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©2011 Waters Corporation 1
Extraction and Cleanup Protocols for LC-MS/MS
Multiresidue Determination of Veterinary Drugs in
Tissue and Milk Samples
Malin Wangler, Waters Sweden
Michael S. Young and Kim vanTran
Waters
Milford
©2011 Waters Corporation 2
Overview
There is a need for multiresidue UPLC-MS methods that can identify and quantify a wide range of veterinary drug residues from many drug classes
Solvent extraction (acetonitrile or methanol) can be effective for many of these compounds in meat and milk— Polar, highly water soluble drugs such as salbutamol are not well recovered
using this approach
Aqueous buffer extraction can also be effective for many compounds— Fat soluble compounds such as dexamethasone are not well recovered
using this approach
Traditional SPE enrichment and cleanup (retention/wash/elution) has limited utility for multi-residue analysis; the range of polarity/solubility among the compounds is very challenging— Dispersive or pass-thru SPE is preferred for multi-residue methods
— Traditional SPE is still a powerful procedure and is preferred for isolation/cleanup of individual compounds or compound classes
©2011 Waters Corporation 3
Optimized Method for TetracyclinesExample of Class Specific Method
Condition/Equilibrate2 mL MeOH, 2 mL water
Load Samplefrom pretreatment
Wash 10.5 mL 5% NH4OH/water
Wash 2 0.5 mL methanol
Elute :0.5 ml 45:55
acetonitrile/75mM oxalic acid
Oasis® MAXSPE protocol
assures analytes are anions
removes neutrals or bases
neutralizes compounds to release from sorbent(oxalic acid stabilizer)
PretreatmentMix 1.5 mL milk with
6 mL pH 4 McIivaine buffer
Centrifuge
Take supernatant/adjust to pH 10
with 0.75 mL 1 M NaOH
100 ppb tetracyclines
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
%
0
100
TCYC021808_01 Sm (SG, 2x2) MRM of 4 Channels ES+ TIC
3.63e5
1. oxytetracycline
2. tetracycline
3. chlortetracycline
4. doxycycline
1
2
3
4UPLC®-MS ConditionsWaters Quattro Premier XEWaters ACQUITY UPLC®
ColumnACQUITY UPLC® BEH C182.1 x 50mm, 1.7 µm
Mobile PhaseA: 0.1 % Formic acid/waterB: Acetonitrile0.4 mL/min flow
Linear GradientTime %A %Binitial 85 152.5 50 503.5 30 703.6 85 154.0 85 15
100 ppb tetracyclines
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
%
0
100
TCYC021808_01 Sm (SG, 2x2) MRM of 4 Channels ES+ TIC
3.63e5
1. oxytetracycline
2. tetracycline
3. chlortetracycline
4. doxycycline
1
2
3
4UPLC®-MS ConditionsWaters Quattro Premier XEWaters ACQUITY UPLC®
ColumnACQUITY UPLC® BEH C182.1 x 50mm, 1.7 µm
Mobile PhaseA: 0.1 % Formic acid/waterB: Acetonitrile0.4 mL/min flow
Linear GradientTime %A %Binitial 85 152.5 50 503.5 30 703.6 85 154.0 85 15
- Recovery 75-90 % at the 100 ng/g level
- Virtually no suppression/enhancement
Method great for tetracyclines butnot suitable for multi-class analysis
©2011 Waters Corporation 4
Goals/Observations
The purpose of this ongoing study is to evaluate simple sample preparation strategies (extraction/cleanup) for multiresidue analysis of veterinary drugs in meat and milk.
No internal standards are used: – recovery is not corrected for by comparison with internal standards– the observed precision is typical for external standard calibration.
Many compounds show significant ion-suppression. This may not be a serious impediment to useful analytical performance if matrix matched standards are employed and the response of the compound is sufficient.
Among the goals of future work will be advances in SPE cleanup to reduce matrix effects.
