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PETRL SOP 521.0 LC/MS/MS: Acepromazine Metabolites (Urine & Plasma) of 21Date Last Revised: 9/14/04

Approved By________Laboratory Director Acknowledged By:________ Quality Assurance Officer Acknowledged By:_______ Lab Manager

_____________________________________________________________________________________________________________________________________

SCREENING AND CONFIRMATION OF 2-1(HYDROXYETHYL) PROMAZINESULFOXIDE BY ION TRAP LC/MS/MS

I. INTRODUCTION

Phenothiazine-derived tranquilizers have been widely used in veterinary medicine sincethe 1950’s. As a class, the phenothiazine derivatives induce tranquilization and blockconditioned responses; but unlike other tranquilizers, the depressant action on thecentral nervous system is mostly sub cortical. These drugs are used in horses torelieve apprehension prior to general anesthesia and as chemical restraint for ease of shipping and handling. Low dose pre-race administration is rumored to occur in order toprevent nervous expenditures of energy prior to racing. Since there is little effect oncoordinated motor responses, energy is conserved for actual competition, withoutnervous exhaustion prior to racing 5,6.

2-(1-hydroxyethyl)promazine sulfoxide

Molecular Formula = C19H24N2O2S

Formula Weight = 344.47106Monoisotopic Mass = 344.155848 Da

Acepromazine

Molecular Formula = C19H22N2OS

Formula Weight = 326.45578Monoisotopic Mass = 326.145283 Da

N

S

O

N

CH3

CH3

CH3

N

S

O

N

CH3

CH3

CH3

N

S

OH

N

CH3

CH3

CH3

O

N

S

OH

N

CH3

CH3

CH3

O

Figure 1. Chemical structures of Acepromazine and its major equine metabolite,2-1(hydroxyethyl) promazine sulfoxide.

II. SCOPE

This standard operating procedure (SOP) describes identification and confirmationprocedures for the presence of 2-1(hydroxyethyl) promazine sulfoxide (HEPS) inplasma and/or urine using the Deca XP Plus Ion Trap LC/MS/MS (Thermo-Finnigan). Additional information will be provided for Acepromazine (ACE), and for all metabolites detectable by this method. The relative abundances of theseanalytes may be affected by drug formulation, route of administration, individual

animal differences, age and condition of samples, and/or method chemistry.Enzyme Linked Immuno-Sorbent Assay (ELISA) presumptive findings using theNeogen Promazine ELISA may not contain Acepromazine or its metabolites. Thistest has a wide range of cross reactivity, and interpretation of results obtained bypromazine ELISA test kit should be correlated to other ELISA test kits (i.e.Tricyclics). Additionally, concentrations predicted by ELISA testing may be highrelative to any single analyte detected in urine or plasma.

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2-

A

d

IIB. BACKGROUND

The Racing Commissioners International (RCI) Uniform Classification Guidelines for Foreign Substances designate Acepromazine as a class three drug 2. TheThoroughbred Owners and Breeders Association (TOBA) Graded Stakes MedicationGuidelines lists the minimum urinary concentrations of the presumed major respective metabolite to be 20 ng/mL 3. The National Thoroughbred RacingAssociation (NTRA) Task Force Guidelines indicate 3 jurisdictions use a 25 ng/mLurine metabolite threshold, 1 jurisdiction uses a 10 ng/mL threshold, 1 jurisdictionuses a 2 ng/mL threshold and 22 jurisdictions use no threshold urinary limit 2,4.

ELISA screening and LCMS(MS) confirmation and quantification sensitivities permitdetection and reporting of urinary concentrations, which could exceed a 72-hour window prior to racing

2,3,4. Potentially trivial urinary findings can be further defined

by monitoring plasma and urine concentrations of all detectable analytes.Additionally, plasma screening by this standard operating procedure would detect

possible attempts to circumvent post-race urine testing by administration of Acepromazine just prior (within 2 hours) to racing.

III. PRIMARY DRUG AND INTERNAL STANDARD REFERENCE MATERIALS

Source of analytical reference and internal standard materials:

Compound Source Cat # Reference Label

(1-hydroxyethyl)promazine sulfoxide NEOGEN Corporation # 110230 R-21OHETHYLPROMS-1

cepromazine Sigma # A-6908 S-Ace-1

3-Codeine Alltech # 1913 R-CodD3-1

10 ug/mL FIA solutions were prepared as follows:

Volume mL 50:50:1

200 uL 3.8 21OHEPSFIA121703

100 uL 9.9

om these, 100 ng/mL LC solutions were prepared as follows:

Volume mL 0.1% F

Fr

A

100 uL 9.9 21OHEPSLC121703

100 uL 9.9 ACELC121703

Using

R-21OHETHPROS-1

ug

2-(1-Hydroxyethyl)Promazine Sulfoxide

Label

ug Using Label

epromazine S-ACE-1

Acepromazine

ACEFIA

Dr

Dr

Ac 121703

ACEFIA121703

2-(1-Hydroxyethyl)Promazine Sulfoxide 21OHEPSFIA121703

Obtain these materials from the pharmacy and record accession on the pharmacy

log sheet.

IV. PREPARATION OF PRIMARY REFERENCE STOCK SOLUTIONS

A. 2-1(hydroxyethyl) promazine sulfoxide is commercially available as 200 ug/mL in methanol byNeoGen Corporation, Catalog Number 110230. (phone: 1-800-447-8201).

