane(p/n51855836)andanalyzed
MRMSetting MRMSetting
Name Name Frag. Frag. Precursor Precursor
ion ion
Production Production CE CE Rt. (min) Rt. (min)
minocycline 120 458 352 35 8.58
441 20
4epitetracycline 120 445 410 20 8.60
427 10
4epioxytetracycline 120 461 426 20 9.47
444 15
Tetracycline 120 445 410 20 9.90
427 15
Oxytetracycline 120 461 426 20 9.95
443 10
Demethylclocycline 120 465 430 25 11.25
448 15
4epichlortetracycline 120 479 444 22 11.59
462 15
Chlortetracycline 120 479 444 22 12.95
462 15
Methacycline 120 443 381 25 13.98
426 15
doxycycline 120 445 154 30 14.08
428 15
a. 4 a. 4 epitetracycline epitetracycline
b. Tetracycline b. Tetracycline
Resultsintherealmilksamples Resultsintherealmilksamples
2 x 1 0
0 . 1 5
0 . 2
0 . 2 5
0 . 3
0 . 3 5
0 . 4
0 . 4 5
0 . 5
0 . 5 5
0 . 6
0 . 6 5
0 . 7
0 . 7 5
0 . 8
0 . 8 5
0 . 9
0 . 9 5
1
1 . 0 5
1 . 1
1 . 1 5
1 . 2
1 . 2 5
1 . 3
1 . 3 5
1 . 4
1 . 4 5 + T I C M R M ( * * - > * * ) s t d 5 - r 0 0 2 . d 1 1
C o u n t s v s . A c q u i s i t i o n T i m e ( m i n ) 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7
a. MilkmatrixBlank
4 x 1 0
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
+ T I C M R M ( * * - > * * ) W o r k l i s t D a t a 6 - r 0 0 4 . d 1 1
C o u n t s v s . A c q u i s i t i o n T i m e ( m i n ) 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7
3 x 1 0
0 0 . 2 5 0 . 5 0 . 7 51 1 . 2 5 1 . 5 1 . 7 52 2 . 2 5 2 . 5 2 . 7 53 3 . 2 5 3 . 5 3 . 7 54 4 . 2 5 4 . 5 4 . 7 55 5 . 2 5 5 . 5 5 . 7 56 6 . 2 5 6 . 5 6 . 7 57 7 . 2 5 + M R M ( 4 4 5 . 0 0 0 0 0 - > 4 2 8 . 0 0 0 0 0 ) W o r k l i s t D a t a 6 - r 0 0 4 . d 1 1
C o u n t s v s . A c q u i s i t i o n T i m e ( m i n ) 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8
b. TIC of 50ppblevel inmilk
c. EIC of 50ppblevel inmilk
Linearity,LODandLOQ Linearity,LODandLOQ
Standardsinsolvent* Standardsinsolvent* StandardsinMilkmatrix* StandardsinMilkmatrix*
Name Name R R
2 2
LOQ LOQ
(S/N=20) (S/N=20)
pgon pgon
column column
LOD LOD
(S/N=3) (S/N=3)
pgoncolumn pgoncolumn
R R
2 2
LOD LOD
(S/N=3) (S/N=3)
pgoncolumn pgoncolumn
minocycline 0.999 41.5 6.2 0.990 16.3
4epitetracycline 0.991 10.8 1.6 0.994 8.7
4epioxytetracycline 0.996 14.7 2.2 0.996 12.8
tetracycline 0.998 9.4 1.4 0.994 10.2
oxytetracycline 0.996 10.7 1.6 0.991 8.6
demethylclocycline 0.999 22.8 3.4 0.993 8.1
4epichlortetracycline 0.986 38.2 5.7 0.987 11.9
chlortetracycline 0.986 8.1 1.2 0.994 7.6
methacycline 0.999 20.8 3.1 0.994 12.3
doxycycline 0.999 32.2 4.8 0.995 11.2
Note:*Thecalibrationcurverangeisfrom1ppb1ppmwithinjectionvolume@5uLandthisis
