is Best Method for Analysis of Drug Residues in Animal ... · kanamycin, 4 gentamicins, neomycin,...

What is the Current Best Method forMulticlass, Multiresidue Analysis of

Veterinary Drug Residues in Animal Tissues?

Steven J. Lehotay, Alan R. Lightfield, Lucía Geis‐Asteggiante, and Marilyn J. Schneider

Violation Rate on Inspector‐GeneratedBovine Slaughter Classes

1498 violations

See OIG Report: www.usda.gov/oig/webdocs/24601-08-KC.pdf

0.00%

0.20%

0.40%

0.60%

0.80%

1.00%

1.20%

1.40%

1.60%

1.80%

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

%Vi

olat

ions

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000

400,000beta-Lactams AminoglycosidesSulfas TetracyclinesMacrolides FlunixinTotal No. Samples

Veterinary Drug Residue Analysis by FSIS

Slide modified from James Withee, FSIS

≈6,300SlaughterHouses

InspectorSamplesn ≈ 150,000/yr≈0.5% of cows

KISTM

orFAST

3 – 6 hrs

–FSIS Lab

St. Louis, MO

+

7‐PlateBioassay

24 – 48 hrs

Shipment24 – 48 hrs

–

> 7 days

–

Analytical Methods*

+

*Confirmation or Determination by Class of Drugs+

QuantitativeBioassay

24 hrs < Tol.

> Tol.

FDA‐CVMEnforcement

Proposed FSIS Residue Monitoring Scheme

≈6,300SlaughterHouses

InspectorSamplesn > 150,000/yr>0.5% of cows

KISTM

orPremi ?

3 hrs

–FSIS LabsSt. Louis, MOAthens, GAAlameda, CA

+

UHPLC‐MS/MS*24 hrs

Shipment24 – 48 hrs

– *Qualitative Screening,Identification, and Semi‐Quantification

QuantitativeBioassay

24 hrs

+

Analytical Confirmation and

Quantification

< 7 days

Bridging

Studies? years

> Tol.

FDA‐CVMEnforcement

4444(+13)(+13) Antibiotics in Initial MMMAntibiotics in Initial MMM(+AMGs)(+AMGs)

Tetracyclines Tetracyclines (3)(3) tetracycline, oxytetracycline, chlortetracycline

AminoglycosidesAminoglycosides (13)(13) spectinomycin, streptomycin, dihydrostreptomycin, amikacin, kanamycin, 4 gentamicins, neomycin, apramycin, hygromycin, tobramycin

MacrolidesMacrolides (8)(8) erythromycin, tilmicosin, lincomycin, tylosin, clindamycin, pirlimycin, tulathromycin, gamithromycin

ββ‐‐LactamsLactams (10)(10) desacetyl cephapirin, amoxicillin, DCCD (cefitofur metabolite), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, nafcillin, dicloxacillin

SulfonamidesSulfonamides (16)(16) sulfamethazine, sulfathiazole, sulfachloropyridazine, sulfadoxine, sulfadimethoxine, sulfapyridine, sulfadiazine, sulfamerazine, sulfaquinoxaline, sulfaethoxypyridazine, sulfanilamide, sulfamethoxazole, sulfamethizole, sulfabromomethazine, sulfanitran, sulfamethoxypyridazine

FluoroquinolonesFluoroquinolones (7)(7) ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, desethylene ciprofloxacin, norfloxacin

1818 Other Drugs Added to the Target ListOther Drugs Added to the Target List

OtherOther (4)(4) melengestrol acetate, zeranol, carbadox, 2‐quinoxaline carboxylic acid (marker residue for carbadox)

PhenicolsPhenicols (3)(3) chloramphenicol, florfenicol, florfenicol amine

ββ‐‐agonistsagonists (5)(5) cimaterol, clenbuterol, ractopamine, salbutamol, zilpaterol

NSAIDsNSAIDs (6)(6) β+dexamethasone, flunixin, oxyphenylbutazone, phenylbutazone, prednisone

Int StdsInt Stds / / QCQC (6)(6) sulfamethazine‐13C6, flunixin‐d3, penicillin G‐d7atrazine, methomyl, imidacloprid