B. Acepromazine is supplied as the maleate salt. Weigh between 5 and 10 mg (X.xx mg) into aglass bottle and label. Dilute to volume using HPLC grade (or better) methanol (Volume Y.yy mL= X.xx mg/Z.ZZ where Z.ZZ is the Salt/Free ratio if applicable). Cap and mix until compoundis completely dissolved in methanol.

C. d3-Codeine is supplied as 100 ug/mL in methanol by Alltech, (1-800-437-3784), Catalog

Number 1913.

V. PREPARATION OF FLOW INJECTION ANALYSIS (FIA) and LIQUID

CHROMATOGRAPHY (LC) COLUMN TEST WORKING STOCK SOLUTIONS

Materials Needed: Primary Stock Solution A & B (IV. A, B above), 50:50:1 (MeCN:Water:Formic Acid)

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Preparation of d3-Codeine Internal Standard Working Stock DilutionMaterials Needed: Primary Stock C

Add 100 uL R-CodD3-1 to 9.9 mL 0.1% Formic Acid to yield 1000 ng/mL and Label withMMDDYY d3COD1000.

VIa. PREPARATION OF CALIBRATION STOCK SOLUTIONS

Working Calibrator Solutions are prepared according to the following table:Using the ACE and HEPS FIA solutions, the following calibrators were prepared as described here:

CAL#Concentration

Used For:

mL of Cal

(Both ACE and

HEPS) Using Cal mL 50:50:1

Matrix Used

for: (U or P)

2 500 1 (2 tot) 10ug/mL 0 U

3 250 0.5 (1 tot) 10ug/mL 1 U

4 100 0.8 2 3.2 U

5 50 0.4 2 3.6 U

6 25 0.2 2 3.8 U

7 10 0.4 4 3.6 P, U8 5 0.2 4 3.8 P, U

9 1 0.4 7 3.6 P, U

10 0.75 0.3 7 3.7 P

11 0.5 0.2 7 3.8 P

12 0.25 0.1 7 3.9 P

13 0.1 0.4 9 3.6 P

Concentration

ng/mL Label

2,500 040604ACE_HEPSCAL2.5K

5,000 040604ACE_HEPSCAL5K

100 040604ACE_HEPSCAL100

1,000 040604ACE_HEPSCAL1K


1.0 040604ACE_HEPSCAL1

7.5 040604ACE_HEPSCAL7.5

5.0 040604ACE_HEPSCAL5

2.5 040604ACE_HEPSCAL2.5




100 uL of CAL# 1-9(above) are added to 0.9 mL Negative Control Urine. 100 uL of these spiked urines are ready

for analysis with 100 uL aliquots of unknown urine sample. D3 Codeine is used as internal standard (see below)

100 uL of CAL# 7-13(above) are added to 0.9 mL Negative Control plasma. D3 Codeine is used as internal standard (see below)

Using 031703d3Cod10, 100 ng/mL solution was prepared

CAL#

Concentration Used

For:

mL of

Stock Using Cal# mL 0.1% FA

14 10 1 031703d3Cod1000 9100 100903d3Cod100

Concentration

ng/mL Label

VIb. PREPARATION OF URINE AND PLASMA CALIBRATORS

Urine analysis utilizes 100 uL of sample.

A 100 uL aliquot of CAL# 1 thru 9 is mixed with 0.9 mL urine in respectively labeledtubes. A 100 uL aliquot of these urine calibrators is then transferred into appropriatelylabeled 16 x 125 screw top culture tubes as calibrators and controls. Prepare duplicate10 and 250 ng/mL QC controls for urine analysis.

Plasma analysis utilizes 1 mL of sample.

A 100 uL aliquot of CAL# 7 thru 13 is mixed with 0.9 mL of plasma in respectivelylabeled tubes. Prepare duplicate 0.5 and 5 ng/mL QC controls for plasma analysis.

100 uL of CAL# 14 (Internal Standard) is added to each tube except NC1 (negativecontrol).

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VII. SAMPLE REQUIREMENTS FOR ANALYSIS

A. Mobile phase blank1. Designate Solvent Blank1…Solvent Blankn.

B. Negative (control) sample1. Designate plasma or urine NC or plasma or urine blank.2. Prepare negative (Control) sample from negative (Control) plasma or urine.

C. Positive control samples1. Designate plasma or urine QC (or PC)1…..QC (or PC)n.2. Prepare positive control samples as described Section VIb.

D. Calibrators

1. Prepare plasma urine calibrators using negative (control) plasma or urine fromstandard working solutions as described in Section VIb.

E. Test samples1. Designated to use the date of which the sample is analyzed and

*.raw data file designated to use sequence row number.

VIIIa. Plasma: Liquid-Liquid Extraction

Safety Requirements: Lab coat, fume hood, eye protection

1. Add 1 mL plasma (controls, calibrators, and samples) into labeled 16 x 125 mm

screw cap culture tubes.2. Add 1 mL 1:1 ammonium hydroxide: water.3. Add 100 uL spiking dilutions to respective calibrator and QC tubes.4. Add 100 uL internal standard (d3-Codeine) to all tubes except NC1 (negative

control - no internal standard).5. Gently vortex all tubes.6. Add 5 mL of 5:1 dichloromethane: chlorobutane into each tube, cap all screw-top

tubes tightly and rotorack for 5 minutes.7. Centrifuge at 2,500 ~ 3,000 rpm (839 ~ 1,409 g) for 5 minutes.8. Decant the (top) organic layer into a labeled fresh test tube for each sample.9. Bring the extracts in test tubes to dryness in a fume hood at approximately 40 °C

under a steady stream of nitrogen.10. Remove test tubes from the drying block, place in a rack, and allow cooling to

room temperature.

