determination of iqb-9302 levels in beagle dog plasma

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DETERMINATION OF IQB-9302 LEVELS IN BEAGLE DOG PLASMA

Sponsor:Laboratorios INIBSA

Crta de Sabadell a Granollers, Km.14.508185 Lliça de Vall (Barcelona)

Spain

T.P.S. Study No.:616C-503-532-98

Contract Laboratory:T.P.S., Inc.

10424 Middle Mt. Vernon Rd.Mt. Vernon, IN 47620

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TABLE OF CONTENTS

Page #TABLE OF CONTENTS 2

SIGNATURE PAGE 3

QUALITY ASSURANCE STATEMENT 4

INTRODUCTION 5

MATERIALS 5

ASSAY PROCEDURE 7

RESULTS AND DISCUSSION 8

REFERENCES 9

TABLE 1 Back Calculated Results and Regression Statistics forIQB-9302 Calibration Standards 10

TABLE 2 Quality Control Results and Statistics 11

TABLE 3 Schedule of Batches Assayed 12

FIGURE 1 Representative Calibration Curve (Batch R09R) 13

FIGURE 2 Representative Chromatograms of Study and QualityControl Samples 14

APPENDIX A IQB-9302 Plasma Concentrations 15

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SIGNATURE PAGE

Work done by: P.A. Downing, B.A. S.E. Blessinger, BSMT(ASCP)

Reported by: Philip A. Downing, B.A. DateAnalytical Chemistry Manager

Approved by: Lisa J. Clare, D.V.M. DateStudy Director

James A. Botta, Jr., D.V.M., Ph.D. DateVice President of Research andDevelopment

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QUALITY ASSURANCE REVIEW STATEMENT

The Study 616C-503-532-98 plasma analysis report has been reviewed for format andcontent by the Quality Assurance Unit. The material contained herein has been accepted asan accurate and true representation of the data and procedures contained in the study.

Quality Assurance audits of this report were conducted by the Quality Assurance Unit andreported to the study director and management.

Deborah G. Fehrenbacher, B.S., MT(ASCP) DateQuality Assurance Auditor

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1.0 INTRODUCTION

The analysis of 430 beagle dog plasma samples for IQB-9302 concentrations hasbeen performed by T.P.S., Inc. Plasma samples from the study (T.P.S. Study No.:616C-503-532-98, Sponsor Study No.: 032b) were collected on 11-10-98 and 12-7-98 and stored frozen until analyzed for IQB-9302 using a validated HPLC method.

2.0 MATERIALS

2.1 Equipment/Materials

2.1.1 Equipment used in the procedure:

A. 1 mL volumetric class “A” pipetB. 5, 10, 25 and 100 mL volumetric class “A” flasksC. 1000 mL volumetric cylinderD. Adjustable Eppendorf pipetE. Adjustable Eppendorf repeating pipetF. Fisher Scientific Accumet® Model 220 pH MeterG. Vac-Elut solid phase extraction vacuum apparatusH. Multi-tube vortexer – Dade International Inc.I. 200 µL limited volume inserts with starburst capsJ. Glass 12x75 mm test tubes

2.1.2 HPLC system included the following components:

A. Waters µBondapak® C18 reverse phase column (3.9 x 300 mm, 10µm particle size)B. Phenomenex® Security GuardTM guard column with C18

(4.0 x 3.0 mm) cartridgeC. Waters Oasis® MCX 1 cc/30 mg SPE cartridgeD. Waters Model 600E multisolvent delivery systemE. Waters Model 712 WISP automatic sample injectorF. Waters Model 490 multi-wavelength detectorG. Chrom Perfect (version 3.08) chromatography software

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2.2 Reagents

A. IQB-9302.HCl, Lot No. 9454/R.E-1, Purity = 100.7%(1.1249 free base correction factor) - Laboratorios INIBSA,Spain

B. Bupivacaine HCl, Lot No. 117H0875, Purity = >99.9% -Sigma Chemical Company

C. Water, HPLC Grade – Fisher ScientificD. Sodium Phosphate Monobasic, ACS Certified – Fisher ScientificE. Sodium Hydroxide Solution, 50% w/w – Fisher ScientificF. Methanol (MeOH), HPLC Grade – Fisher ScientificG. Acetonitrile (ACN), HPLC Grade – Fisher ScientificH. Ammonium Hydroxide (NH4OH), ACS Certified – Fisher

ScientificI. o-Phosphoric Acid 85%, HPLC Grade – Fisher ScientificJ. 0.1N Hydrochloric Acid Solution – Fisher ScientificK. Blank dog plasma – T.P.S., Inc. (collected in-house from

untreated beagle dogs)

2.3 Calibration Standards

Calibration standards (Lot No. CC01) were prepared on 11-09-99 atconcentrations of 50, 100, 250, 500, 1000, 2000, 5000 ng/mL (free base)by fortifying blank dog beagle plasma with IQB-9302. Aliquots of eachlevel were frozen following preparation.