62(+13)62(+13) Drugs in Initial Method(s) Drugs in Initial Method(s) +6+6 ISIS//QCQC

Developed, Validated, and TransferredDeveloped, Validated, and TransferredMethod for Method for Aminoglycosides (AMGs)Aminoglycosides (AMGs)in Bovine Kidney, Liver, and Musclein Bovine Kidney, Liver, and Muscle

Mix a 2.0 g sample with 10 mM NH4OAc / 0.4% EDTA / 1% NaCl / 2% TCA buffer in stomacher.Centrifuge and decant supernatant.Adjust pH to 6.5 with NaOH and/or HCl solutions.Sample clean‐up / analyte recovery performed using disposable pipette extraction (loose 50 mg WCX sorbent in 5 mL tip) on manifold extractor apparatus.Elute the analytes using 1.0 mL of 10% formic acid in H2O

4‐fold higher sample throughput thanprevious USDA‐FSIS method

UHPLC‐MS/MS Chromatographic Profile of 13 Aminoglycosides

2.5 min

>10‐fold faster than previous USDA‐FSIS method

HPLC‐MS/MS ChromatographicProfile of Veterinary Drugs in Tissues

20 min

UHPLC‐MS/MS Chromatographic Profile of Veterinary Drugs in Tissues

Mobile Phase: A – 95% water / 5% MeCN / 0.1% formic acidB – 100% MeCN / 0.1% formic acid

7.5min

GoalGoal:: Identify in Kidney at Identify in Kidney at ½½ ““ToleranceTolerance”” LevelsLevels½ “Tol.” (ng/g) Analytes No.

3 ββ‐‐Agonists, zeranol, chloramphenicolAgonists, zeranol, chloramphenicol 7

5 amoxacillin, ampicillin, cloxacillin 3

10 melengestrol acetate 1

12.5 flunixin 1

15 carbadox, 2‐quinoxaline carboxylic acid 2

25 Fluoroquinolones, penicillin G 8

50 Sulfonamides, remaining NSAIDs, Macrolides, ββ‐‐LactamsLactams 31

60 tilmicosin 1

100 florfenicol, tylosin 2

150 florfenicol amine 1

250 pirlimicin 1

500 oxytetracycline, tetracycline 2

1000 chlortetracycline, tulathromycin 2

400 Fixed IS/QCs 6

1)Mol et al. (Rikilt – The Netherlands)2)Martos et al. (U. Guelph – ON, Canada)3)Mastovska et al. (USDA‐ARS – Wyndmoor, PA)4) Leepipatpiboon et al. (Chulalongkorn U., Thailand)5) Stubbings et al. (FERA – York, UK)6) Kaufmann et al. (Switzerland) – Too Tedious!

Comparison of 6 Vet. Drug MMMs

All methods gave similar qualitative MS/MS screening capabilities with nearly all of the drug analytes meeting identification criteria at ½ “tolerance” level in kidney.

Speed, cost, ease of use and ruggedness become the differentiating aspects.

Comparison of 5 Vet. Drug MMMs½x, 1x, and 2x “Tolerance” Levels (n= 6 each) in Kidney, No I.S.

0%

20%

40%

60%

80%

100%

120%

Avg. R

ecoveries

Mol Martos Mastovska Leepipatpiboon Stubbings6

β‐Lactam

s7 Fluoro‐

Quinolones

8 Growth

Prom

oters

7Macrolides

14Sulfonamides

3Tetracyclines

Evaluation of Incurred Samples

• FSIS provided 10 kidneys found in their monitoringprogram to contain drug residues.

•We analyzed the samples in blind fashion(unknown drugs and unknown levels).

• These were each analyzed in duplicate using thedifferent features of Mol, Martos, and Mastovska (et al.) 3 MMMs to compare and assess their performances on real samples.