11. Reconstitute the residues with 100 μL of 0.1% formic acid (aqueous) for plasmaanalysis.

12. Transfer the above solution into a 200 μL insert and load in the auto sampler vials. All the samples are now ready for LC/MS/MS analysis.

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VIIIb. Urine: Liquid-Liquid Extraction

Safety Requirements: Lab coat, fume hood, eye protection

1. Add 0.9 mL negative control urine into labeled 16 x 125 mm screw cap culturetubes for calibrators and controls. (Enzyme Hydrolysis is not required for HEPSin urine, although hydroxyl metabolites will be better detected.).

2. Add 100 uL spiking dilutions to respective calibrator and QC tubes and gentlymix.

3. Transfer 100 uL of the calibrators and controls into respectively labeled 16 x 125mm screw cap culture tubes

4. Transfer 100 uL unknown urine to appropriately labeled tubes.5. Add 4 mL pH 10 saturated borate buffer to all tubes.6. Add 100 uL internal standard (d3-Codeine) to all tubes except NC1 (negative

control no internal standard).

7. Gently vortex all tubes.8. Add 5 mL of 5:1 dichloromethane: chlorobutane into each tube, cap all screw-top

tubes tightly and rotorack for 5 minutes.9. Centrifuge at 2,500 ~ 3,000 rpm (839 ~ 1,409 g) for 5 minutes.10. Decant the (top) organic layer into a labeled fresh 16 x 100 culture tube for each

sample.11. Bring the extracts in test tubes to dryness in a fume hood at approximately 65 °C

under a steady stream of nitrogen.12. Remove test tubes from the drying block, place in a rack, and allow cooling to

room temperature.

13. Reconstitute the residues with 200 μL of 0.1% formic acid (aqueous).

14. Transfer the above solution into a 200 μL insert and load in the auto sampler.vials. All the samples are now ready for LC/MS/MS analysis.

Urine: Enzyme Hydrolysis Procedure (optional)

1. To 0.1 mL urine (samples, calibrators, and controls), add 1 mL 1 M sodium

acetate (pH 5), 1 mL β-glucuronidase (patella vulgata - 5000 units/mL).2. Cap and incubate at 65oC for 3 hours.3. Cool tube and adjust to pH 9.0 with ammonium hydroxide: water (1:1).4. Extract with 5 mL dichloromethane: isopropanol (10:1).5. Centrifuge and aspirate aqueous layer to waste.

6. Extract organic layer remaining from step 5 with 3 mL 0.1 N sulfuric acid.7. Centrifuge and aspirate organic layer to waste.8. Adjust aqueous layer remaining from step 7 to pH 9.0 with ammonium hydroxide:

water (1:1).9. Extract with 5 mL dichloromethane: isopropanol (10:1).10. Centrifuge and aspirate aqueous layer to waste.11. Proceed from step 5 above (urine liquid-liquid extraction).

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VIIIc. Sample Extraction Validation

2-1(hydroxyethyl)promazine sulfoxide and Acepromazine pH efficiency (DCM)

0

20

40

60

80

100

120

4 5 6 7 8 9 10 11 12 13

pH

N o r m a l i z e d %

HEPS

ACE

Figure 2. Optimum Extraction pH for Acepromazine (ACE) and 2-1(hydroxyethyl) promazinesulfoxide (HEPS)

2-1HEPS & Ace Solvent Comparison at pH 10

0

10

20

30

40

50

60

70

80

90

100

Diethyl Ether 6:1 Methyl tertbutyl

ether

Toluene DCM Chlorobutane 10:1 EtAc

Solvent


2-1(hydroxyethyl)promazine sulfoxide

Acepromazine

2-1HEPS & Ace Solvent Comparison at pH 10

0

10

20

30

40

50

60

70

80

90

100

Diethyl Ether 6:1 Methyl tertbutyl

ether

Toluene DCM Chlorobutane 10:1 EtAc

Solvent


2-1(hydroxyethyl)promazine sulfoxide

Acepromazine

Figure 3. Extraction solvent comparison for ACE-HEPS. Note: No single solvent was optimum for bothcompounds.

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Solvent Efficiency Fine Tune Mix (at pH 12 ammonium hydroxide)

0

20

40

60

80

100

120

D C M

C B T

E t O A C

E t O A C : C B T_

1 : 1

E t O A C : C B T_

2 : 1

E t O A C : C B T_

3 : 1

E t O A C : C B T_

5 : 1

D C M : C B T_

1 : 1

D C M : C B T_

2 : 1

D C M : C B T_

3 : 1

D C M : C B T_

5 : 1

Solvent

% P C E f f i c i e n c y

HEPS

ACE

Solvent Efficiency Fine Tune Mix (at pH 12 ammonium hydroxide)

0

20

40

60

80

100

120

D C M

C B T

E t O A C

E t O A C : C B T_

1 : 1

E t O A C : C B T_

2 : 1

E t O A C : C B T_

3 : 1

E t O A C : C B T_

5 : 1

D C M : C B T_

1 : 1

D C M : C B T_

2 : 1

D C M : C B T_

3 : 1

D C M : C B T_

5 : 1

Solvent

% P C E f f i c i e n c y

HEPS

ACE

Figure 4. pH 12 1:1 ammonium hydroxide extraction solvent comparison and fine tuning. 5:1 DCM:CBTwas selected as the extraction solvent for both plasma and urine.

