Compendium of Analytical Laboratory Methods for Food

and Feed Safety: Chemical Analytical Manual (CAM)

METHOD NUMBER: C-005.03

POSTING DATE: original posting 2/1/2019; Chemistry Research Coordination Group

posting extension, 12/2020.

POSTING EXPIRATION DATE: 1/31/2023

PROGRAM AREA: Chemical Contaminants and Toxins

METHOD TITLE: Determination of pentobarbital in ingredients of animal origin and in

finished pet foods using liquid chromatography tandem mass spectrometry.

VALIDATION STATUS: Level 2, Single laboratory validation

AUTHOR(S): Tara J. Nickel and Christine R. Casey, US FDA Office of Regulatory

Affairs/Office of Regulatory Science/Office of Food and Feed Laboratory Operations/ Denver
Laboratory

METHOD SUMMARY/SCOPE:
Identification and quantitative determination of pentobarbital in tallow and other
grease products of animal origin using Liquid Chromatography-Tandem Mass Spectrometry
(LC-MS/MS), using the negative electrospray ionization (ESI) mode. Pentobarbital is extracted
from a homogenous portion of tallow, wet dog food, dry dog food, or horsemeat using
acetonitrile. After centrifugation, the supernatant is diluted 1:1 with water and analyzed via
LC-MS/MS using a solvent standard curve. Deuterated pentobarbital (pentobarbital-D5) is used
as an internal standard (I.S.) to correct for sample matrix suppression and/or loss of analyte.
Identification of pentobarbital in a sample is based on both correlation of pentobarbital
chromatographic retention time (RT) with that of a standard and ion ratio match.
Analytes(s): Pentobarbitol
Matrices: Tallow and other grease products of animal origin, horsemeat, wet (canned) dog
food, and dry dog food, wet (canned) and dry cat food, meat/bone meal, and meats including
horse, beef, lamb, pork, goat, venison, bison, elk, rabbit, liver, and kidney.

REVISION HISTORY: Revised 7/18/2019. Revision expanded the scope of method from
tallow and grease products to include horsemeat, wet (canned) dog food, and dry dog food.
Revised 2/1/2020: Scope was expanded to include wet (canned) and dry cat food; meat/bone
meal, and meats including horse, beef, lamb, pork, goat, venison, bison, elk, rabbit, liver, and
kidney.
 FOOD AND DRUG ADMINISTRATION Document Number:
Revision #: 04
OFFICE OF REGULATORY AFFAIRS WI-000140
Revised: Nov. 2019
Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 1 of 34
foods using liquid chromatography tandem mass spectrometry.

Sections in This Document

1. Purpose ................................................................................................................................. 2
2. Scope .................................................................................................................................... 2
3. References ............................................................................................................................ 2
4. Procedure .............................................................................................................................. 3
4.1. Summary of Method .................................................................................................... 3
4.2. Preparation of Samples ............................................................................................... 4
4.3. Preparation of Standards............................................................................................. 5
4.4. Reagents ..................................................................................................................... 8
4.5. Equipment (equivalent equipment may be substituted)................................................ 8
4.6. Instrumentation (equivalent instrumentation may be substituted) ................................ 9
Analyte ......................................................................................................................................... 9
4.7. Extraction .................................................................................................................. 11
4.8. Data Analysis and Quality Acceptance ...................................................................... 12
5. Glossary .............................................................................................................................. 16
6. Records ............................................................................................................................... 16
7. Document History ................................................................................................................ 17
8. Change History .................................................................................................................... 17
9. Attachments......................................................................................................................... 17
Attachment A - Representative chromatograms ................................................................... 18
The chromatograms are graphical images are generated in the SCIEX MultiQuant software summary
report. There is a chromatogram for each of the pentobarbital transitions. The first column is
the pentobarbital quantitation ion transition (most abundant ion) chromatogram (m/z 225→182),
identified as Pentobarbital 1. The second column is the chromatogram for the second most
abundant ion transition (m/z 225→85), identified as Pentobarbital 2 and is used as a qualifier ion
transition. The third column is the least abundant ion (m/z 225→ 138), identified as
Pentobarbital 3 and is used as a qualifier transition. For the bone meal chromatograms (Figures
A10 and A11, a fourth transition is included. It is directly below the Pentobarbital 1 figure. It is
identified as Pentobarbital 4 and is the chromatogram for the transition m/z 225→ 42. It is more
abundant than the pentobarbital quant transition and can be used as an alternative qualifier ion
in cases of matrix interference peaks.

For the most current and official copy, check QMiS.

 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 2 of 34
foods using liquid chromatography tandem mass spectrometry.

1. Purpose
This work instruction describes the application of LIB 4648 for the
determination of pentobarbital in ingredients of animal origin and in finished pet
foods using liquid chromatography tandem mass spectrometry.

2. Scope
For routine analysis of regulatory samples of numerous matrices of animal
origin including: tallow (animal fat/grease), wet (canned) dog and cat food, dry
dog and cat food, meat/bone meal, and meats including horse, beef, lamb,
pork, goat, venison, bison, elk, rabbit, liver, and kidney using LC-MS/MS.
Complete method performance details and validation data is summarized in
the FDA QMiS Denver Lab (DENL) validation summary reports (RPRT- 00055,
RPRT- 000060, RPRT- 00089, RPRT-000125, RPRT-000126).
3. References
Determination of Pentobarbital in Pet Food Using Liquid Chromatography
Tandem Mass Spectrometry, Tuner, James A., Mohrhaus, Angie S., FCC
SOP T064 Version 1, 3/1/2018.
A. LC-HRMS Screen CALIFORNIA ANIMAL HEALTH &FOOD SAFETY
LABORATORY, DTOX-02-877 Rev. 6. November 3, 2017.
B. Pentobarbital in Beef Tallow March 2018, Personnel Correspondence
with UC Davis, “Pentobarbital in Beef Tallow March 2018 (004) UC
Davis extraction March 2018” l
C. U.S. Food and Drug Administration, Office of Foods, Guidelines for the
Validation of Chemical Methods for the FDA Foods Program, 2nd Edition
2015.
https://www.fda.gov/downloads/scienceresearch/fieldscience/ucm27341
8.pdf
D. ORA Laboratory Manual, Volume II-Methods, Method Verification and
Validation ORA-LAB.5.4.5
https://www.fda.gov/downloads/ScienceResearch/FieldScience/Laborat
oryManual/UCM092147.pdf
E. DENL QMS # 18-3 and QMiS RPRT-000060 (Determination of
pentobarbital in Tallow, wet pet food, and dry pet food using liquid
chromatography tandem mass spectrometry).