thecalculate results
jiaxit umeisu - 7 Lev els, 7 Lev els Used, 21 Points, 21 Point s Used, 0 QCs
Conc entrat ion(g/ L)
-50 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050
R
esponses5 x10
-0.1
0
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
y =143. 4386*x -442. 6564
R^ 2 =0.99960865
Methacycline R Methacycline R
2 2
=0.9996 =0.9996
6 6 repeatinjectionsoverlapped repeatinjectionsoverlapped
+ M R M (4 4 3 .0 -> 4 2 6 . 0 ) st d 1 1 -r0 0 2 . d
Ac q u isit io n T ime (min )
1 2 1 3 1 4 1 5 1 6
C
ounts4 x1 0
-0 . 1
-0 .0 5
0
0 .0 5
0 . 1
0 .1 5
0 . 2
0 .2 5
0 . 3
0 .3 5
0 . 4
0 .4 5
0 . 5
0 .5 5
0 . 6
0 .6 5
0 . 7
0 .7 5
0 . 8
0 .8 5
0 . 9
0 .9 5
1
1 4 . 0 2 1
+ M R M : 1 0 (7 .8 4 6 -9 .2 7 2 min , 2 2 sc a n s) (4 4 5 . 0 ->
M a ss-to -Ch a rg e (m/ z )
1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0
C
ounts2 x1 0
0
0 . 2 5
0 . 5
0 . 7 5
1
1 . 2 5
1 . 5
1 . 7 5
2
2 . 2 5
2 . 5
2 . 7 5
3
3 . 2 5
3 . 5
3 . 7 5
4
4 . 2 5
4 . 5
4 . 7 5
5
5 . 2 5
5 . 5
5 . 7 5
4 1 0 .0
1 5 4 . 0
4 4 5 . 0
A c q u isit io n T ime (min )
1 2 1 3 1 4 1 5 1 6
R
elativeA
bundance(%
)2 x1 0
-0 . 1
-0 . 0 5
0
0 . 0 5
0 . 1
0 . 1 5
0 . 2
0 . 2 5
0 . 3
0 . 3 5
0 . 4
0 . 4 5
0 . 5
0 . 5 5
0 . 6
0 . 6 5
0 . 7
0 . 7 5
0 . 8
0 . 8 5
0 . 9
0 . 9 5
1
1 . 0 5
1 . 1
1 . 1 5
1 . 2
1 . 2 5
4 4 3 . 0 -> 4 2 6 . 0 , 4 4 3 . 0 -> 3 8 1 .0
R a t io =1 2 . 7
Ratioofthequalifierionandquantizationionautomaticallyca Ratioofthequalifierionandquantizationionautomaticallycalculatedand lculatedand
graphicallyrepresentedbyMASSHUNTERSoftware graphicallyrepresentedbyMASSHUNTERSoftware
Recoveryandrepeatability Recoveryandrepeatability
Theresult wereobtainedin milkmatrix
Name Name Recoveryin Recoveryin
milk milk
(Conc. 50ppb, (Conc. 50ppb,
N=6) N=6)
RSD % RSD %
(Signal (Signal
response response
n=6) n=6)
RSD % RSD %
(Ionratio (Ionratio
n=6) n=6)
Recovery Recovery
inmilk inmilk
(Conc. 100ppb, (Conc. 100ppb,
n=6) n=6)
RSD % RSD %
(Signal (Signal
response response
n=6) n=6)
RSD % RSD %
(Ionratio (Ionratio
n=6) n=6)
minocycline 96.5 4.9 2.1 101.4 1.6 1.0
4epitetracycline 89.2 3.8 1.5 96.3 1.6 0.9
4epioxytetracycline 84.4 5.4 1.3 88.2 0.9 0.6
tetracycline 86.1 2.5 1.2 90.7 1.1 1.2
oxytetracycline 77.6 3.8 1.6 82.5 1.2 0.9
demethylclocycline 79.2 2.0 3.1 84.7 0.9 0.6
4epichlortetracycline 76.4 5.5 5.4 84.3 1.1 0.5
chlortetracycline 94.3 4.5 1.5 100.9 1.8 1.1
methacycline 86.3 1.0 1.9 91.2 1.2 0.8
doxycycline 78.7 3.6 6.7 82.4 1.0 0.8
Tetracyclinescaneasilydegradeunderconditionsofweakacid, Tetracyclinescaneasilydegradeunderconditionsofweakacid,strongacid,strong strongacid,strong