HEPS Method Extraction Efficiency

y = 2E+07x

y = 2E+07x

0

50000000

100000000

150000000

200000000

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

ng/mL

I n t e g r a t i o n

Series1

Series2

Figure 4b. Final extraction efficiency (recovery) of HEPS (~ 100 %)

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IX. SEQUENCE ORDER FOR ANALYSIS

A. The sequence order for screening and confirmation is the same, except for unknown samples. Screening samples are sequentially ordered by track andsample, with blanks bracketing the individual track sets. Confirmation isindependent, repeat preparation of all QC and calibrators, with target samplesprepared in triplicate, and triplicates bracketed by blanks.

1. Blank2. Column Test3. Blank4. Negative Control5. Negative Control +Internal Standard6. QC17. QC28. QC3

9. Blank10. C111. C212. C313. C414. C515. C616. C7

Screening analysis uses no waste injection, to allow for repeat analysis in case of power failure, sequence error, retention drift,or other unforeseen need for reanalysis. Confirmation uses partial loop injection due to lower sample throughput and better repeatability statistics at these concentrations.

17. Blank18. Track A, Samples 1 thru N

19. Blank

BlankTrack A, Sample X1

Track A, Sample X2 Track A, Sample X3 Blank

Repeat as needed for the number of samples for confirmation

20. Repeat 17 thru 19 as needed

21. Blank22. QC123. QC224. QC325. Blank26. C127. C2

28. C329. C430. C531. C632. C733. Blank34. Blank-Standby Method

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X. LIQUID CHROMATOGRAPHIC/MASS SPECTRAL IDENTIFICATION ANDESTIMATION OF 2-1(hydroxyethyl) promazine sulfoxide

A. Instrumentation

1. Thermo-Electron Deca XP Plus ion trap mass spectrometer with Xcalibur V1.3 for system controland data acquisition and processing.

2. Thermo-Electron Surveyor quaternary HPLC pump, autosampler, column compartment and on-line degasser. Autosampler uses no waste injection in screen mode and partial loop injectionmode in confirmation mode.

B. HPLC conditions

1. Ace C18 Analytical Column, 3.0 x 50 mm, 5 micron particle size (Part # ACE –a) 111-0503, Mac-Mod Analytical, 127 Commons Court, PO Box 2600, Chadds Ford, PA

19317).2. LC Guard Column

a) Type: Ace 3 C18 (Part No. ACE-111-0103GD, Mac-Mod Analytical, 127 Commons Court, POBox 2600, Chadds Ford, PA 19317).

b) Dimension: 2.1 × 12.5 mmc) Particle size: 5 micrond) Temperature: ambient

3. Pre-Column Filter Column-Saver (Part # MMCCS210 – Mac-Mod Analytical, 127 CommonsCourt, PO Box 2600, Chadds Ford, PA 19317)

in

LC Conditions

m %A %C μL/min

0 0 80 20 20

1 0.2 80 20 20

2 0.3 80 20 200

3 0.5 80 20 200

4 3 5 95 200

5 4.5 5 95 200

6 4.51 80 20 400

7 5.9 80 20 400

8 5.91 80 20 20

9 6.2 80 20 20

A. 2.3 mM pH 5 Formic Acid

C. Acetonitrile 0.1% Formic Acid

Column MacMod Ace 3μ C-18 50x3 mm PN 111-0503

C. Mass Spectrometer Conditions

1. Positive Ion Electrospray Mode2. Deca XP Plus Acquisition Parameters (W=Wideband Activation)

Segment 1 MSMS % CollisionIsolation

Width Q Time ms Scan Range Monitor Ions

HEPS Scan Event 1 345.2 34 1.3 0.25 30 150-350 300,314

d-3 Codeine Scan Event 2 303.2 33 1.3 0.25 30 150-310 285,243,215

Segment 2

ACE Scan Event 1 327.2 31 1.3 0.25 30 150-335 282,254,240

Other Detected Metabolites

MSMS % Collision

Isolation

Width Q Time ms Scan Range Monitor Ions

343.2 39W 1.3 0.25 30 150-350 298

343.2 39W 1.3 0.25 30 150-350 270

ACE Sulfoxide

Hydroxy ACE

359.2 45W 1.3 0.25 30 150-365 328

361.2 31W 1.3 0.25 30 150-370 330Hydroxy HEPS

Hydroxy ACE Sulfoxide

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XIa. METHOD STATISTICS - STABILITY

Freeze-

0

20

40

60

80

100

0 1 2 3

P e r c e n t R e c o v e r e d

Thaw Plasma (1 ng/mL) Stability of HEPS and ACE

4 5 6 7 8

Cycles

HEPS

ACE

Freeze Thaw Stability of ACE-HEPS Urine @ 10 ng/mL

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

Cycles

R e c o v e r e d

P e r c e n t

HEPS

ACE

Figure 5. Freeze Thaw stability (1 ng/mL in plasma - left) and (10 ng/mL urine - right)for ACE and HEPS through six freeze-thaw cycles.