For the most current and official copy, check QMiS.

 FOOD AND DRUG ADMINISTRATION Document Number:
Revision #: 04
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Revised: Nov. 2019
Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 3 of 34
foods using liquid chromatography tandem mass spectrometry.

F. DENL QMS #18-4 and QMiS RPRT-000055 (determination of

pentobarbital in tallow, wet pet food, and dry pet food using liquid
chromatography tandem mass spectrometry).
G. DENL QMS #18-4a and QMiS RPRT-000089 (determination of
pentobarbital in wet dog food, and dry dog food using liquid
chromatography tandem mass spectrometry).
H. DENL QMS #18-4b and QMiS RPRT-000125(determination of
pentobarbital in bone meal using liquid chromatography tandem mass
spectrometry).
I. DENL QMS #18-4c and QMiS RPRT-0000126 (determination of
pentobarbital in meats, wet cat food, and dry cat food using liquid
chromatography tandem mass spectrometry).
J. U.S. Food and Drug Administration (2003) Guideline for Industry: Mass
Spectrometry for Confirmation of the Identity of Animal Drug Residues,
Fed. Regist. 68, 25617–25618.
https://www.fda.gov/downloads/AnimalVeterinary/GuidanceCompliance
Enforcement/GuidanceforIndustry/ucm052658.pdf
K. CVM # GFI 118. Guidance for Industry Mass Spectrometry for
Confirmation of the Identity of Animal Drug Residues
https://www.fda.gov/media/70154/downloadhttp://www.fda.gov/downloads/
AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/UCM0526
58.pdf
L. DENL QMS #18-4a and QMiS RPRT-000089 (determination of
pentobarbital in horsemeat, wet pet food, and dry pet food using liquid
chromatography tandem mass spectrometry).\
M. LIB 4648: Determination of pentobarbital in tallow using liquid
chromatography tandem mass spectrometry (LC-MS/MS) CARTS:
IR01702
http://inside.fda.gov:9003/downloads/PolicyProcedures/Laboratories/La
boratoryInformationBulletins/UCM632922.pdf
4. Procedure
4.1. Summary of Method
Pentobarbital is extracted from a homogenous portion of sample using
acetonitrile. After centrifugation, the supernatant is diluted 1:1 with water and
analyzed via LC-MS/MS using a multi-point solvent standard curve.
Deuterated pentobarbital (pentobarbital-D5) is used as an internal standard

For the most current and official copy, check QMiS.

 FOOD AND DRUG ADMINISTRATION Document Number:
Revision #: 04
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Revised: Nov. 2019
Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 4 of 34
foods using liquid chromatography tandem mass spectrometry.

(I.S.) to correct for sample matrix suppression and/or loss of analyte.

Identification of pentobarbital in a sample is based on both correlation of
pentobarbital chromatographic retention time (RT) with that of a standard(s)
and ion ratio match.
4.2. Preparation of Samples
4.2.1. For tallow, at minimum a 25-gram sample portion is necessary. If sample
appears heterogeneous, stir the 25 g sample manually with a spatula or
spoon to ensure homogeneity.
4.2.2. For meat, at minimum a 50-gram sample portion is necessary. Tissue
should be cut into small pieces, trying to avoid including any overly fatty
portions, and then ground with dry ice in an appropriate blender. Resulting
composite should be a fine powder.
4.2.3. For canned pet food, an entire can (approximately 150 grams or more) of
the canned pet food is necessary. Wet pet food should be ground and
mixed in in an appropriate blender. Do not add water. Dry ice may be
used if necessary.
4.2.4. For dry pet food, approximately 150 grams is necessary. Dry pet food
should be ground and mixed in an appropriate blender. Dry ice may be
used if necessary (effective for products containing chewy ‘bits’).
4.2.5. For bone/meat meal, at minimum 50 grams is necessary. Bone meal
should be ground and mixed in an appropriate blender. Dry ice may be
used if necessary.
4.2.6. If dry ice is used in sample preparation, allow carbon dioxide gas to
sublime in a freezer prior to removing aliquot for analysis.
4.2.7. If a sample appears heterogeneous, it should be mixed manually, or via a
blender to ensure a homogenous sample prior to removing a portion for
analysis.
4.2.8. A 2.00-gram portion is used for each analysis. Two portions of each
sample should be analyzed.

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 5 of 34
foods using liquid chromatography tandem mass spectrometry.