©2011 Waters Corporation 5
Veterinary Drug Classes in This Study
tetracycline
Tetracycline Fluoroquinolone
enrofloxacin
Sulfonamide
sulfamethazine
Macrolide
erythromycin
Beta Lactam
oxacillin
NSAID
phenylbutazone
Steroid
dexamethasone
Beta-adrenergic
salbutamol
©2011 Waters Corporation 6
Instrumentation for This Study
Xevo™ TQ ACQUITY UPLC® ACQUITY® TQD
Used for meat analysis Used for milk analysis
©2011 Waters Corporation 7
Column: ACQUITY CSH™ C18 1.7µm
100 x 2.1 mm
Mobile phase
— A: 0.1% formic in water
— B: 0.1% formic acid acetonitrile
Injection volume: 7 µL
Injection mode: partial loop injection
Sample diluent: 20:80 ACN:water
Column temperature 30 °C
Weak Needle Wash: 10:90
acetonitrile:water (600 μL)
Strong Needle Wash: 50:30:40 water:acetonitrile:IPA (200 μL)
Seal wash: 10:90 acetonitrile: water
Gradient Table
Veterinary Drugs ACQUITY UPLC® Conditions
615850.47.0
69550.43.9
69550.44.9
615850.45.0
40
15
% B
6600.42.5
Initial850.4Initial
Curve% AFlow
(mL/min)
Time
(min)
615850.47.0
69550.43.9
69550.44.9
615850.45.0
40
15
% B
6600.42.5
Initial850.4Initial
Curve% AFlow
(mL/min)
Time
(min)
©2011 Waters Corporation 8
MS ConditionsPrincipal MRM Transitions
Polarity ES+
(Except Chloramphenicol ES-)
Capillary (kV) 2.80
Extractor (V) 3.00
Source Temperature (°C) 150
Cone Gas Flow (L/Hr) 30
Desolvation Temperature (°C) 500
Desolvation Gas Flow (L/Hr) 1000
Collision Gas Flow (mL/Min 0.15
MassLynx V4.
Waters Xevo TQ MS:
meat analysis – conditions shown
Waters ACQUITY® TQD :
milk analysis – similar MS conditions and same transitions
Compound Principal MRM Cone CID
Amoxicillin 366>113 15 20
Carbadox 263>231 25 15
Ciprofloxacin 332>288 28 18
Chloramphenicol 321>152 10 15
Chlortetracyline 479>444 25 25
Dexamethasone 393>355 20 15
Enrofloxacin 360>316 30 25
Erythromycin 734>158 30 25
Lincomycin 407>126 30 25
Oxacillin 402>160 15 15
Oxytetracycline 461>426 22 20
Penicillin-G 335>160 20 15
Phenylbutazone 309>160 20 15
Ractopamine 302>107 22 25
Salbutamol 240>148 20 25
Sulfamerazine 265>92 25 25
Sulfamethazine 279>92 32 30
Sulfanilamide 173>156 25 10
Tetracycline 445>154 25 25
Tylosin 916>174 50 30
©2011 Waters Corporation 9
LC/MS Functions
Function 1SulfanilamideSalbutamol Ractopamine Lincomycine
Function 2Carbadox Sulfamerazine Sufamethazine Erthromycin Tylosin
Function 3PhenylbutazonePenicillin Dexamethasone Oxacillin
Function 4CiprofloxacinEnrofloxacin
Function 5Tetracyline Oxytetracycline Chlortetracyline
Function 6Chloramphenicol
Dwell time 0.010 sec. Two transitions per analyteMinimum of 10 datapoints across each chromatographic peak
©2011 Waters Corporation 10
Typical LC/MS/MS ResponseErythromycin (10 ng/g meat sample)
Time1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90
%
0
100
1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90%
0
100VET02FA060711_87 2: MRM of 10 Channels ES+
734.93 > 158.15 (Erthromycin)
4.85e4S/N:RMS=3825.18
2.65
VET02FA060711_87 2: MRM of 10 Channels ES+
734.93 > 576.54 (Erthromycin)4.55e3S/N:RMS=430.62
2.34
TimeTime1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90
%
0
100
1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90
%
0
100
1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90%
0
100VET02FA060711_87 2: MRM of 10 Channels ES+
734.93 > 158.15 (Erthromycin)
4.85e4S/N:RMS=3825.18
2.65
VET02FA060711_87 2: MRM of 10 Channels ES+
734.93 > 576.54 (Erthromycin)4.55e3S/N:RMS=430.62
2.34
Comparison of response for principal (top) and confirmatory transitions for erythromycin (Waters Xevo TQ)
©2011 Waters Corporation 11
Analytical OptionsSample Preparation Strategies
Perform both aqueous and acetonitrile extraction on the same
tissue or milk sample
— Tedious and time consuming multi-step extraction
o strategy results in two extracts requiring separate workup
before combining for one LC/MS analysis
o provides best recovery of the widest range of compounds
Perform extraction using a mixed acetonitrile/aqueous system
— Less steps required for analysis
o strategy results in one rather messy extract that requires
workup
o workup is straightforward
o reasonable recovery and cleanup is possible for a wide range of
compounds
©2011 Waters Corporation 12
Definitions
Ion-suppresion or enhancement (Matrix effect)- A decrease (suppression) or increase (enhancement) in the
MS response for an analyte caused by the presence of interfering substances resulting from the sample matrix.