Figure 6. Temperature stability (1 ng/mL in plasma - left) and (10 ng/mL urine - right)for ACE and HEPS. The ranges through 45oC simulate conditions potentiallyencountered during shipping, handling, and storage of the unknown samples. Thehigher temperature challenges model forced thawing and hydrolysis procedures. ACEappears to be subject to degradation, with the more extreme decline in plasma partiallydue to plasma protein denaturation.

ACE-HEPS Plasma Stability (1 ng/mL) at Temperature (3 hrs)

0

20

40

60

80

100

120

-70 0 5 25 37 65 80

Degrees Centigrade

P e r c e n t R e c o v e r y

ACE-HEPS Urine Stability (10 ng/mL) at Temperature (3 hrs)

0

20

40

60

80

100

120

-70 0 5 25 37 65 80

Degrees Centigrade

R e c o v e r e d

%

HEPS

ACE

ACE-HEPS Urine Stability (10 ng/mL) at Temperature (3 hrs)

0

20

40

60

80

100

120

-70 0 5 25 37 65 80

Degrees Centigrade

R e c o v e r e d

%

HEPS

ACE

HEPS

ACE

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XIb. METHOD STATISTICS – PRECISION AND ACCURACY

Hydroxyethyl Promazine Sulfoxide Within Run Precision as % Difference From Mean

-40.000

-30.000

-20.000

-10.000

0.000

10.000

20.000

30.000

40.000

0 2 4 6 8

ng/mL

% D i f f e r e n c e F r o m M e a n

10

Hydroxyethyl Promazine Sulfoxide Between Run Precision as % Difference From Mean

-25

-20

-15

-10

-5

0

5

10

15

20

25

30

0 2 4 6 8

ng/mL

%

D i f f e r e n c e F r o m

M e a n

10

Figure 7. HEPS plasma within run (top) and between run (bottom) precision (σ=3).

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Within Run Precision and Accuracy

n=6

ng/mL CV Accuracy

0.10 25.13 91.45

0.25 17.65 100.62

0.50 13.84 102.20

0.75 6.41 98.19

1.00 8.46 113.79

5.00 7.12 98.61

10.00 5.05 102.39

Between Run Precision and Accuracy

n=7

ng/mL CV Acc

0.10 22.53 88.79

0.25 17.68 102.13

0.50 11.23 100.51

0.75 13.05 108.98

1.00 7.86 107.85

5.00 5.89 97.13

10.00 4.28 99.92

Figure 10. Within Run (left) and Between Run Precision and Accuracy

Pre-Run Versus Post Run Calibrators

y = 0.9827x

0.000

2.000

4.000

6.000

8.000

10.000

0.000 2.000 4.000 6.000 8.000 10.000

Pre-Run Calibration ng/mL

P o s t R u n C a l i b r a t i o n

n g / m L

Figure 8. Youden Plot of Plasma calibrator pairs from analyses of greater than 40 plasma injections (excluding calibrators,

blanks and controls. Plot indicates a representation of combined inter-assay and intra-assay variation (range represents ~3σ).

PETRL QUANTITATION PROCEDURE2-(1-hydroxyethyl)promazine sulfoxide plasma 0.5 ng/mL Control

95% Interval

9 / 2 6 / 0 3

1 0 / 1 2 / 0 3

1 0 / 1 7 / 0 3

1 2 / 4 / 0 3

1 2 / 1 2 / 0 3

1 2 / 1 5 / 0 3

1 2 / 1 5 / 0 3

3 / 1 2 / 0 4

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

( n g / m L )

Control Mean

Historical Control Mean

Cum Control L95% CI

Cum Control U95% CI

Figure 9. Control Plot of 0.5 ng/mL plasma control data (n=4; mean of 2 calibrators, 2 controls) from analyses of greater than30 plasma injections (excluding calibrators, blanks and controls). Dispersion is reduced due to averaging of replicates. Plot

indicates a representation of Type A method uncertainty (95% ~2σ) at this concentration.

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XIc. METHOD STATISTICS – LIMITS

This SOP is intended strictly for screening and confirmation purposes, but quantitativeconsiderations are included to characterize method performance, deterioration of system responsiveness, and as value added estimates for positive test results. Thepositive threshold for this SOP is the limit of confirmation of the principle analytehydroxyethyl promazine sulfoxide (HEPS) in plasma. This SOP may be used for confirmation of any urine analyte reported, but quantitative estimates are limited toAcepromazine (ACE) and HEPS due to the availability of standard reference material.

A. Limit of detection in plasma of HEPS and ACE is less than 50 pg/mL, andless than 100 pg/mL in urine. Limit of detection is defined as the ability of automatic integration to report estimated concentrations that represent achromatographic signal to noise level greater than 5:1 for the respectivequantifying ions (or sum of quantifying ions).

B. Limit of quantitation of HEPS and ACE is100 pg/mL in plasma, and 250

pg/mL in urine. Limit of quantitation is defined as +/- 25% residual for therespective calibrator. The low end calibrators primarily serve as anindicator of system integrity and response.

C. Limit of confirmation of HEPS and ACE in plasma is ~100 pg/mL, and~300 pg/mL in urine. Limit of confirmation is defined as all qualifying anddiagnostic ions present +/- 30% relative abundance, with no interferingions > than 20%.