4.3. Preparation of Standards

4.3.1. Pentobarbital and pentobarbital-D5 are ordered premade with
concentration 1000 µg/mL (1 mg/mL) in methanol.
4.3.2. Prepare a pentobarbital standard at 2,500 ng/mL with 25 µL of the 1000
µg/mL stock diluted to 10.0 mL with acetonitrile.
4.3.3. Prepare a pentobarbital ICV standard at 2,500 ng/mL with 25 µL of the
1000 µg/mL stock, from a different source or different ampoule, diluted
to10.0 mL with acetonitrile.
4.3.4. Prepare a pentobarbital-D5 internal standard spiking solution at 2,500
ng/mL with 25 µL of the 1000 µg/mL pentobarbital-D5 stock diluted to 10.0
mL with acetonitrile.
4.3.5. All prepared solutions have an expiration date of one year from the
preparation date when stored at 4oC. If a component of the solution expires
prior to one year of the preparation date, the earliest expiry date becomes
the solution’s expiration date.
4.3.6. Table 4.3.8 and 4.3.9 are examples of the preparations of working solutions
and the solvent calibrants. Table 4.3.10 demonstrates the equivalent
concentrations of the calibrants to the concentration in the samples for use
in the processing method.
4.3.7. All eight calibration standards shown in Table 4.3.10 are not required for
regulatory sample analysis; however, a minimum of 5 calibration standards
are used with every batch of samples with a 10.0 ng/g standard (in sample
amount), which is equivalent to an in vial concentration of 1.0 ng/mL as the
lower limit of quantitation (LLOQ).
4.3.8. Working Solution Preparation (in acetonitrile)
starting
Final vol Final
concentration Standard added
Standard Name concentration
(mL)
(mL) (ng/mL)
(µg/mL)

Pentobarbital-2500 1,000 0.025 10.0 2,500

Pentobarbital ICV 1,000 0.025 10.0 2,500

Pent-D5 Spiking
1,000 0.025 10.0 2,500
(ISTD)

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 6 of 34
foods using liquid chromatography tandem mass spectrometry.

4.3.9. Solvent Calibration Standard Preparation (in 50:50 acetonitrile:water)

Initial conc volume of Volume D5- Final Final Conc Final Conc
Calibration
pentobarbital pentobarbital std ISTD Added Volume pentobarbital d5-Pent
Curve
(ng/mL) added (mL) (2500 ng/mL) (mL) (ng/mL) (ng/mL)

Cal-1 2,500 0.020 0.100 50.0 1.00 5.00

Cal-2 2,500 0.010 0.040 20.0 1.25 5.00
Cal-3 2,500 0.010 0.020 10.0 2.50 5.00
Cal-4 2,500 0.020 0.020 10.0 5.00 5.00
Cal-5 2,500 0.040 0.020 10.0 10.00 5.00
Cal-6 2,500 0.100 0.020 10.0 25.00 5.00
Cal-7 2,500 0.200 0.020 10.0 50.00 5.00
Cal-8 2,500 0.400 0.020 10.0 100.00 5.00
ICV 2,500 0.040 0.020 10.0 10.00 5.00

4.3.10. Conversion of Solvent Standards to In Sample Concentration

Equivalent
Equivalent
Volume of d5-pent
In Vial Final Sample weight. pentobarbital in
Calibration ACN (ISTD) In
Concentratio Dilution matrix
Curve extraction matrix
n (ng/mL) (g) concentration
(mL) concentration
(ng/g)
(ng/g)

Cal-1 1.00 2.00 10 2.00 10.0 50.0

Cal-2 1.25 2.00 10 2.00 12.5 50.0
Cal-3 2.50 2.00 10 2.00 25.0 50.0
Cal-4 5.00 2.00 10 2.00 50.0 50.0
Cal-5 10.0 2.00 10 2.00 100 50.0
Cal-6 25.0 2.00 10 2.00 250 50.0
Cal-7 50.0 2.00 10 2.00 500 50.0
Cal-8 100.0 2.00 10 2.00 1000 50.0
ICV 10.0 2.00 10 2.00 100 50.0

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 7 of 34
foods using liquid chromatography tandem mass spectrometry.

4.3.11. Fortified (spiked) sample preparation (demonstrates typical fortification

levels for pentobarbital in sample)
initial
concentration volume used sample final concentration in
Spike Level (ng/g) (ng/mL) (mL) weight (g) sample (ng/g)

10 ng/g 2500 0.008 2.00 10.0

50 ng/g 2500 0.040 2.00 50.0
250 ng/g 2500 0.200 2.00 250

4.3.1. Example spiking calculation:

40 μL 2500 ng = 50.0 ng

2.00 grams of negative control mL g

4.3.2. Example calculation of in vial versus in sample concentration

50 ng 2 g sample amount 0.500 mL extract = 5.00 ng in vial

g 10 mL extraction volume 1.00 mL final volume mL

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 8 of 34
foods using liquid chromatography tandem mass spectrometry.

4.4. Reagents
4.4.1. Water, Fisher, LC-MS grade
4.4.2. Acetonitrile, Fisher, LC-MS Grade
4.4.3. Pentobarbital, Cerilliant, 1.000 ± 0.005 mg/mL in methanol, 1 mL ampoule,
part # P-010
4.4.4. Pentobarbital-D5, Cerilliant, 1.000 ± 0.005 mg/mL in methanol, 1 mL
ampoule, part #: P-013
4.4.5. Diluent for standards, 50/50 water/acetonitrile (v/v), 500 mL. Combine 250
mL water + 250 mL acetonitrile in a 500 mL graduated cylinder. Cap and
invert to mix.
4.5. Equipment (equivalent equipment may be substituted)
4.5.1. Vortexer/ Mixer, Troemner, (500-2500 rpm)
4.5.2. SPEX Geno/Grinder 2000 (500 rpm)
4.5.3. Sonicator (Branson 2510 or 8510)
4.5.4. Appropriate mixers, blenders, food processors, etc. used to homogenize
sample matrix if necessary
4.5.5. Centrifuge capable of 6000 rpm with refrigeration (4 °C) for 50 mL tubes
4.5.6. Plastic centrifuge tubes with caps, 15 mL and 50 mL
4.5.7. Microcentrifuge tubes, at least 1 mL capacity
4.5.8. Nylon syringe filters, PALL Life Science Acrodisc 13 mm Syringe Filters 0.2
µm
4.5.9. Luer slip 1 mL syringes
4.5.10. 2 mL glass amber autosampler vials and pre-slit snap caps (#66030-608)
4.5.11. Calibrated pipettes and volumetric glassware

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 9 of 34
foods using liquid chromatography tandem mass spectrometry.