2.4 Quality Control Samples

Quality control (QC) samples (Lot No. QC01) were prepared on 11-09-99at three different levels (150, 750, 2500 ng/mL – free base) by fortifyingblank dog beagle plasma with IQB-9302. Aliquots of each level werefrozen following preparation.

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3.0 ASSAY PROCEDURE

3.1 Sample Preparation

A. Transferred 0.50 mL of each standard, QC, and sample toseparate labeled glass test tubes.

B. Pipetted 25 µL of 2.0% phosphoric acid and 0.50 mL ofinternal standard (2 µg/mL) to each tube and briefly vortexed.(Note: 0.5 mL of water was added to the plasma blankstandards instead of internal standard.)

C. Conditioned SPE cartridges with 2 (1 mL) aliquots ofMeOH followed by 2 (1 mL) aliquots of water.

D. Transferred samples to SPE cartridges.D. Washed remaining sample from each test tube using 1 mL

of 0.1N HCl and then transferred wash to SPE cartridges.F. Washed SPE cartridges with 1 mL of 0.1N HCl.G. Washed SPE cartridges with 2 (1 mL) aliquots of MeOH.H. Extracted analytes with 2 (1 mL) aliquots of 5% NH4OH in

MeOH. Extract was collected in clean glass test tubes.I. Extract was dried under nitrogen at approximately 50°C.

J. Reconstituted with 150 µL of mobile phase A and vortexedon a multi-tube vortexer for approximately 15 minutes.

K. Transferred samples to 200 µL limited volume inserts withstarburst caps.

3.2 Chromatographic Conditions

IQB-9302 was separated on a Waters µBondapak® C-18 reverse-phasecolumn with a mobile phase of MeOH:ACN:0.02M phosphate buffer,pH=6, (15:40:45, v/v/v) at a flow rate of 1.2 mL/min at ambientconditions. Injections of 100µL were made by the WISP and a run time of25 minutes per injection was used. The 600 series pump was programmedto wash the column for approximately 2.5 minutes using 85%ACN inwater following the elution of the internal standard. The detector was setat a wavelength of 210 nm and an AUFS of 0.30

3.3 Data handling

All chromatographic data was collected using Chrom Perfect (version3.08) chromatographic software. A method, sequence file, and calibrationfile were constructed and downloaded prior to the injection of the firstsample. For each assay day, a standard curve was constructed by plotting

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peak area ratios for IQB-9302/Internal Standard versus the respectivestandard concentration. Sample concentrations were determinedmathematically by comparison of the peak area ratios in the sample to theweighted (1/x) regression equation obtained from the standards data.

4.0 RESULTS AND DISCUSSION

4.1 Standards

Table 1 contains results of the standard curves obtained during theanalysis of study samples. Calibration standards where analyzed induplicate (singlet when the number of study samples was less than 25)Correlation coefficients ranged from 0.997-1.00). The mean calculatedrelative standard deviation (% RSD) for the back-calculatedconcentrations of the calibration standards ranged from 2.9 to 8.3. Thepercent deviation (% Dev.) was determined be comparing the back-calculated concentrations to the theoretical concentration for eachrespective standard. The mean percent deviations ranged from –2.2 to 1.7.Figure 1 shows a representative standard calibration curve plotted usingChrom Perfect software from Batch R09R.

4.2 Quality Control Samples

Results of the analysis of QCs are shown in Table 2. QC samples whereanalyzed in duplicate (singlet when the number of study samples was lessthan 25) at 3 levels. The mean calculated relative standard deviation (%RSD) for the back-calculated concentrations of the low, middle, and highQC pools were 6.0, 4.6, 3.8, respectively. The percent deviation (% Dev.)was determined be comparing the back-calculated concentrations to thetheoretical concentration for each respective standard. The mean percentdeviation for the low, middle, and high QC pools were 2.8, 2.3, and 0.5,respectively.