XId. Criteria for Identification of HEPS from Equine Urine and Plasma

Identification of HEPS and ACE

The diagnostic ions for HEPS are 314, 300, 256, and 238 m/z and the retentiontime of HEPS for the suspect sample, calibrator, and QC control must agree to+/- 0.15 minutes.

The diagnostic ions for ACE are 282, 254, and 240 m/z and the retention time of ACE for the suspect sample, calibrator, and QC control must agree to +/- 0.15minutes.

Confirmation of HEPS and ACE

All diagnostic ions for HEPS and ACE are present in the full scan MSMSspectrum of the suspect sample ,+/- 25% relative, of that ion compared with

calibrator and control spectra, with no interfering ions > than 20%. Spectra maybe averaged and/or subtracted.

Primary quantification of HEPS will be based on plasma. Urine quantificationdata is included for informational (or requested) purposes only. ACE is onlydetectable within 4 hours post administration in plasma, and is quantifiable andconfirmable within 2 hours post administration (25 mg Acepromazine, IV). The

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presence of confirmable plasma ACE indicates administration within 2 hours of sample collection.

XII. SEQUENCE OF POSITIVE SAMPLE DATA PACKET

A. SAMPLE TRANSFER SHEET (WS#32)B. SAMPLE USAGE SHEET (FORM #7)C. SAMPLE LISTD. COLUMN TEST CHROMATOGRAME. LC METHODF. MS METHODG. EXTRACTED ION CHROMATOGRAM COMPARISONH. SPECTRA COMPARISONI. CONFIDENCE REPORTJ. QUANTIFICATION REPORTK. QUANTIFICATION CALIBRATION CURVE

Other Required Documentation

In addition to the positive data packet, the following documentations are alsorequired:

Sequence Sample list print-outs are maintained in the Deca XP three ringbinder.Instrument usage logbook completion (and maintenance log if needed),indicating date and project.

Data packets for samples determined to be negative will contain the followingelements:

a. Sample Transfer Sheet (WS # 32)b. Sample Usage Sheet (Form #7)c. Confidence Determination Reportd. Quantification Report

XV. REAGENTS

A. Methanol, Optima grade (Cat. No. A 454-4, Fisher Scientific.)B. Acetonitrile, Optima grade (Cat. No. A 996-4, Fisher Scientific.)C. Water, Optima grade (Cat. No. W7-4, Fisher Scientific.)D. Dichloromethane, HPLC grade (Cat. No. 9315-03 , J.T. Baker)E. Formic Acid, SupraPur (Cat. No. 11670-1, EM Science)F. Ammonium hydroxide 28% (Cat. No. AX1303-3,EM Science)G. Water, HPLC grade (Cat. No. 4218-03, J.T. Baker)H. Chlorobutane, HPLC grade (Cat. No. CX0914-1 , EM Science)

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XVI. FORMULAE

Safety Requirements: Lab coat, fume hood, gloves, safety glasses. CAUTION: Strong alkali solutions generate heat upon mixing.

A. 0.1% Formic Acid: (Using E. and C. above), add 1000 uL Formic Acid (E.) to1000 L of Water (C.). Mix thoroughly. Cap, label.

B. 2.3 mM Formic Acid: (Using E. and C. above), add 400 uL Formic Acid (E.) to1000 mL of Water (C.). Mix thoroughly. Check pH. Adjust to pH 5.0 with 1:1Ammonium Hydroxide: Water. Cap, label, and record pH.

C. 1:1 Ammonium Hydroxide: Water: (Using F. and G. above), add 500 mLAmmonium Hydroxide (F.) to 500 mL HPLC grade water (G.). Mix thoroughly,Cap, label.

D. 50:50:1: (Using B, C, and E above), add 10 mL Formic Acid (E.) to 500 mLWater (C.) and Acetonitrile (B.).

E. Saturated Sodium Borate, pH 10: (Using H., G., and F. above), add sufficientSodium Borate (G.) to 1 L HPLC grade water (H.), till no more dissolves. Heatwith stirring. Continue to add (G.) until no more dissolves. Cool mixture. Adjustto pH 10 with 50% Sodium Hydroxide (F.).

XVII. MATERIALS

A. 16 × 100 mm test tubes.

B. 16 × 125 mm screw-top test tubes.

C. 16 × 150 mm screw-top test tubesD. Polypropylene CapsE. Test tube rackF. Pipettes and tips.G. RotorackH. CentrifugeI. Vortex mixer (Scientific Industries, Inc.)J. Branson Ultrasonic Water Bath, 8510 (Fisher Scientific or equivalent)K. pH meter ( IQ Scientific Instruments )L. Sample Concentrator (Dri-Block DB-3, Techne)M. IEC HN-SII Centrifuge (International Equipment Company)N. Rotorack (Speci-Mix, Thermolyne)

O. 2 mL autosampler vials and capsP. 200 uL Insert

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XVIII. Clearance Profiles of ACE and HEPS

Aepromazine Plasma Clearance 25 MG IV

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0 5 10 15 20

Hours

n g / m L

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Hours

n g / m L

2-1(hydroxyethyl) promazine sulfoxide Plasma Clearance25 MG IV Acepromazine Maleate