4.6. Instrumentation (equivalent instrumentation may be substituted)

4.6.1. Agilent 1200 HPLC system, Combi Pal autosampler, with an AB SCIEX
5500 QTRAP MS instrument operated in the negative mode with an ESI
source using Multiple Reaction Monitoring (MRM). AB SCIEX 1.6.2
software was used for instrument control and MultiQuant 3.0 was used for
data processing.
4.6.2. AB SCIEX QTRAP 5500 source/parameter settings:
A. Curtain gas: 30 psi
B. GS1: 50 psi
C. GS2: 60 psi
D. Collision gas: medium
E. Ion spray voltage (IS): -3500V
F. Source temperature: 400°C
G. Entrance Potential (EP): -10V
4.6.3. Pentobarbital MS parameters: Retention times (RT), transitions,
declustering potential (DP), collision energy (CE), cell exit potential (CXP),
and the resulting typical ion ratios for the product ions of each analyte from
the ABI SCIEX 5500 QTRAP analysis.

Typical Average
RT Transition DP CE CXP ion ratio,
qual/quant
(min) (m/z) ISTD (V) (V) (V) %
Analyte
182 -19 -13 100

85 -100 -18 -9 15
pentobarbital 4.20 225 → Pent-D5
138 -21 -10 7

42* -46 -7 275*

pentobarbital-D5 4.20 230 → 187 -100 -17 -10 N/A

*additional transition of 225→42 was added as additional/alternative confirmation criteria due to matrix
interference peak for 225→85 transition in some bone meal sources at <10 ppb level. The 225→42 transition is
more abundant than the 225→82 transition, giving the ion ratio of >100%.

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 10 of 34
foods using liquid chromatography tandem mass spectrometry.

4.6.4. Combi Pal autosampler settings

A. sample injection volume: 5 µL
B. autosampler tray temperature: 15ºC
C. Combi Pal Injector wash solution 1: 95% water/5% acetonitrile
D. Combi Pal Injector wash solution 2: 95% acetonitrile/5% water
4.6.5. Agilent 1200 HPLC settings
A. HPLC column – Agilent Zorbax Eclipse Plus C18, 2.1 x 50 mm, 1.8
micron size column (part # 959757-902)
B. A divert valve directed column effluent to waste before (0-0.5 minutes),
and during the system re-equilibration time (5.0-8.50)
C. Column temperature was 40oC
D. LC flow rate was 0.350 mL/min.
E. The mobile phase was water (A) and acetonitrile (B), and the LC
gradient is described in Table 4.6.5.F below
F. LC gradient for pentobarbital
@Step Total Time Flow Rate A (%) B (%)
(min) (µl/min) (Acetonitrile)
(Water)

0 0.00 350 95.0 5.0

1 3.50 350 5.0 95.0

2 4.50 350 95.0 5.0

4 8.50 350 95.0 5.0

LC note: It has been observed that any mobile phase modifiers (formic acid,
ammonium formate, etc) have a significant negative effect on pentobarbital
response. Ensure LC lines have been thoroughly cleaned and purged with
water and acetonitrile before analysis.

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 11 of 34
foods using liquid chromatography tandem mass spectrometry.

4.7. Extraction
A. Weigh 2.00± 0.05 g of each homogenized sample into a 50 mL centrifuge
tube. For each unknown sample, weigh out two portions.
B. For each batch, include an empty tube to serve as Reagent Blank (RB).
C. Weigh out three portions of negative control material to serve as negative
control (NC), matrix spike (SPK), and matrix spike duplicate (DUP).
D. For all samples in the batch, including RB, NC, SPK, and DUP, add 40 µL
of 2500 ng/mL pentobarbital-D5, resulting in a 50 ng/g d5-pentobarbital
concentration.
E. Fortify spike (SPK) and duplicate (DUP) portions with 40 µL of 2500 ng/mL
pentobarbital spiking standard, resulting in a 50 ng/g pentobarbital
concentration in sample. (Fortification level may be adjusted as necessary,
as long as the concentration falls within the calibration curve).
F. If a sample appears to be significantly different than an available control
matrix, an additional portion of sample should be spiked to assess
extraction efficiency and matrix effects compared to the most similar control
matrix.
G. Add 10 mL of acetonitrile to each sample tube.
H. Cap tubes tightly and shake @ 500 rpm for 5 minutes on the geno grinder.
I. Sonicate sample tubes for 30 minutes.
J. Vortex sample tubes for 30 seconds.
K. Centrifuge sample tubes at 6000 x g and 4°C for 10 minutes.
L. Combine 500µL of sample supernatant with 500µL of water in a
microcentrifuge tube; vortex to mix, then filter using a 0.2 µm Nylon syringe
filter into a 2 mL amber LC vial and cap.
M. Analyze via LC-MS/MS.

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Determination of pentobarbital in ingredients of animal origin and in finished pet Page 12 of 34
foods using liquid chromatography tandem mass spectrometry.

4.8. Data Analysis and Quality Acceptance

4.8.1. Calibration curves are established from a multi-point solvent calibrant
standard curve, typically ranging from 1-50 ng/mL in vial, equivalent 10-500
ng/g in sample, with the concentration on the x-axis and internal standard
corrected peak response on the y-axis. Suggested seven-point curve for
routine sample analysis is 1.00, 1.25, 2.5, 5.0, 10.0, 25.0, 50.0 ng/mL
(equivalent to 10.0, 12.5, 25, 50, 100, 250, 500 ng/g in sample).
4.8.2. Quantitative results will be reported for samples with responses that fall
within the standard curve range and meet identity confirmation criteria.
4.8.3. The calculated method limit of detection (MDL) and calculated method limit
of quantitation (LOQ), and typical linearity (r2 ) are indicated in Table 4.8.4.