4.3 Assay Performance

Samples were analyzed over 10 acceptable runs (the tenth run includedrepeat samples). Acceptability of each run was determined as follows:four of the six QC samples must pass a 20 % maximum bias acceptancecriteria for the low, middle, and high pools with no more than one QCsample at any concentration failing the acceptance criteria. Calibrationstandards must also pass the 20 % maximum bias acceptance criteria foreach level with no more than 25 % of the standards failing the criteria (SeeTable 3). Ten runs passed the above mentioned acceptance criteria. One

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run (R04R) was rejected due to pump failure during the run and samplesre-extracted and analyzed in run R04XR.

4.5 Sample Analysis

Study samples were randomized prior to analyzing. Samples were storedfrozen and allowed to thaw-out at room temperature prior to aliquoting.Samples were re-frozen after aliquoting. Eleven samples were re-analyzeddue to improper integration.

Figure 2 contains representative chromatograms of study samples and QC:Group #4 – Day 28 - predose (Animal BKG4M03), Group #3 - Day 1 –1.5 hour (Animal BKG3F01), Group # 3 - Day 1 - 5 min. (AnimalBKG3F01), and mid-level QC (750 ng/mL).

5.0 REFERENCES

Downing, P.A., Validation of a High Performance Liquid Chromatographic Assayfor IQB-9302 in Dog Plasma, T.P.S., Inc., June 16. 1999

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TABLE 1

Back Calculated Results and Regression Statistics for IQB-9302 CalibrationStandards

Batch STD#1

STD#2

STD#3

STD#4

STD#5

STD#6

STD#7

Y-intercept r-factor

R01R 54.969.0*

100101

244253

481517

9061018

19231973

49405240

-0.009 0.998

R02R 53.549.7

10195.4

251254

467704*

10021021

19721999

48645171

-0.004 0.999

R03R 52.153.5

97.0106

241245

485493

9911012

17592065

50365164

-0.003 0.998

R04XR 48.950.8

91.8126*

245272

478545

9411118

19231865

52304892

-0.004 0.997

R05R 15.4*53.8

10099.8

249245

498519

985966

18022060

50535119

-0.0002 0.998

R06R 41.056.4

80.687.1

297293

404488

11251111

19312169

47934922

-0.003 0.997

R07R 51.746.2

101101

244247

503514

10001027

1613*3540*

47535211

-0.002 0.997

R08R 53.364.3*

10993.1

239248

495499

9871013

19391970

50015103

-0.011 1.00

R09R 43.453.7

92.6103

251268

495528

9921018

19152020

49455075

-0.001 1.00

R10R 50.7 101 247 498 999 1971 5033 -0.009 1.00N 16 18 19 18 19 17 19

Nominal(ng/mL)

50 100 250 500 1000 2000 5000

Mean 50.9 97.8 254 495 1012 1956 5029Std. Dev. 4.21 6.80 16.5 29.5 55.4 97.0 145% RSD 8.3 7.0 6.5 6.0 5.5 5.0 2.9% Dev. 1.7 -2.2 1.7 -1.0 1.2 -2.2 0.6

* Standard dropped due to out-of-range value (±20 %)

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TABLE 2

Quality Control Results and Statistics

Batch LQC MQC HQCR01R 147

164765741747

24652462

R02R 160152

755775

25312613

R03R 147146

770771

24722570

R04XR 150164

712760

23802484

R05R 148173

805817

26222774

R06R 139219*

708737

24852618

R07R 148157

841787

25492502

R08R 165163

819801

23872420

R09R 142159

720752772

24672428

R10R 151 761 2525N 18 21 19

Nominal (ng/mL) 150 750 2500Mean 154 767 2513

Std. Dev. 9.28 35.3 96.0% RSD 6.0 4.6 3.8% Dev. 2.8 2.3 0.5

* QC dropped due to out-of-range value (±20 %)

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TABLE 3

Schedule of Batches Assayed

Batch # Date Extracted Status

R01R 11-15-99 AcceptedR02R 11-17-99 AcceptedR03R 11-22-99 AcceptedR04R 11-24-99 Failed *

R04XR 11-30-99 AcceptedR05R 12-6-99 AcceptedR06R 12-8-99 AcceptedR07R 12-10-99 AcceptedR08R 12-13-99 AcceptedR09R 12-15-99 AcceptedR10R 12-17-99 Accepted

* Batch R04R was rejected due to pump failure during the run. Samples were re-extractedand assayed in Batch R04XR.