0.000

1.000

2.000

3.000

4.000

5.000

6.000

7.000

0 10 20 30 40 50 60 70 80

Hours

n g / m

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

20 30 40 50 60 70 80

1000 uL Plasma Sample Preparation

L

2-1(hydroxyethyl) promazine sulfoxide Urine Clearance 100 uL

25 MG IV Acepromazine Maleate

0.000

100.000

200.000

300.000

400.000

500.000

600.000

700.000

800.000

900.000

0 10 20 30 40 50 60 70 8

Hours

n g / m

0

L

0.000

5.000

10.000

15.000

20.000

25.000

30.000

35.000

40.000

45.000

20 30 40 50 60 70 80

Figure 10. Plasma Clearance of ACE (top panel), Plasma Clearance of HEPS (middle panel), and urineclearance of HEPS (bottom panel) following 25 MG IV

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_____________________________________________________________________________________________________________________________________

5 MG IV Acepromazine 2-1 HEPS Plasma Clearance

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.160

0.180

0.200

0 5 10 15 20 25 30 35 40 45

Hours Post Administration

n g / m L

5 MG IV Acepromazine 2-1 HEPS Urine Clearance

0.000

5.000

10.000

15.000

20.000

25.000

30.000

35.000

40.000

45.000

50.000

0 5 10 15 20 25 30 35 40 45


n g / m L

Figure 11. Plasma Clearance of ACE (top panel), Plasma Clearance of HEPS (middle panel), and urineclearance of HEPS (bottom panel) following 5 MG IV

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_____________________________________________________________________________________________________________________________________

Acepromazine IV Administrations 2-1 HEPS Urine Clearance

0.001

0.010

0.100

1.000

10.000

100.000

1000.000

0 10 20 30 40 50 60 70


n g / m L

5 MG Plasma

5 MG Urine

25 MG Plasma

25 MG Urine

Figure 12. Combined log plot of plasma and urine clearance of HEPS from 5 MG(blue) and 25 MG (red) IV administration.

Urine Acepromazine Analytes Retention Times and Spectra

RT: 0.00 - 5.50 SM: 3B

0 1 2 3 4 5

Time (min)

0

50

100

0

50

1000

50

100

0

50

100

0

50

100

0

50

100

2.07

2.33

4.58

2.39

2.42

4.04

4.12

4.53

NL: 6.10E5

m/z= 297.50-298.50+315.50-316.50+329.50-330.50F: + c ESI w Full ms2 [email protected] [

95.00-380.00] MS

102003_acenbc_p05_031020124831

NL: 2.35E5

m/z= 287.50-288.50+310.50-311.50+340.50-341.50

F: + c ESI w Full ms2 [email protected] [

95.00-370.00] M S 102003_acenbc_p08

NL: 3.33E7

m/z= 299.50-300.50+313.50-314.50 F: + c ESI Full

ms2 [email protected] [ 95.00-350.00] MS

102003_acenbc_p08

NL: 2.49E5

m/z= 294.50-295.50+311.50-312.50 F: + c ESI Full

ms2 [email protected] [ 90.00-350.00] MS

102103_AceNBC_eh04

NL: 2.85E6

m/z= 255.50-256.50+269.50-270.50+297.50-298.50

F: + c ESI w Full ms2 [email protected] [

90.00-350.00] MS 102103_AceNBC_eh04

NL: 1.88E6

m/z= 281.50-282.50 F: + c ESI Full m s2

[email protected] [ 150.00-335.00] MS

101803_acenbc_p02

OH-HEPS

OH-AS

HEPS

OH-A

Ace

AS

Primary Analyte

Figure 13. Detected urine metabolites of Acepromazine elution profile (above) andcorresponding MSMS spectra (below)

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_______ ______________________________________________________________________________________________________________________________

220 240 260 280 300 320 340 360 380

m/z

7

0

5

4

0

2

0.6

0.0

0.2

0.4

100

0

50

R e l a t i v e A b u n d a n c e

0.7

0.0

1.7

0.0

0.5

1.0

330

298316259 361283254 272 299

224 246

328

288 311257 314297359254 272 332286 335228 327241

314

300

256238

345282243 283 328212 223 279

298

238 281254 312255

208 295 325242212 265226

270298

256

255307 343265

282

240254

327239281256

102003_acenbc_p05_031020124831#18

5 RT: 2.07 AV: 1 NL: 4.72E5 F: + c ESI

w Full ms2 [email protected] [

95.00-380.00]

102003_acenbc_p08#219 RT: 2.36

AV: 1 NL: 1.92E5 F: + c ESI w Full ms2

[email protected] [ 95.00-370.00]

102003_acenbc_p08#220 RT: 2.36

AV: 1 NL: 2.77E7 F: + c ESI Full ms2


102103_AceNBC_eh04#229 RT: 2.44



102103_AceNBC_eh04#418 RT: 4.04

AV: 1 NL: 1.15E6 F: + c ESI w Full ms2


101803_acenbc_p02#502 RT: 4.52



OH-HEPS

OH-AS

HEPS

OH-A

Ace

AS

Primary Analyte

Plasma Acepromazine Analytes Retention Times

RT: 0.00 - 5.52 SM: 3B

0 1 2 3 4 5

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

2.284.03

2.39

2.55

4.56

4.55

NL: 3.15E5

m/z=

297.50-298.50+315.50-316.50+329.50-330.50 F: +

c ESI w Full ms 2 [email protected] [ 95.00-380.00]