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 13 of 34
foods using liquid chromatography tandem mass spectrometry.

4.8.4. MDL, LOQ, Linearity Table

Average
MDL (ng/g) LOQ (ng/g)
Matrix Linearity
n=7* n=7*
(r2)

Finished Pet Food

Dry Dog Food 2.9 9.3 0.9989

Wet Dog Food 2.5 7.8 0.9994

Dry Cat Food 1.8 5.8 0.9999

Wet Cat Food 1.0 3.2 1.0000

Rendering Streams
Tallow 3.6 11.6 0.9991

Bone Meal * n=9 2.3 8.0 0.9991

Meat Products
Beef 1.6 5.2 0.9997

Bison 1.0 3.2 0.9998

Elk 1.2 3.9 0.9994

Goat 1.3 4.2 0.9998

Horse 0.9 2.9 0.9991

Lamb 0.9 3.0 0.9998

Pork 0.9 3.0 1.0000

Rabbit 1.6 5.2 0.9999

Venison 0.8 2.6 1.0000

Kidney (Bison) 1.8 5.7 0.9999

Liver (Beef) 1.6 5.0 0.9997

Average for all matrices: 1.6 5.3 0.9996

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 14 of 34
foods using liquid chromatography tandem mass spectrometry.

4.8.5. The tallow MDL and LOQ data in LIB 4648 were originally calculated from
12.5 ng/g spikes (n=9), resulting in calculated MDL of 2.4 ng/g and LOQ of
8.2 ng/g). Additional tallow spikes (n=7) were analyzed at 5 ng/g and 10
ng/g – and the MDL and LOQ were re-calculated using that data, which is
in Table 4.8.4 above.
4.8.6. All calibration curves were generated with the AB SCIEX MultiQuant
software. Depending on the range of the calibration curve, a linear fit with
no weighting or with 1/x weighting (not forced through zero) can be used for
all recovery calculations. If a smaller dynamic range is used, a linear curve
with no weighting should be used. All calibration curves should have r2
≥0.995.
4.8.7. Precision and accuracy general guidance (performance may vary with
sample matrix, especially for different tallow/animal fat matrices):
A. The FDA OFVM specifies that analyte recovery should be within the
range 60%-115% corresponding to concentration from 10-100 mg/kg
(ppb), with RSDr ≤ 22%. Recoveries for elk (123%) and beef liver
(121%) were >115% at the 10 ng/g spiking level. Higher recoveries
at the low spiking level may be due to the use of a linear curve
without weighting.
B. The aforementioned ranges are for guidance on data acceptability.
Ranges for precision and accuracy should be determined in house
after a sufficient number of data points are obtained.
C. Reagent blank (RB) and negative control (NC) should have
responses below the lowest calibration standard.
D. Validation data demonstrated satisfactory quantitative analysis for
pentobarbital determined in all matrices at spiking levels 10, 50, and
250 ng/g, with method accuracy generally ranging from 85-123%,
with RSDr ≤ 16%.
E. Any QC failures must be investigated prior to reporting results.

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Revised: Nov. 2019
Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 15 of 34
foods using liquid chromatography tandem mass spectrometry.

F. If a sample demonstrates a response above the highest calibration

point the sample can be further diluted with the 50:50
acetonitrile:water diluent to approximately the midpoint of the solvent
curve. The vials (calibration curve, quality control samples, and
diluted sample(s)) can then be reinjected and the data can be
processed without the internal standard correction. Appropriateness
of this approach should be evaluated on a case by case basis. Care
must be taken to ensure the concentration range of the solvent
curve reflects the dilution of the sample. (Data was evaluated with
and without the use of internal standard during validation for some
matrices). It is advised to use the internal standard correction for
extracts that fall within the routine calibration curve range.
4.8.8. Positive confirmation of identity for positive samples, and spiked samples
A. Signal to noise must be ˃3:1 (The AB SCIEX MultiQuant software is
used to calculate signal to noise, if required.)
B. Retention time must match the comparison standard(s) within 5%.
C. Ion ratios must match the comparison standard(s) by an absolute
value of ±20%. (MultiQuant software uses the average ion ratio of all
standards in the calibration curve). If the ratio of either of the
qualifier transitions fails due to instrument sensitivity, the other two
transitions may be used with a ± 10% tolerance (absolute) on the
ratio when compared to the average standard ratio.
4.8.9. Sample Result Reporting
A. Quantitative sample results greater than the lower limit of
quantitation (LLOQ) of 10 ng/g shall be reported.
B. Samples with calculated amounts less than the lower limit of
quantitation (LLOQ) of 10 ng/g, but greater than the MDL shall be
reported as “pentobarbital detected at <10 ng/g, but greater than
MDL”, along with the value for the MDL for that matrix.
C. Samples with calculated amounts < MDL shall be reported as
“pentobarbital not detected at or above MDL”.

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 16 of 34
foods using liquid chromatography tandem mass spectrometry.