- The matrix effect is calculated by comparison of analyte response for a standard prepared in sample matrix with analyte response for a standard prepared in pure solvent.
Recovery- The amount of an analyte recovered by the analytical method. - Recovery of an analyte from a sample is calculated by
comparison of the response shown for an analyte fortified into the sample matrix prior to all sample preparation with response shown for a blank matrix sample fortified with the analyte after all sample preparation steps.
©2011 Waters Corporation 13
Part 1
Milk Analysis
©2011 Waters Corporation 14
Milk Composition
Typical Cow’s Milk
—Approximately 14 % solids
o 4 % fat
o 4 % protein
o 5 % sugar (lactose)
o 85 % water
©2011 Waters Corporation 15
Veterinary Residues in MilkMultiresidue LC/MS Analysis
Typical Sample Preparation Strategies
— Precipitation/extraction with strong buffer (McIlvaine pH 4) followed by SPE
o good for tetracyclines, beta-adrenergics, polar sulfonamides, fair for fluoroquinolones,
o poor recovery of most other compounds
— Precipitation/extraction with 3:1 acidic acetonitrile with SPE cleanup
o excellent protein precipitation
o poor recovery of tetracyclines, beta-adrenergics, polar sulfonamides
o good recovery of most other compounds
— This study: two-step precipitation/extraction
o good recovery of a wide range of compounds
©2011 Waters Corporation 16
Typical Recoveries From MilkComparison of Precipitation/Extraction Techniques
Drug Class 3:1 ACN Aq Buffer 1:1 ACN*
Beta-adrenergic <10 ~100 >80
Tetracycline <25 >70 >25
Fluoroquinolone >50 >50 >50
Macrolide >60 <35 >60
Beta-Lactam >70 <30 >70
Steroid >70 <10 >70
*Conclusion:-Procedure chosen for this study
-Extraction/precipitation of milk with an equal volume of acetonitrile provides recovery of the widest range of compounds
However - insufficient protein precipitation
See also: Stolker et. al., Anal. Bioanal. Chem. 391, 2309 (2008)
©2011 Waters Corporation 17
Analytical Method For This StudyMilk – 2 mL Sample
Initial Extraction/Precipitation
Pipet 2 mL sample into centrifuge tube
Add 2 mL acetonitrile
Centrifuge @ 8000 x g
Take 2 mL supernatant
Protein Precipitation
Add 3 mL acetonitrile(0.2% formic acid)
Centrifuge @ 8000 x g
Take 1 mL supernatant
SPE CleanupSep-Pak C18 (1 cc, 100 mg) Evaporate and reconstitute
provides good recovery of most compounds
minimal extraction of fat
much protein in extract
secondary protein precipitation step
removes most residual protein without significant loss of polar analytes
©2011 Waters Corporation 18
SPE Cleanup Sep-Pak C18 (pass-thru mode)
Condition1 mL 80:20 acetonitrile/water
Pass-Thru/Collect1 mL protein ppt sample
1 cc 100 mg
install collection tubes
Rinse/Collect0.5 mL 80:20 acetonitrile/water
Evaporate/Reconstitute 0.2 mL 25:75 acetonitrile/buffer
(25 mM ammonium formate buffer @ pH 4.5)
add 0.25 mL 200 mM ammonium formate in 50:50 ACN/methanol*
* buffers sample to protect acid labile analytes
©2011 Waters Corporation 19
Effect of Buffering Prior to Evaporation
Analyte Recovery Without Buffer Recovery With Buffer
Sulfamerizine 70-80 70-80
Lincomycin <25 80-100
Erythromycin <25 60-80
Penicillin <40 75-85
©2011 Waters Corporation 20
Results: Milk0.67 x MRL Level
Compound MRL Spiked %REC (%RSD) %Suppression
Level (ppb) n=3
Carbadox 67.0 27 (27)* 43 (enhance)
Chloramphenicol(P) 6.7 94 (16) 10.0
Chlorotetracyline (T) 67.0 22 (20) 7.0
