Extraction and Cleanup Protocols for LC-MS/MS Multiresidue ... · Malin Wangler, Waters Sweden...

©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


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


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


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.


Veterinary Drug Classes in This Study

tetracycline

Tetracycline Fluoroquinolone

enrofloxacin

Sulfonamide

sulfamethazine

Macrolide

erythromycin

Beta Lactam

oxacillin

NSAID

phenylbutazone

Steroid

dexamethasone

Beta-adrenergic

salbutamol


Instrumentation for This Study

Xevo™ TQ ACQUITY UPLC® ACQUITY® TQD

Used for meat analysis Used for milk analysis


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)


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


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


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)


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


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.


Part 1

Milk Analysis


Milk Composition

Typical Cow’s Milk

—Approximately 14 % solids

o 4 % fat

o 4 % protein

o 5 % sugar (lactose)

o 85 % water


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


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)


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)


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


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


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


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


Part 2

Pork Muscle Analysis


Pork Muscle Composition

Typical Pork Muscle

—Approximately 30 % solids

o 5-20 % fat

o 15-25 % protein

o ~70% water


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


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


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


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


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


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


Thank´s for your attention!

Extraction and Cleanup Protocols for LC-MS/MS Multiresidue ... · Malin Wangler, Waters Sweden...

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Transcript of Extraction and Cleanup Protocols for LC-MS/MS Multiresidue ... · Malin Wangler, Waters Sweden...