MS 102203_acenbc_pl10

NL: 3.15E5

m/z=287.50-288.50+310.50-311.50+327.50-328.50 F: +


MS 102203_AceNBC_pl14_031023074056

NL: 7.00E6

m/z= 299.50-300.50+313.50-314.50 F: + c ESI Full

ms 2 [email protected] [ 95.00-350.00] MS

102203_AceNBC_pl14_031023074056

NL: 3.15E5

m/z= 297.50-298.50+311.50-312.50 F: + c ESI w

Full ms2 [email protected] [ 90.00-350.00] MS

102203_AceNBC_pl10_031023075611

NL: 3.15E5

m/z= 269.50-270.50+297.50-298.50 F: + c ESI w

Full ms2 [email protected] [ 90.00-350.00] MS102203_acenbc_pl06

NL: 3.15E5

m/z=

239.50-240.50+253.50-254.50+281.50-282.50 F: +

c ESI Full ms 2 [email protected] [ 90.00-350.00] MS

102203_AceNBC_pl08NL: 3.15E5

m/z=

241.50-242.50+255.50-256.50+283.50-284.50 F: +


MS 102203_AceNBC_pl14_031023074056

OH-HEPS

OH-AS

HEPS

OH-A

Ace

AS

HEP

Primary Analyte

Figure 14. Extracted ion chromatograms of Acepromazine plasma metabolitesfollowing base extraction conditions previously described (NOTE: Acepromazine isdetected only at low pg/mL plasma levels for no more than 4 hours). Proposedmetabolic pathway (below) based on detected analytes. (NOTE: HEP detected inGC/MS analysis appears to be a thermal artifact of HEPS.)

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OH-HEPS

OH-AS

OH-A Ace

ASHEP

HEPS

S

N

2HC CH2

CH2

N

CH3

CH3

C

O

CH3

S

N

2HC CH2

CH2

N

CH3

CH3HC

OH

CH3

S

N

2HC CH2

CH2

N

CH3

CH3

C

O

CH3

O

S

N

2HC CH2 CH2 N

CH3

CH3HC

OH

CH3

O

S

N

2HC CH2

CH2

N

CH3

CH3

C

O

CH3

HO

S

N

2HC CH2

CH2

N

CH3

CH3

C

O

CH3

HO

O

S

N

2HC CH2 CH2 N

CH3

CH3HC

OH

CH3

O

HO

Low plasma levels, rapidly cleared,

poorly reabsorbed

HEPS primary plasma and urine analyte,

proceeding via AS

No detectable HEP in urine or pla sma,

presence in GC/MS due to thermolytic

decomposition of HEPS

Acepromazine Oxidation ProductsRT: 0.45 - 5.50 SM: 5B

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100


2.47

4.52

2.46

NL: 4.49E8

m/z= 342.50-343.50 F:

+ c ESI Full ms [

300.00-380.00] MS

040604_HEPSACE_Re

cov_13_040407121630

1

2

3

MS of 343 m/z

TIC of MSMS 343 m/z

NL: 3.27E8

TIC F: + c d Full m s2

[email protected] [

80.00-355.00] MS

040604_HEPSACE_Re

cov_13_040407121630

Figure 15. Peaks detected by MS Data Dependant analysis of degraded Acepromazinecontrol stock solution

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_____________________________________________________________________________________________________________________________________

200 220 240 260 280 300 320 340

m/z

8

0

2

4

6

1.7

0.0

0.5

1.0


100

0

20

40

60

80

312

295298

256

238 343255 280270208 281

268212

326249224 311298

295 312

256 343

238 270

255

281254 269225 311212208 326 344

240

312

040604_HEPSACE_Recov_13

_040407121630#428 RT: 2.46

AV: 1 NL: 5.89E7 F: + c d Full

ms2 [email protected] [

80.00-355.00]


_040407121630#716 RT: 3.82

AV: 1 NL: 1.00E6 F: + c d Full

ms2 [email protected] [

80.00-355.00]


_040407121630#871-879 RT:

4.56-4.59 AV: 4 NL: 4.84E6 F:

+ c d Full ms 2 [email protected] [

80.00-355.00]

1

2

3

Figure 16. Spectra obtained by MS Data Dependent analysis of degradedAcepromazine control stock solution

Acepromazine instability indicated by studies cited previously appears to be primarilyoxidative in nature, with S-oxide, N-oxide, and N-hydroxyl as possibilities.

REFERENCES:

1. Analysis of Acepromazine and Metabolite in Horse Serum, Gargi Choudhary,Thermo Electron, Wayne Skinner and Scott Stanley, University of California Davis,

Thermo Electron Application Note

2. Uniform Classification Guidelines for Foreign Substances and RecommendedPenalties and Model Rule, Rev January 3, 2003, Association of RacingCommissioners International Inc., RCI Drug Testing Standards and Practices Program

3. NTRA Racing Integrity and Drug Testing Task Force Report, September 2003,National Thoroughbred Racing Association

4. Proposal For a National Policy on Drug Testing and Therapeutic Medication, October 2001, National Horsemen’s Benevolent & Protective Association, Inc.

5. The pharmokinetics, pharmacological responses and behavioral effects of

acepromazine in the horse, S. Ballard, T. Shultes, J. Blake, T. Tobin, J. Vet.Pharmacol. Therap. 5,21-31, 1982

6. Atypical conditions for quantitative recovery of Acepromazine andchlorpromazine from plasma, S. Ballard, T. Tobin, J. Tox. & Environ Health , 7, 745-751, 1981

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