5. Glossary
A. RB: Reagent Blank. Used to verify reagents are uncontaminated by
interfering components, the reagent blank is an extract that contains no
sample matrix. Carried thorough the extraction as if it were a sample,
one must be extracted with each batch and display no interference
peaks at the reference times of interest at or above lowest calibration.
H. NC: Negative Control. Used to verify the lack of matrix effects, the
control is an aliquot of matrix material known to contain no analytes of
interest. One must be extracted with each batch for each matrix type,
and must display no interference peaks at reference times of interest at
or above lowest calibration.
I. SPK/DUP: Matrix spike/matrix spike duplicate. Used to demonstrate
effective and reproducible extraction, the matrix spike and duplicate are
two aliquots of negative control matrix material, each fortified at a level
near the midpoint of the curve. A pair of matrix spikes must be extracted
and analyzed with each batch for each matrix type.
J. ICV: Independent Calibration Verification. Used to assure the accuracy
of the calibration curve, the ICV is a solvent standard prepared from a
secondary standard source.
K. CCV: Continuing Calibration Verification. Used to check the calibration
during a run, the CCV is a re-injection of a midpoint solvent standard
curve. A CCV is injected after every ten sample extracts and at the end
of the analytical sequence.
L. MDL = Method Detection Limit = 𝜎𝜎(𝑡𝑡(𝑑𝑑𝑑𝑑=𝑁𝑁−1,1−∝ =0.99,𝑜𝑜𝑜𝑜𝑜𝑜 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠)
M. Where: σ = standard deviation (of 5 ng/g spikes, N=7)
N. t = Student’s T value for df = N-1 at the 99% confidence level, one
sided
O. LOQ = Limit of Quantitation = 𝜎𝜎(10)
P. Where: σ = standard deviation (of 10 ng/g spikes, N=7)
Q. LLOQ = Lower Limit of Quantitation, lowest standard in calibration
curve. For this analysis, the LLOQ should be equal to 10 ng/g in
sample (or lower).
6. Records
A. Analyst Worksheets
B. Batch Records

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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 17 of 34
foods using liquid chromatography tandem mass spectrometry.

C. Equipment FVPMS

7. Document History
Revision Status* Approving Official Name
Date Author Name and Title
# (D, I, R) and Title
00 I 11/30/2018 Tara Nickel, Chemist R. Stadtmuller, Quality System
Christine Casey, Chemist Manager
01 R 12/4/18 Tara Nickel, Chemist R. Stadtmuller, Quality System
Manager
02 R 03/06/19 Tara Nickel, Chemist R. Stadtmuller, Quality System
Manager
03 R 5/1/2019 Tara Nickel, Chemist R. Stadtmuller, Quality System
Manager
04 R 11/13/2019 T. Nickel, Chemist R. Stadtmuller, Quality System
Manager
* - D: Draft, I: Initial, R: Revision

8. Change History
Revision
Change
#
00 Original
01 Change 10 uL to 20 uL on 2.7.2. Add reporting statement on 2.8.9.3 for
amounts below 10ng/g and >MDL.
02 Minor corrections for spelling, grammar, and formatting
03 Add dry dog food, wet dog food, horsemeat information throughout document
04 Add information for additional matrices throughout document. Copy to ORS
template.

9. Attachments
Attachment A - Representative chromatograms................................................... 18-29
Attachment B – Instrument Acquisition method.................................................... 30-34

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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 18 of 34
foods using liquid chromatography tandem mass spectrometry.

Attachment A - Representative chromatograms

The result summary table states the analyte peak precursor name, observed analyte retention
time (RT), expected analyte retention time (RT) time, calculated concentration (ng/g), analyte
response (peak area), calculated Ion Ratio (ration of qualifier ion peak area to quantifier ion
peak area), and Ratio Confirmation (a checkmark indicates the ratio matches the comparison
standards ±20%).

The chromatograms are graphical images are generated in the SCIEX MultiQuant software
summary report. There is a chromatogram for each of the pentobarbital transitions. The
first column is the pentobarbital quantitation ion transition (most abundant ion)
chromatogram (m/z 225→182), identified as Pentobarbital 1. The second column is the
chromatogram for the second most abundant ion transition (m/z 225→85), identified as
Pentobarbital 2 and is used as a qualifier ion transition. The third column is the least
abundant ion (m/z 225→ 138), identified as Pentobarbital 3 and is used as a qualifier
transition. For the bone meal chromatograms (Figures A10 and A11, a fourth transition is
included. It is directly below the Pentobarbital 1 figure. It is identified as Pentobarbital 4 and
is the chromatogram for the transition m/z 225→ 42. It is more abundant than the
pentobarbital quant transition and can be used as an alternative qualifier ion in cases of
matrix interference peaks.

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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 19 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A1 Result Summary for: Solvent Calibrant # 3- equivalent to ng/g in sample

(In vial concentration: 5.0 ng/mL pentobarbital, 5.0 ng/mL d5-pentobarbital)
Calculated
Calculated
Analyte Expected Concentration Analyte Ratio
Analyte Peak Name Ion Ratio
RT RT Response Confirms
(Expected Value)
(ng/g)

Pentobarbital 1 (225->182.0) 4.21 4.20 52.50 170000.0

Pentobarbital 2 (225->85.0) 4.21 4.20 26600 15.6% (15.8%) 

Pentobarbital 3 (225->138.0) 4.21 4.20 11500 6.8% (7.2%) 

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 20 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A2: Result Summary for Tallow Negative Control, Source T1

Calculated
Calculated
Analyte Expected Concentration Analyte Ratio
Analyte Peak Name Ion Ratio
RT RT Response Confirms
(ng/g) (Expected Value)

Pentobarbital 1 (225->182.0) 3.90 4.20 <0 3190.0

Pentobarbital 2 (225->85.0) 4.22 4.20 1920 60.1% (15.8%)

Pentobarbital 3 (225->138.0) 4.06 4.20 395 12.4% (7.2%) 

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 21 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A3: Result Summary for Tallow - Fortified at 50 ng/g

Calculated Calculated
Analyte Expected Concentration Analyte Ion Ratio Ratio
Analyte Peak Name
RT RT Response (Expected Confirms
(ng/g) Value)