Analysis of Incurred Kidney (2 g)

Pirlimycin in Incurred Kidney (no IS)

0

50

100

150

200

250

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)

5 min shake; w/o acid5 min shake; w/ acid30 min shake; w/o acid30 min shake w/ acid60 min heat; w/o acid60 min heat; w/ acid


Sulfamethazine in Incurred Kidney (no IS)

020406080

100120140160180

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)


Beta/Dexamethasone in Incurred Kidney (no IS)

050

100150200250300350400450

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)


Beta/Dexamethasone was missed by FSIS



Flunixin in Incurred Kidney (no IS)

0

100

200

300

400

500

600

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)


Flunixin vs. Flunixin-d3 IS in Incurred Kidney

0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)

5 min shake; w/o acid

5 min shake; w/ acid


30 min shake w/ acid

60 min heat; w/o acid

60 min heat; w/ acid


Penicillin G vs. PenG-d7 IS in Incurred Kidney

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)

5 min shake; w/o acid 5 min shake; w/ acid30 min shake; w/o acid 30 min shake w/ acid60 min heat; w/o acid 60 min heat; w/ acid

Spikes were ok, but incurredpenicillins can’t tolerate acids

No need for long shake nor heat,so fast and cool is fine!


““Goldilocks?Goldilocks?”” MMM for Vet. DrugsMMM for Vet. Drugs

extraction

clean‐up

add 10 mL of 4/1 (v/v) MeCN/watervortex briefly, shake for 5 mincentrifuge for 5 min >3500 rcf

add IS mix (SMZ‐IS; flunixin‐d3; PenG‐d7)

2 g tissue in a 50 mL tube

supernatant + 500 mg C18 + 10 mL hexane sat’d w/MeCN; mix for 30 s, centrifuge for

5 min > 3500 rcf; aspirate hexane to waste

evaporate 5 mL extract to 1 mL final vol.

filter extract with the Mini‐UniPrepTM

UHPLC‐MS/MS analysis

Modified from: K. Mastovska, A.R. Lightfield, J. Chromatogr. A 1202 (2008) 118‐123.

Method LogisticsMethod Logistics

1 chemist was able to process 60 pre1 chemist was able to process 60 pre‐‐homogenized homogenized samples in an 8samples in an 8‐‐hr day for an overnight sequencehr day for an overnight sequence

(longest step was 1 hr to evaporate MeCN)(longest step was 1 hr to evaporate MeCN)

No glassware to be cleaned afterwardsNo glassware to be cleaned afterwards

Cost of materials Cost of materials ≈≈ $3/sample (using bulk C18)$3/sample (using bulk C18)

Waste = 10 mL hexane and 5 mL MeCNWaste = 10 mL hexane and 5 mL MeCN(and two 50 mL and one 15 mL PP tubes)(and two 50 mL and one 15 mL PP tubes)

Streamlined Method ValidationNeedsNeeds::•• Trueness (Recoveries at Trueness (Recoveries at ≥≥3 Levels, n=10)3 Levels, n=10)•• Precision (Repeatability & Reproducibility)Precision (Repeatability & Reproducibility)•• Ruggedness (MultiRuggedness (Multi‐‐day, Multiday, Multi‐‐Analyst, etc.)Analyst, etc.)•• Selectivity (Interferences in Blanks?)Selectivity (Interferences in Blanks?)•• Range (Calibration and Matrix Effects)Range (Calibration and Matrix Effects)•• Detection Limits (LOI < Detection Limits (LOI < ½½ ““Tol.Tol.””?)?)•• Qualitative (False Negatives/Positives)Qualitative (False Negatives/Positives)

Can We Meet All Needs in 3Can We Meet All Needs in 3‐‐5 Days?5 Days?

55‐‐Day Validation ExperimentDay Validation ExperimentDay 1Day 1::

•• Analyst 1 in hot lab, Reagents A, 10 matrix blanks Analyst 1 in hot lab, Reagents A, 10 matrix blanks from different sources, 6 spikes at 3 levels each in from different sources, 6 spikes at 3 levels each in 6 matrices + 4 spikes each at same levels in mixed 6 matrices + 4 spikes each at same levels in mixed matrices (1 in glass tubes); 5matrices (1 in glass tubes); 5‐‐point calibration point calibration each in mixed matrix and reagenteach in mixed matrix and reagent‐‐only stds; only stds; reagent blk = 0reagent blk = 0‐‐Std injStd inj’’d after high std to check for d after high std to check for carrycarry‐‐over over

