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 1
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 2
Optimized Method for Tetracyclines Example of Class Specific Method Oasis MAX SPE protocol Condition/Equilibrate 2 ml MeOH, 2 ml water Load Sample from pretreatment Wash 1 0.5 ml 5% NH 4 OH/water Wash 2 0.5 ml methanol Elute : 0.5 ml 45:55 acetonitrile/75mm oxalic acid Pretreatment Mix 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 assures analytes are anions removes neutrals or bases neutralizes compounds to release from sorbent (oxalic acid stabilizer) - Recovery 75-90 % at the 100 ng/g level - Virtually no suppression/enhancement Method great for tetracyclines but not suitable for multi-class analysis 2011 Waters Corporation 3
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 4
Veterinary Drug Classes in This Study Tetracycline Fluoroquinolone Sulfonamide Macrolide tetracycline enrofloxacin sulfamethazine erythromycin Beta Lactam NSAID Steroid Beta-adrenergic salbutamol oxacillin phenylbutazone dexamethasone 2011 Waters Corporation 5
Instrumentation for This Study Xevo TQ ACQUITY UPLC ACQUITY TQD Used for meat analysis Used for milk analysis 2011 Waters Corporation 6
Veterinary Drugs ACQUITY UPLC Conditions 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 Time (min) Gradient Table Flow % A (ml/min) % B Curve 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 Initial 2.5 3.9 4.9 5.0 7.0 0.4 0.4 0.4 0.4 0.4 0.4 85 60 5 5 85 85 15 40 95 95 15 15 Initial 6 6 6 6 6 2011 Waters Corporation 7
MS Conditions Principal 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 8
LC/MS Functions Function 1 Sulfanilamide Salbutamol Ractopamine Lincomycine Function 2 Carbadox Sulfamerazine Sufamethazine Erthromycin Tylosin Function 3 Phenylbutazone Penicillin Dexamethasone Oxacillin Function 4 Ciprofloxacin Enrofloxacin Function 5 Tetracyline Oxytetracycline Chlortetracyline Function 6 Chloramphenicol Dwell time 0.010 sec. Two transitions per analyte Minimum of 10 datapoints across each chromatographic peak 2011 Waters Corporation 9
% % Typical LC/MS/MS Response Erythromycin (10 ng/g meat sample) Comparison of response for principal (top) and confirmatory transitions for erythromycin (Waters Xevo TQ) VET02FA060711_87 100 S/N:RMS=3825.18 2: MRM of 10 Channels ES+ 734.93 > 158.15 (Erthromycin) 4.85e4 2.65 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 VET02FA060711_87 S/N:RMS=430.62 2: MRM of 10 Channels ES+ 734.93 > 576.54 (Erthromycin) 4.55e3 2.34 0 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 Time 2011 Waters Corporation 10
Analytical Options Sample 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 11
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 12
Part 1 Milk Analysis 2011 Waters Corporation 13
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 14
Veterinary Residues in Milk Multiresidue 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 15
Typical Recoveries From Milk Comparison 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 16
Analytical Method For This Study Milk 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 provides good recovery of most compounds minimal extraction of fat much protein in extract Protein Precipitation Add 3 ml acetonitrile(0.2% formic acid) Centrifuge @ 8000 x g Take 1 ml supernatant secondary protein precipitation step removes most residual protein without significant loss of polar analytes SPE Cleanup Sep-Pak C18 (1 cc, 100 mg) Evaporate and reconstitute 2011 Waters Corporation 17
SPE Cleanup Sep-Pak C18 (pass-thru mode) Condition 1 ml 80:20 acetonitrile/water Pass-Thru/Collect 1 ml protein ppt sample install collection tubes 1 cc 100 mg Rinse/Collect 0.5 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 18
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 19
Results: Milk 0.67 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 20
Part 2 Pork Muscle Analysis 2011 Waters Corporation 21
Pork Muscle Composition Typical Pork Muscle Approximately 30 % solids o 5-20 % fat o 15-25 % protein o ~70% water 2011 Waters Corporation 22
Analytical Method For This Study Pork 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/water Vortex, shake 30 min Centrifuge @ 10000 x g Take 0.60 ml supernatant provides good recovery of most compounds much fat in extract SPE Cleanup Sep-Pak tc18 (40 mg 96 well plate) Evaporate and reconstitute 2011 Waters Corporation 23
SPE Cleanup Sep-Pak tc18 Plate (pass-thru mode) Condition 500 µl 80:20 acetonitrile/water Pass-Thru/Collect 600 µl sample extract install collection plate 40 mg/well Rinse/Collect 600 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 2011 Waters Corporation 24
Results: Pork Muscle 1 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 25
Conclusions Sample 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 26
Conclusions Sample 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 27
Thank s for your attention! 2011 Waters Corporation 28