Pentobarbital 1 (225_>182.0) 4.21 4.20 51.65 129000.0

Pentobarbital 2 (225->85.0) 4.21 4.20 20500 15.8% (15.8%) 

Pentobarbital 3 (225->138.0) 4.21 4.20 8930 6.9% (7.2%) 

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 22 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A4: Result Summary for Wet Dog Food Negative Control, Source W2

Calculated Calculated
Analyte Expected Concentration Analyte Ion Ratio Ratio
Analyte Peak Name
RT RT Response (Expected Confirms
(ng/g) Value)

Pentobarbital 1 (225->182.0) 3.90 4.20 <0 1326.0

Pentobarbital 2 (225->85.0) 3.85 4.20 824 62.2% (14.3%)

Pentobarbital 3 (225->138.0) 4.29 4.20 243 18.4% (6.8%) 

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 23 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A5: Result Summary for Wet Dog Food Fortified at 50 ng/g, Source W2

Calculated Calculated
Analyte Expected Concentration Analyte Ion Ratio Ratio
Analyte Peak Name
RT RT Response (Expected Confirms
(ng/g) Value)

Pentobarbital 1 (225->182.0) 4.19 4.20 56.02 53007.0

Pentobarbital 2 (225->85.0) 4.19 4.20 6812 12.9% (14.3%) 

Pentobarbital 3 (225->138.0) 4.19 4.20 3619 6.8% (6.8%) 

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Revised: Nov. 2019
Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 24 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A6: Result Summary for Dry Dog Food Negative Control, Source D3

Calculated Calculated
Analyte Expected Concentration Analyte Ion Ratio Ratio
Analyte Peak Name
RT RT Response (Expected Confirms
(ng/g) Value)

Pentobarbital 1 (225->182.0) N/A 4.20 N/A 1326.0 N/A

Pentobarbital 2 (225->85.0) 3.95 4.20 824 0.0% (14.3%)

Pentobarbital 3 (225->138.0) 3.92 4.20 243 0.0% (6.8%)

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Revised: Nov. 2019
Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 25 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A7: Result Summary for Dry Dog Food Fortified at 50 ng/g, Source D3

Calculated Calculated
Analyte Expected Concentration Analyte Ion Ratio Ratio
Analyte Peak Name
RT RT Response (Expected Confirms
(ng/g) Value)

Pentobarbital 1 (225->182.0) 4.22 4.20 51.26 68852.0

Pentobarbital 2 (225->85.0) 4.22 4.20 9158 13.3% (14.3%) 

Pentobarbital 3 (225->138.0) 4.22 4.20 5022 7.3% (6.9%) 

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 26 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A8: Result Summary for Horsemeat Negative Control

Calculated
Calculated
Analyte Expected Concentration Analyte Ratio
Analyte Peak Name Ion Ratio
RT RT Response Confirms
(ng/g) (Expected Value)

Pentobarbital 1 (225->182.0) N/A 4.69 N/A N/A

Pentobarbital 2 (225->85.0) 4.53 4.69 2165 0.0% (14.9%)

Pentobarbital 3 (225->138.0) 4.54 4.69 780 0.0% (6.9%)

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Revised: Nov. 2019
Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 27 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A9: Result Summary for Horsemeat Fortified at 50 ng/g

Calculated Calculated
Analyte Expected Concentration Analyte Ratio
Analyte Peak Name RT Ion Ratio
RT Response Confirms
(ng/g) (Expected Value)

Pentobarbital 1 (225->182.0) 4.69 4.69 50.29 70189.0

Pentobarbital 2 (225->85.0) 4.53 4.69 11187 15.9% (14.9%) 

Pentobarbital 3 (225->138.0) 4.54 4.69 4374 6.2% (6.9%) 

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 28 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A10: Result Summary for Bone Meal Negative Control (source 2)
Calc Calculated
Analyte Expecte Conc. Analyte Ratio
Analyte Peak Name Ion Ratio
RT d RT Response Confirms
(ng/g) (Expected Value)

Pentobarbital 1 (225->182.0) N/A 4.23 N/A N/A

Pentobarbital 2 (225->85.0) 4.25 4.23 7.07e3 0.0% (15.9%)

Pentobarbital 3 (225->138.0) N/A 4.23 N/A 0.0% (6.1%)

Pentobarbital 4 (225->42.0) 4.23 4.23 5.24e3 0.0% (281.7%)

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

Pentobarbital 4

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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 29 of 34
foods using liquid chromatography tandem mass spectrometry.

Figure A11: Result Summary for Bone Meal Fortified at 50 ng/g

Calc Calculated
Analyte Expecte Conc. Analyte Ratio
Analyte Peak Name Ion Ratio
RT d RT Response Confirms
(ng/g) (Expected Value)

Pentobarbital 1 (225->182.0) 4.25 4.23 45.76 1.93e5

Pentobarbital 2 (225->85.0) 4.25 4.23 4.87e4 25.3% (15.9%) 

Pentobarbital 3 (225->138.0) 4.24 4.23 1.54e4 8.0% (6.1%) 

Pentobarbital 4 (225->42.0) 4.24 4.23 5.16e5 267.4% (281.7%) 

Chromatograms – Bars on peaks are expected ion ratio ± 20% of comparison standards

Pentobarbital 1 Pentobarbital 2 Pentobarbital 3

Pentobarbital 4

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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 30 of 34
foods using liquid chromatography tandem mass spectrometry.