Days 2 & 3 (repeated on Days 4 & 5)Days 2 & 3 (repeated on Days 4 & 5)::

•• Analysts 2 & 3 in cooler labs repeat using Analysts 2 & 3 in cooler labs repeat using Reagents B & C with different sources of matricesReagents B & C with different sources of matrices

QuantitativeQuantitative Method ValidationMethod Validation

A) Trueness: Recoveries of Spiked Samples

B) Precision: Repeatability and Reproducibility

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐The MMM cannot be used for enforcement actions if

there is a tolerance value unless the method is bridged to the FDA‐approved method (or it becomes an AOAC Official Method). The method may be

determinative if there is no tolerance value for the analyte, or an enforcement action is not being taken.

Recoveries of Tylosin (100Recoveries of Tylosin (100‐‐400 ng/g)400 ng/g)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Day 1 Day 2 Day 3 Day 4 Day 5 Average

Rec

over

y

Stev

e

Luci

a

Ala

n

Mar

ilyn

Chr

istin

e

n = 30 each dayn = 30 each day(10 reps x 3 levels)(10 reps x 3 levels)

n =

150

Avg. Repeatability = 12% RSD

Reproducibility = 21% RSD

Average Recoveries of the 62 DrugsAverage Recoveries of the 62 Drugs

0

10

20

30

40

50

60

70‐100 50‐69 <50

%Recovery

No. of D

rug Ana

lytes

MatrixMatrix‐‐matched stdsmatched stdsw/o internal stdsw/o internal stdsn = 150 per drug n = 150 per drug

(10 reps x 3 levels x 5 days)(10 reps x 3 levels x 5 days)20 different kidneys20 different kidneys

Precision in the Analysis of the 62 DrugsPrecision in the Analysis of the 62 Drugs

Using matrixUsing matrix‐‐matched stds w/o normalization to internal stdsmatched stds w/o normalization to internal stds

0

10

20

30

40

50

60

≤15 16‐20 21‐25 >25%RSD

No. of D

rug Ana

lytes Reproducibility Repeatability

n = 150 per drug n = 150 per drug (10 reps x 3 levels x 5 days)(10 reps x 3 levels x 5 days)

20 different kidneys20 different kidneys

QualitativeQualitative Method ValidationMethod Validation

A) Screening: Capability to replace the 7‐plate bioassay in FSIS

B) Identification: Capability to replace the single class LC/MS‐MS “confirmation” methods

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐Then, the NADA (FDA‐approved enforcement method)

is used for both quantitative determination and qualitative confirmation of drug violations.

ScreeningScreening Criteria and ResultsCriteria and Results

USDA‐FSIS Criteria:Presence of at least one fragment ionSignal/Noise > 3Retention time match within ±5%

54/62 analytes met screening criteria at ½ “Tol.” levelChloramphenicol met screening criteria at 2x “Tol.” level

8 drugs to be addressed in future work:desacetyl cephapirin, florfenicol amine, clenbuterol,phenylbutazone, oxyphenylbutazone, tulathromycin,cimaterol, amoxacillin

1. Retention time (tR) is within ±4 SD of average tRand peak shape matches that of reference std

2. tR and peak shape of qual. ion(s) matches those of the quantification ion

3. 2 qual. ions ≤|20%| or 1 qual. Ion ≤|10%| of avg ion ratio from contemporaneous reference stds

4. Absence of positive findings in blanks

5. Signal > ½ “Tol.” calibration stds in matrix

Our LC‐MS/MS Identification Criteria

Ret. Time Consistency for IdentificationRet. Time Consistency for Identification

0

2

4

6

8

10

12

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Avg tR (min)

4 x

Std

Dev

(s)

Combined, n=250Solvent Stds, n=50Matrix Stds, n=50

Desacetyl cephapirin and Florfenicol amine need sorted out

Tulathromycin A ?