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APPENDIX A

IQB-9302 Plasma Concentrations

Dosage Key

GroupNumber

TestArticle

Dose(mg/kg/day)

Dose Volume(mL/kg)

Number ofAnimals

Males FemalesBKG1* Vehicle 0 1 5 5BKG2 IQB-9302 1 1 5 5BKG3 IQB-9302 2 1 5 5BKG4 IQB-9302 3 1 5 5

* Only predose samples were collected from Group BKG1

Report Code Key

B = Below Quantifiable Limits (< 50 ng/mL)N = None Detected

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Group BKG1 (0 mg/kg/day): IQB-9302 Plasma Concentrationsin Male Beagle Dogs

Day 1

Collection Interval Animal Number Concentration (ng/mL)

Predose BKG1M 1 N BKG1M 2 N BKG1M 3 N BKG1M 4 N BKG1M 5 N

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Day 1



5 min BKG2M 1 1762 BKG2M 2 1943 BKG2M 3 1249 BKG2M 4 1142 BKG2M 5 2045



1 hour BKG2M 1 173 BKG2M 2 143 BKG2M 3 125 BKG2M 4 123 BKG2M 5 131

1.5 hour BKG2M 1 B BKG2M 2 B BKG2M 3 B BKG2M 4 57.1 BKG2M 5 51.3

3 hour BKG2M 1 N BKG2M 2 N BKG2M 3 N BKG2M 4 N BKG2M 5 N

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Day 1







1.5 hour BKG3M 1 272 BKG3M 2 150 BKG3M 3 B BKG3M 4 67.1 BKG3M 5 80.8


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Day 1







1.5 hour BKG4M 1 108 BKG4M 2 71.3 BKG4M 3 299 BKG4M 4 193 BKG4M 5 287

3 hour BKG4M 1 N BKG4M 2 N BKG4M 3 N BKG4M 4 N BKG4M 5 B

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Group BKG1 (0 mg/kg/day): IQB-9302 Plasma Concentrationsin Female Beagle Dogs

Day 1


Predose BKG1F 1 N BKG1F 2 N BKG1F 3 N BKG1F 4 N BKG1F 5 N

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Day 1



5 min BKG2F 1 1651 BKG2F 2 1227 BKG2F 3 1356 BKG2F 4 1682 BKG2F 5 1793



1 hour BKG2F 1 124 BKG2F 2 62.2 BKG2F 3 143 BKG2F 4 136 BKG2F 5 110

1.5 hour BKG2F 1 N BKG2F 2 N BKG2F 3 B BKG2F 4 B BKG2F 5 B

3 hour BKG2F 1 N BKG2F 2 N BKG2F 3 N BKG2F 4 N BKG2F 5 N

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Day 1






1 hour BKG3F 1 244 BKG3F 2 280 BKG3F 3 218 BKG3F 4 301 BKG3F 5 285

1.5 hour BKG3F 1 107 BKG3F 2 84.2 BKG3F 3 94.2 BKG3F 4 129 BKG3F 5 97.8


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Day 1







1.5 hour BKG4F 1 133 BKG4F 2 242 BKG4F 3 430 BKG4F 4 120 BKG4F 5 296

3 hour BKG4F 1 N BKG4F 2 N BKG4F 3 64.7 BKG4F 4 N BKG4F 5 54.1

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Day 28







1.5 hour BKG2M 1 61.3 BKG2M 2 B BKG2M 3 58.4 BKG2M 4 54.6 BKG2M 5 54.7


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Day 28







1.5 hour BKG3M 1 310 BKG3M 2 124 BKG3M 3 63.4 BKG3M 4 81.3 BKG3M 5 120

3 hour BKG3M 1 53.2 BKG3M 2 N BKG3M 3 N BKG3M 4 N BKG3M 5 N

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Day 28







1.5 hour BKG4M 1 176 BKG4M 2 211 BKG4M 3 438 BKG4M 4 261 BKG4M 5 320

3 hour BKG4M 1 N BKG4M 2 N BKG4M 3 N BKG4M 4 N BKG4M 5 50.8

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Day 28






1 hour BKG2F 1 102 BKG2F 2 79.5 BKG2F 3 140 BKG2F 4 103 BKG2F 5 157

1.5 hour BKG2F 1 N BKG2F 2 N BKG2F 3 61.6 BKG2F 4 N BKG2F 5 B


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Day 28







1.5 hour BKG3F 1 67.6 BKG3F 2 205 BKG3F 3 126 BKG3F 4 123 BKG3F 5 112


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Day 28







1.5 hour BKG4F 1 162 BKG4F 2 348 BKG4F 3 419 BKG4F 4 223 BKG4F 5 330

3 hour BKG4F 1 N BKG4F 2 59.5 BKG4F 3 101 BKG4F 4 N BKG4F 5 51.1

determination of iqb-9302 levels in beagle dog plasma

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