Ciprofloxacin (F) 67.0 67 (20) 32.0
Dexamethasone (St) 67.0 87 (6) 8 (enhance)
Enrofloxacin (F) 134.0 76 (11) 26.0
Erythromycin (M) 6.7 59 (10) 5.0
Lincomycin(M) 33.0 102 (9) 25.0
Oxacillin (B-L) 67.0 79 (12) 9 (enhance)
Oxytetracycline (T) 67.0 24 (16) 9 (enhance)
Penicillin (B-L) 33.0 73 (8) 8(enhance)
Phenylbutazone (NSAID) 67.0 67 (18) 20.0
Ractopamine 200.0 65 (14) 0.0
Salbutamol 67.0 80.4 (3) 96.0
Sulfamerazine (S) 67.0 71 (4) 16 (enhance)
Sulfamethazine (S) 67.0 71 (6) 74 (enhance)
Sulfanilamide (S) 67.0 110 (30)* 60.0
Tetracycline (T) 67.0 31 (18) 21 (enhance)
* Response Near Detection Limit
©2011 Waters Corporation 21
Part 2
Pork Muscle Analysis
©2011 Waters Corporation 22
Pork Muscle Composition
Typical Pork Muscle
—Approximately 30 % solids
o 5-20 % fat
o 15-25 % protein
o ~70% water
©2011 Waters Corporation 23
Analytical Method For This StudyPork Muscle – 5 g Sample
Extraction/Precipitation
Weigh homogenized sample into 50 mL centrifuge tube
Add 10 mL of 0.2 % formic acid in 80:20 ACN/waterVortex, shake 30 min
Centrifuge @ 10000 x g
Take 0.60 mL supernatant
SPE CleanupSep-Pak tC18 (40 mg 96 well plate)
Evaporate and reconstitute
provides good recovery of most compounds
much fat in extract
©2011 Waters Corporation 24
SPE Cleanup Sep-Pak tC18 Plate (pass-thru mode)
Condition500 µL 80:20 acetonitrile/water
Pass-Thru/Collect600 µL sample extract
install collection plate
Rinse/Collect600 µL 80:20 acetonitrile/water
Evaporate/Reconstitute 250 µL 20:80 acetonitrile/water
add 200 μL 200 mM ammonium formate in 50:50 ACN/methanol*
* buffers sample to protect acid labile analytes
40 mg/well
©2011 Waters Corporation 25
Results: Pork Muscle1 x MRL Level
Compound MRL Spiked %REC (%RSD) %Suppression
Level (ppb) n=5
Carbadox 100 8.9 (36)* 62.7
Chloramphenicol(P) 10 57.5 (20) 7.1
Chlorotetracyline (T) 100 41.9(11) 5.7
Ciprofloxacin (F) 100 130 (21) 85.6
Dexamethasone (St) 100 70.2 (7) 36.9
Enrofloxacin (F) 200 106 (4) 70.3
Erythromycin (M) 10.0 36.1 (9) 4.2
Lincomycin(M) 50.0 64.5 (17) 93.4
Oxacillin (B-L) 100 51.5 (4) 25.2
Oxytetracycline (T) 100 51.1 (8) 9.4
Penicillin (B-L) 50.0 46.8 (7) 11.3
Phenylbutazone (NSAID) 100 15.9 (16) 53.3
Ractopamine 300 73.7 (7) 81.1
Salbutamol 100 70.8 (14) 97.4
Sulfamerazine (S) 100 63.4 (5) 56.7
Sulfamethazine (S) 100 67.1 (5) 53.6
Sulfanilamide (S) 100 74.4 (21)* 71.8
Tetracycline (T) 100 58.3 (10) 0.4
Tylosin 20.0 46.6 (11) 8.4
* Response Near Detection Limit
©2011 Waters Corporation 26
ConclusionsSample Preparation
Methods were demonstrated for determination of
multiclass/multiresidue veterinary drugs in milk and meat
A two step extraction/protein precipitation procedure was
demonstrated for milk analysis
— Recoveries averaged 67% (22-110) with the lowest values for
tetracyclines
— Matrix effects, though significant, were less than for meat samples
A single step meat extraction/protein precipitation procedure
was evaluated
— Recoveries averaged 60% (9-106) with the lowest values for
carbadox and phenyl butazone
— Matrix effects were significant
©2011 Waters Corporation 27
ConclusionsSample Cleanup
Pass-thru cleanup using C18-silica was used in this study
— Effective for removal of residual fats from both types of extracts
— Not effective for reduction of matrix effects
There is much opportunity for development of improved cleanup
procedures for these types of analysis
©2011 Waters Corporation 28
Thank´s for your attention!