Clenbuterol

Ion Ratios in Spks, n=50 each

0%

20%

40%

60%

80%

100%

120%

5 10 20Spk Conc. (ng/g)

Ion

Rat

io

1/2x Spk (2/1 ions) 1x Spk (3/1 ions) 2x Spk (3/2 ions)

Ion Ratios for Ampicillin in MatrixIon Ratios for Ampicillin in Matrix

Ion ratio variability decreases vs. Ion ratio variability decreases vs. ↑↑ conc.conc.

1 false neg @ 5 ng/g (2%); no false positives, n=501 false neg @ 5 ng/g (2%); no false positives, n=50

(10 x 5 days)

Ion Ratio Criteria in 2002/657/EC (EU)Ion Ratio Criteria in 2002/657/EC (EU)

Rel. Abundance Rel. Abundance Acceptable Diff. vs. Ref.Acceptable Diff. vs. Ref.vsvs. Base Peak. Base Peak APIAPI‐‐MSMS

>50%>50% ±±20% RSD20% RSD>20>20‐‐50% 50% ±±25% RSD25% RSD>10>10‐‐20%20% ±±30% RSD 30% RSD ≤≤110%0% ±±50% RSD50% RSD

Ref. RatioRef. Ratio EU RangeEU Range** FSIS (1 ion)FSIS (1 ion) (2 ions)(2 ions)70%70% 56% – 84% 60% – 80% 50% – 90%24% 18% – 30% 14% – 34% 4% – 44%12% 8.4% – 15.6% 3% – 23% >0% – 33%4% 2% – 6% >0% – 14% >0% – 24%

* 2 ion transitions needed to achieve 3 ident. points in MS/MS

Ion Ratios of Spks, n=30 each

0%

20%

40%

60%

80%

100%

120%

140%

160%

Ions 2/1 Ions 3/1 Ions 3/2

Ion

Rat

io

Day 1 Day 2 Day 3 Day 4 Day 5

Ion Ratios for Ciprofloxacin in KidneyIon Ratios for Ciprofloxacin in Kidney

Conc.,

ng/g EU FSIS

25 (n=50) 54% 2%

50 (n=50) 40% 0%

100 (n=50) 26% 0%

False Negatives

No False Positives, n=50 (EU or FSIS)

(10 x 3 levels)

Ion Ratios for Lincomycin in KidneyIon Ratios for Lincomycin in KidneyIon Ratios of Spks, n=30 each

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%


Ion

Rat

io


Conc.,

ng/g EU FSIS

25 (n=50) 26% 0%

50 (n=50) 8% 0%

100 (n=50) 2% 0%

False Negatives

No False Positives, n=50 (EU or FSIS)

(10 x 3 levels)

Ion Ratios in Stds, n=10 each

0%

100%

200%

300%

400%

500%

1.5 3.0 6.0 12 18Conc. (ng/g)

Ion

Rat

ioSolvent 2/1 Matrix 2/1Solvent 3/1 Matrix 3/1Solvent 3/2 Matrix 3/2

Ion Ratios for CimaterolIon Ratios for Cimaterol

Quant. ion gave 78Quant. ion gave 78±±14 14 %recoveries, n=150%recoveries, n=150

Kidney matrix interferencesKidney matrix interferences<20 ng/g for ions 2 & 3<20 ng/g for ions 2 & 3

1 false positive in1 false positive in50 matrix blanks50 matrix blanks

Summary of Identification ResultsSummary of Identification Results

41/61 drug analytes met ident. criteria at all levels

3 analytes met ident. criteria at the 1x “Tol.” level:desethylene ciprofloxacin, cefazolin, florfenicol

5 analytes met ident. criteria at the 2x “Tol.” level:sulfaniliamide, sarafloxacin, sulfanitran, cloxacillin, melengesterol acetate

12 drugs did not meet ident. criteria at the spk levels

An UHPLC‐MS/MS instrument upgrade would lowerlimits of identification through dilution of extracts.

Veterinary Drug Residues ConclusionsVeterinary Drug Residues Conclusions

•• The streamlined method has met validation The streamlined method has met validation criteria for criteria for ≥≥49/61 drugs in a 549/61 drugs in a 5‐‐day validation for day validation for qualitative identification screening purposes.qualitative identification screening purposes.