Attachment B: AB SCIEX QTRAP 5500 data acquisition method

HPLC column – Agilent Zorbax Eclipse Plus C18, 2.1 x 50 mm, 1.8 micron size column

Mobile Phase. A: Water

Mobile Phase. B: Acetonitrile

Synchronization Mode: LC Sync

Auto-Equilibration: Off
Acquisition Duration: 8min31sec
Number Of Scans: 1854
Periods In File: 1
Acquisition Module: Acquisition Method
Software version Analyst 1.6.2

MS Method Properties:
Period 1:
--------------
Scans in Period: 1854
Relative Start Time: 1000.00 msec
Experiments in Period: 1

Period 1 Experiment 1:
----------------------------
Scan Type: MRM (MRM)
Scheduled MRM: No
Polarity: Negative
Scan Mode: N/A
Ion Source: Turbo Spray
Resolution Q1: Unit
Resolution Q3: Unit
Intensity Thres.: 0.00 cps
Settling Time: 0.0000 msec
MR Pause: 5.0070 msec
MCA: No

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 31 of 34
foods using liquid chromatography tandem mass spectrometry.

Step Size: 0.00 Da

@Q1 Mass (Da)Q3 Mass (Da) Dwell(msec) Param Start Stop ID

225.000 182.000 50.00 CE -19.00 -19.00 Pentobarbital 1
CXP -13.00 -13.00

@Q1 Mass (Da) Q3 Mass (Da) Dwell(msec) Param Start Stop ID

225.000 85.000 50.00 CE -18.00 -18.00 Pentobarbital 2
CXP -9.00 -9.00

@Q1 Mass (Da) Q3 Mass (Da) Dwell(msec) Param Start Stop ID

225.000 138.000 50.00 CE -21.00 -21.00 Pentobarbital 3
CXP -10.00 -10.00

@Q1 Mass (Da) Q3 Mass (Da) Dwell(msec) Param Start Stop ID

225.000 42.000 50.00 CE -46.00 -46.00 Pentobarbital 4
CXP -7.00 -7.00

@Q1 Mass (Da) Q3 Mass (Da) Dwell(msec) Param Start Stop ID

230.000 187.000 50.00 CE -17.00 -17.00 d5-Pentobarbital
CXP -10.00 -10.00

Parameter Table(Period 1 Experiment 1):

CAD: Medium
GS1: 50.00
GS2: 60.00
CUR: 30.00
TEM: 400.00
IS: -3500.00
DP -100.00
EP -10.00

Valco Valve Diverter

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 32 of 34
foods using liquid chromatography tandem mass spectrometry.

Total Time (min) Position

1 0.0 B
2 3.0 A
3 5.0 B
Agilent LC Pump Method Properties
Pump Model: Agilent 1260 Binary Pump
Minimum Pressure (psi): 0.0
Maximum Pressure (psi): 8702.0
Dead Volume (µl): 40.0
Maximum Flow Ramp (ml/min²): 100.0
Maximum Pressure Ramp (psi/sec): 290.0
Max Flow Ramp Up (ml/min²): 100.0
Max Flow Ramp Dn (ml/min²): 100.0

Step Table:
@Step Total Time(min) Flow Rate(µl/min) A (%) B (%)
0 0.00 350 95.0 5.0
1 3.50 350 5.0 95.0
2 4.50 350 95.0 5.0
3 8.50 350 95.0 5.0

Left Compressibility: 50.0

Right Compressibility: 115.0
Left Dead Volume (µl): 40.0
Right Dead Volume (µl): 40.0
Left Stroke Volume (µl): -1.0
Right Stroke Volume (µl): -1.0
Left Solvent: A2
Right Solvent: B1

Agilent LC Pump Method Properties

Pump Model: Agilent 1260 Binary Pump
Minimum Pressure (psi): 0.0
Maximum Pressure (psi): 8702.0
Dead Volume (µl): 40.0
Maximum Flow Ramp (ml/min²): 100.0
Maximum Pressure Ramp (psi/sec): 290.0
Max Flow Ramp Up (ml/min²): 100.0

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 FOOD AND DRUG ADMINISTRATION Document Number:
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Denver Laboratory Compendium method C-005.xx

Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 33 of 34
foods using liquid chromatography tandem mass spectrometry.

Max Flow Ramp Dn (ml/min²): 100.0

Step Table:
@Step Total Time(min) Flow Rate(µl/min) A (%) B (%)
0 0.00 0 50.0 50.0
1 8.50 0 50.0 50.0

Left Compressibility: 50.0

Right Compressibility: 115.0
Left Dead Volume (µl): 40.0
Right Dead Volume (µl): 40.0
Left Stroke Volume (µl): -1.0
Right Stroke Volume (µl): -1.0
Left Solvent: A2
Right Solvent: B2

Agilent Column Oven Properties

Left Temperature (°C): 40.00
Right Temperature (°C): 40.00
Temperature Tolerance +/- (°C): 1.00
Start Acquisition Tolerance +/- (°C): 1.00
Time Table (Not Used)
Column Switching Valve Installed 10Port2Pos
Position for first sample in the batch: Left
Use same position for all samples in the batch

CTC PAL Autosampler Method Properties

Loop Volume1 (µl): 20
Loop Volume2 (µl): 20
Injection Volume (µl): 5.000
Barcode Reading: Disabled

Method Description:

Syringe: 100ulDLW

Cycle date: 9/9/2010 3:26:06 PM

Cycle name: Analyst LC-Inj DLW Fast_Rev05

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Title:
Determination of pentobarbital in ingredients of animal origin and in finished pet Page 34 of 34
foods using liquid chromatography tandem mass spectrometry.

Airgap Volume (µl) 3

Front Volume (µl) 5
Rear Volume (µl) 5
Filling Speed (µl/s) 5
Pullup Delay (ms) 3
Inject to LC Vlv1
Injection Speed (µl/s) 5
Pre Inject Delay (ms) 500
Post Inject Delay (ms) 500
Needle Gap Valve Clean (mm) 3
Valve Clean Time Solvent 2 (s) 3
Valve Clean Time Solvent 1 (s) 4
Post Clean Time Solvent 1 (s) 3

For the most current and official copy, check QMiS.