base,heating,andoxidation base,heating,andoxidation
UsingZorbaxRX UsingZorbaxRX C8,2.1 C8,2.1 150mm,5 150mm,5 mcolumncaneasilyseparatethe mcolumncaneasilyseparatethe
isomers isomers
No. No. Name Name CASNo. CASNo. Structure Structure
1 Minocycline 10118908
2 Oxytetracycline 6153646
3 Tetracycline 60548
4 Demeclocycline 127333
5 Chlortetracycline 57625
6 Methacycline 914001
7 Doxycycline 564250
8 4epitetracycline 64755
9 4epioxytetracycline 35259393
10 4epichlortetracycline 14297939
Thisstudyshowsthatrobust,sensitive,andrepeatableresultsareobtainedusing
goodsamplepreparation,goodchromatographyandtheAgilent6410Triple
Quadrupolefortheanalysisoftetracylinedrugresiduesinmilk
TheChinagovernmentStandardrequirement(GB/T213172007)setatanMRLof
50ppbcaneasilybemetwiththismethod
Likeotherveterinarydrugs,tetracyclinesareeasilydegradedinvitroandvivo.
Thismethodshowsasimplewaytodetectandmonitorthedegradedisomerswith
confidenceusingthehighlyselectiveLC/MS/MStechnologymakingtheresultsmore
reliable
Inordertoremovethematrixeffectsonthequantitativeresults,thecalibration
curveisestablishedusingrealantibioticfreemilk
Additionalworkneedstobeconductedtodetermineifionsuppressioneffectsand
bereduced
Futureworkusinginternalstandards(ISTDmethods)shouldbeconductedto
determineitsabilitytoaddressmatrixeffects,accuracy,andprecision
SamePrecursorions
Sameproductions
Therearetotal3pairs isomers inthis analysis
1. Tetracyclineand4Epitetracycline
2. Oxytetracyclineand4epioxytetracycline
3. Chlortetracyclineand4epichlortetracycline
TheLC/MS/MSmethodisoptimizedbaseduponthegood TheLC/MS/MSmethodisoptimizedbaseduponthegood
LCseparation LCseparation
LC/MS/MSresultsprovidesconfirmationandsensitivity LC/MS/MSresultsprovidesconfirmationandsensitivity
EU requirement: tetracyclineinmilkMRL 100ug/kg;
US FDArequirement: tetracyclineinmilkMRL 300ug/kg
China GovStandard: tetracyclineinmilk: 50ug/kg
Acknowledgment Acknowledgment
Andy ZHAI andYunZOU are greatfully acknowledgedforsample p Andy ZHAI andYunZOU are greatfully acknowledgedforsample preparation reparation
Ionsuppressionobservedinmilkmatrixfortetracycline Ionsuppressionobservedinmilkmatrixfortetracycline
0
20000
40000
60000
80000
100000
120000
140000
160000
0 200 400 600
Tetracycline
1
2
Note: Note: Thestandardin solvent is 1 ,and2is thesamecompoundinmilkmatrix
Fromtheabove,itisobviousthattherealmatrixhastheionsuppression
effects.UsingtheESTDmethodforcalibration,matrixmatchedstandards
shouldbepreparedinantibioticfreemilk.