•• Sample throughput is 60 samples/day by 1 chemist Sample throughput is 60 samples/day by 1 chemist for UHPLCfor UHPLC‐‐MS/MS analysis.MS/MS analysis.

•• The method is being implemented for routine The method is being implemented for routine monitoring of cattle (so far) by the USDA labs.monitoring of cattle (so far) by the USDA labs.

•• Quantification is acceptable for Quantification is acceptable for ≈≈60% of the drugs, 60% of the drugs, but enforcement still requires the NADA method. but enforcement still requires the NADA method.

•• Is the new MMM Is the new MMM ““just rightjust right”” to deserve the name to deserve the name ““GoldilocksGoldilocks””??

Acknowledgments

USDA FSIS:Terry DutkoLouis BluhmChilton Ng

Pat McCaskeyet al.

US‐Israel Binational Agricultural Research

and Development Grant US‐4273‐09

*Currently at CovanceGreenfield, Indiana

KaterinaMastovska*

ChristineHaines

Tanks Berry Mulch!Tanks Berry Mulch!

+ +

+ + ! !

Thanks very much!Thanks very much!

Contacts:

[email protected]

[email protected]

Outline of the TalkOutline of the Talk

I. Current Situation and Proposed ChangesA. Feasibility StudyB. Choice of Target Drugs and LevelsC. Aminoglycosides Method

II. Comparison of MMMs and OptimizationA. Spiked SamplesB. Incurred Samples

III. Validation (Quantitative and Qualitative)IV. Conclusions

Ion Ratios for SulfapyridineIon Ratios for SulfapyridineIon Ratios of Spks, n=30 each

0%

50%

100%

150%

200%

250%

300%


Ion

Rat

io


Ion source was cleaned Ion source was cleaned between Days 2 & 3 between Days 2 & 3 ––ion ratios changed for ion ratios changed for

some analytessome analytes

DefinitionsDefinitions

•• IndicationIndication = = result of a screening methodresult of a screening method((i.e.i.e. ““presumedpresumed”” positive or negative)positive or negative)

•• DeterminationDetermination = = result from an analytical determinative result from an analytical determinative method (method (e.g.e.g. GC/PFPD, LC/UV)GC/PFPD, LC/UV)

•• IdentificationIdentification = = qualitative result from a highly selective qualitative result from a highly selective method (method (e.g.e.g. GCGC‐‐MS, LCMS, LC‐‐MSMSnn))

•• ConfirmationConfirmation = = result from 2 or more independent result from 2 or more independent analyses in agreement (ideally, one of which uses a analyses in agreement (ideally, one of which uses a

different chemical mechanism or approach)different chemical mechanism or approach)

Syringeless FiltersSyringeless FiltersMini-UniPrepTM (Whatman)

1) Place unfiltered sample (max. 0.5 mL) in chamber.

2) Compress filter plunger into sample chamber. Clean filtrate fills reservoir bottom up.

3) Place the Mini-UniPrepTM vial in an autosampler.

aqueous samples: PVDF(polyvinylidenefluoride) filter

LC-MS/MS InstrumentationWaters Acquity TQD = UPLC-MS/MS system

UPLC (Ultra-Performance Liquid Chromatography)Pressures up to 15,000 psiLow extra-column volumesCompatible with sub-2 μm particle columns

TQD - Fast triple quadrupole MSRapid data acquisition (5 ms dwell time)Rapid positive/negative ion mode switching (20 ms)

• Fast analysis with good resolution• Lower solvent consumption, less waste• Narrow peaks – fast data acquisition needed


Penicillin G in Incurred Kidney (no IS)

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10

Kidney Sample

Con

cent

ratio

n (n

g/g)



Sulfadimethoxine in Incurred Kidney (no IS)

0

50

100

150

200

250

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)


Analysis of Incurred Kidney (2 g)Tilmicosin vs. SMZ-d6 IS in Incurred Kidney

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)








Tetracyclines (Sum) in Incurred Kidney (no IS)

0

1000

2000

3000

4000

5000

6000

1 2 3 4 5 6 7 8 9 10Kidney Sample

Con

cent

ratio

n (n

g/g)


Uncertainty (U) Limitation in the MethodUncertainty (U) Limitation in the Method• n = 200 extractions among 20 kidneys over 5 days (n = 5)

Method Step QC‐StdAvg. %

RecoveryAvg.%RSD % of U

UHPLC‐MS/MS Atrazine 90* 11 100

Cleanup Methomyl 92 9 0

Extraction Flunixin‐d3 94 10 0

Homogenization Imidacloprid 79† 10 0

* Should be 100% (‐10% bias in spks vs. stds?)† ≈11‐15% probably lost during homogenization

Ion Ratios for Tulathromycin AIon Ratios for Tulathromycin A

Ion Ratios in Stds, n=10 each

0%

20%

40%

60%

80%

100%

120%

140%

160%

500 1000 2000 4000 6000Conc. (ng/g)

Ion

Rat

ioSolvent 2/1 Matrix 2/1Solvent 3/1 Matrix 3/1Solvent 3/2 Matrix 3/2

(UHP)LC(UHP)LC‐‐MS(/MS) CapabilitiesMS(/MS) Capabilities

Simultaneous identification and quantificationSimultaneous identification and quantification

Sensitive determination in complex matrices Sensitive determination in complex matrices

Wide scope (analyte, matrices) Wide scope (analyte, matrices)

Multiclass, multiresidue methods (MMMs)Multiclass, multiresidue methods (MMMs)

Identification/Confirmation

Lab‐Based Screening

Fast, wide-scope sample preparation is the key!

Slide modified from Kate Mastovska

Tier 1: Screening Test Performed by the USDA‐FSIS Inspectors in the Slaughterhouse.

Currently with Microbial Inhibition Tests

Tier 2: Presumptive Positive Samples Sent to the USDA‐FSIS Laboratory for Quantitative and Qualitative Analysis.

Currently with 7‐plate bioassay, but we propose to use UHPLC‐MS/MS at least for lab‐based screening and antibiotic identifications

Two‐Tiered Approach for Antibiotics

Major Classes of Antibiotics Major Classes of Antibiotics

AminoglycosidesAminoglycosides

SulfonamidesSulfonamides TetracyclinesTetracyclinesββ‐‐LactamsLactams

MacrolidesMacrolides QuinolonesQuinolones

Penicillin G Sulfadimethoxine Tetracycline

Gentamicin C1 EnrofloxacinErythromycin

Currently, 219 vet. drugs (including 94 antibiotics)Currently, 219 vet. drugs (including 94 antibiotics) are on our MMM list are on our MMM list

FieldField‐‐Based Microbial Inhibition AssaysBased Microbial Inhibition Assays

FAST Premi® KISTM

Pictures by Paul Pierlott

FAST, PremiFAST, Premi®® & KIS& KISTMTM ComparisonComparisonSpiked Samples of Kidney (Liquid) Spiked Samples of Kidney (Liquid)

Threshold concentration (µg/mL)

Antibiotic FAST Premi® KISTM

U.S. tolerance(µg/g)

Penicillin G 0.4 0.005 0.005‐0.01 0.05

SDMX 1.4 0.12 0.01‐0.1 0.1

OTC 1.5 2.1 0.5‐1.5 12

Tylosin 1.2 0.09 0.1‐0.2 0.2

Danofloxacin 1.7 4.5 4‐>6 0.2 (L)

Streptomycin 1.4 9.0 4‐10 2.0

Neomycin 0.04 1.2 0.4‐0.6 7.2

Spectinomycin 66 3.5 5‐6 4.0

M.J. Schneider, S.J. Lehotay, Anal. Bioanal. Chem. 390 (2008) 1775‐1179

vs. SMZ‐d6

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20%

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beta

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Mol

Martos

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Stubbings

IS

Comparison of 6 Vet. Drug MMMs

Old School Veterinary Drug AnalysisOld School Veterinary Drug Analysis

Single analyteresidue methods

Single‐classresidue methods

Selective multi‐class residue methods

Slide by Kate Mastovska

New School Veterinary Drug AnalysisNew School Veterinary Drug Analysis

Multiclass, MultiresidueMethods (MMMs)

(UHP)LC(UHP)LC‐‐MS(/MS)MS(/MS)



Sulfamethazine vs. SMZ-d6 IS in Incurred Kidney

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1 2 3 4 5 6 7 8 9 10Kidney Sample

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cent

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g/g)







IS

7‐Plate Bioassay (Lab Screen)

Pictures from FSIS

Microbial Inhibition AssaysMicrobial Inhibition Assays

Relatively inexpensive and easyRelatively inexpensive and easy

2424‐‐hr sample turnaround timehr sample turnaround time

Not sensitive for all antibiotic classes Not sensitive for all antibiotic classes

Cannot distinguish particular analytes or mixtures Cannot distinguish particular analytes or mixtures

Prone to unidentifiable microbial inhibitions (Prone to unidentifiable microbial inhibitions (≈≈3%)3%)

>60% of 7‐plate samples in FSIS are positives and need to be confirmed by chemical‐based

(LC‐MS/MS) methods anyway

>60% of 7‐plate samples in FSIS are positives and need to be confirmed by chemical‐based

(LC‐MS/MS) methods anyway


Antibiotics – with US tolerance Number

• Dihydrostreptomycin ‐ 6‐14,000 ng/mL 7

• Streptomycin ‐ 75 ng/mL 1

• DCCD ‐ 2‐28 ng/mL 10

• Florfenicol amine ‐ 33 ng/mL 1

• Oxytetracycline ‐ 3‐57 ng/mL 13

• Penicillin G ‐ 2‐3 ng/mL 3

• Pirlimycin ‐ 2‐29 ng/mL 5

• Sulfamethazine ‐ 2‐140 ng/mL 2

Drug Residues Identified inDrug Residues Identified in235 Kidney (Exudate) Samples235 Kidney (Exudate) Samples

Feasibility StudyFeasibility Study:: Vet. Drug Residues in Vet. Drug Residues in Bovine Serum and Kidney (Exudate)Bovine Serum and Kidney (Exudate)

Analysis of 121 drug residues, including 65 antibiotics

Two sample preparation and LC‐MS/MS methods1) aminoglycosides (ion‐pairing LC)2) MMM for veterinary drugs (reversed‐phase LC)

Screened / analyzed 235 serum and kidney (exudate) culled dairy cow samples from a slaughterhouse

M.J. Schneider, K. Mastovska, S.J. Lehotay, A.R. Lightfield, B. Kinsella, C.E. Shultz, Anal. Chim. Acta 637 (2009) 290‐297

Addition of Quality Control Spikes, tooAddition of Quality Control Spikes, too

1)1) Imidacloprid added during sample homogenizationImidacloprid added during sample homogenization

2)2) I.S. compounds added prior to extraction I.S. compounds added prior to extraction ‐‐sulfamethazinesulfamethazine‐d6; flunixin‐d3; Pen.G‐d7

3) Methomyl added prior to the cleanup step

4) Atrazine added to final extracts

RSDoverall = √(RSD12 + RSD2

2 + RSD32 + RSD4

2)

…And Then There Were Three…Leepipatpiboon et al. – TCA & Precipitation!Stubbings et al. – Use of PSA Lowered RecoveriesKaufmann et al. – WAY too Long and Intensive

• Mol et al. – 30 min Shake w/ Acid• Martos et al. – 60 min Heat w/ Acid Addition• Mastovska et al. – 5 min Shake w/o Acid

MMM

Antibiotics – no US tolerance No. of CowsGentamicin (serum) 8 ng/mL 1Kanamycin (serum) 1 ng/mL 1Lincomycin (kidney) 4 ng/mL 1

Other vet drugs in kidney (exudate)Flunixin, >300 (≥1,000) ng/mL 23 (17)Beta/dexamethasone, 20‐75 ng/mL 7 Prednisone, <10 ng/mL 3

Drug mixtures (not counting flunixin) 12

Drug Residues Found by LCDrug Residues Found by LC‐‐MS/MS inMS/MS inAnalysis of 235 Culled Dairy CowsAnalysis of 235 Culled Dairy Cows

is Best Method for Analysis of Drug Residues in Animal ... · kanamycin, 4 gentamicins, neomycin,...

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