Study Title 

Determining the Partition Coefficient (n-Octanol/Water) of EFKA 8530 

by the Flask-Shaking Method Following OECD Guideline 107 

Data Requirement 

OECD Guideline 107 

Author

Karen L. Proctor 

Study Completed On 

15 April 2008 

Submitted To 

Ciba Specialty Chemicals Company 

540 White Plains Road 

Tarrytown, New York 10591 

Performing Laboratory 

Springborn Smithers Laboratories 

790 Main Street 

Wareham, Massachusetts 02571-1037 

Laboratory Project ID 

Springborn Smithers Study No. 13658.6319 

Page 1 of 43 


Springborn Smithers Study No. 13658.6319 Page 4 

KEY STUDY PERSONNEL 

The following Springborn Smithers personnel were responsible for the conduct of the work and 

reporting of the study results. 

Karen L. Proctor Study Director 

Rebecca J. Smith Chemistry Technician II 

James D. Hatch Chemist I 

David O. Duval Chemistry Technician I 

Andrea M. Soares Technical Report Writer 

Ronald C. Biever Vice President, Operations 
Springborn Smithers Study No. 13658.6319 Page 5 

TABLE OF CONTENTS 

Page

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT..........................................2

QUALITY ASSURANCE UNIT STATEMENT............................................................................3

KEY STUDY PERSONNEL...........................................................................................................4

TABLE OF CONTENTS.................................................................................................................5

SUMMARY.....................................................................................................................................7

1.0 INTRODUCTION..................................................................................................................8

2.0 MATERIALS AND METHODS...........................................................................................8

2.1 Protocol....................................................................................................................8

2.2 Test Substance and Standard Reagents....................................................................8

2.2.1 Test Substance .............................................................................................8

2.2.2 Standard Reagents........................................................................................9

2.3 n-Octanol-Saturated Water and Water-Saturated n-Octanol Solution 

Preparation ...............................................................................................................9

2.4 Stock Solution Preparation ....................................................................................10

2.5 Preparation of Quality Control Samples................................................................10

2.6 Preparation of Calibration Standards.....................................................................11

2.7 Test Apparatus.......................................................................................................11

2.8 Test Solution Preparation.......................................................................................11

2.8.1 Preliminary Test.........................................................................................11

2.8.2 Definitive Test ...........................................................................................11

2.9 Test Procedure .......................................................................................................12

2.9.1 Preliminary Test.........................................................................................12

2.9.2 Definitive Test ...........................................................................................12

2.10 Chemical Analysis .................................................................................................13

3.0 CALCULATIONS ...............................................................................................................14

4.0 RESULTS.............................................................................................................................14

4.1 Preliminary Results................................................................................................14

4.2 Definitive Results...................................................................................................14

PROTOCOL DEVIATION ...........................................................................................................16

REFERENCES ..............................................................................................................................17

Table 1. Results of the preliminary n-octanol/water partition coefficient test 

with EFKA 8530........................................................................................18
Springborn Smithers Study No. 13658.6319 Page 6 

Table 2. Results of the definitive n-octanol/water partition coefficient test 

with EFKA 8530........................................................................................19

Table 3. Results of the analysis of the EFKA 8530 n-octanol stock solution 

and quality control samples. ......................................................................20

Figure 1. HPLC chromatogram of an EFKA 8530 water-saturated n-octanol 

phase test solution from the determination of the partition 

coefficient. .................................................................................................21

Figure 2. HPLC chromatogram of a quality control sample (1032 mg a.i./L) 

from the determination of the partition coefficient for EFKA 8530..........22

Figure 3. HPLC chromatogram of a calibration standard (25.0 mg a.i./L) from 

the determination of the partition coefficient for EFKA 8530. .................23

APPENDIX 1 - STUDY PROTOCOL..........................................................................................24

APPENDIX 2 - ANALYTICAL METHODOLOGY....................................................................34

Table 1A. Analytical results for the recovery of EFKA 8530 from n-octanol 

during the method validation study............................................................39

Figure 1A. Representative chromatogram of a 10.0 mg a.i./L calibration 

standard used for quantification during the method validation study 

with EFKA 8530........................................................................................40

Figure 2A. Representative chromatogram of a 1032 mg a.i./L recovery sample 

fortified with EFKA 8530..........................................................................41

Figure 3A. Representative chromatogram of a control sample during the method 

validation study with EFKA 8530. ............................................................42

Figure 4A. A typical regression analysis for the calibration standards used to 

quantitate EFKA 8530. ..............................................................................43
Springborn Smithers Study No. 13658.6319 Page 7 

SUMMARY 

Determining the Partition Coefficient (n-Octanol/Water) of EFKA 8530 

by the Flask-Shaking Method Following OECD Guideline 107 

STUDY SPONSOR: Ciba Specialty Chemicals Company 

PROTOCOL TITLE: Determining the Partitioning Coefficient (n-Octanol/Water) of a 

Test Substance by the Flask-Shaking Method Following OECD 

Guideline 107, Springborn Smithers Laboratories Protocol No.: 

040805/OECD/Kow 

SPRINGBORN SMITHERS 

STUDY NUMBER: 13658.6319 

TEST SUBSTANCE: EFKA 8530, Lot No. 15306, reported to have a purity of 93.56%, 

was received from Ciba Specialty Chemicals Company on 

31 October 2007. 

DEFINITIVE 

TEST DATE: 11 January to 4 February 2008 

RESULTS: Analyses of the definitive test samples determined that the mean 

partition coefficient of the test substance was 24.9 

(log Pow = 1.31). 
Springborn Smithers Study No. 13658.6319 Page 8 

1.0 INTRODUCTION 

The objective of this study was to determine the n-octanol/water partition coefficient (Pow) for 

EFKA 8530 by the flask-shaking method. The n-octanol/water partition coefficient (Pow) is the 

equilibrium ratio of the concentration of EFKA 8530 in n-octanol and water in a dilute solution. 

The test substance was analyzed by gel permeation chromatography with evaporative light 

scattering detection (GPC/ELSD). 

The study was initiated on 19 December 2007, the day the Study Director signed the protocol, 

and terminated the day the Study Director signed the final report. The definitive test was 

conducted from 11 January to 4 February 2008 at Springborn Smithers Laboratories (SSL) 

located in Wareham, Massachusetts. All raw data, the protocol and the final report produced 

during this study are stored in Springborn Smithers' archives at the above location. 

2.0 MATERIALS AND METHODS 

2.1 Protocol 

The procedures followed during this study are detailed in the Springborn Smithers Laboratories 

protocol entitled "Determining the Partitioning Coefficient (n-Octanol/Water) of a Test 

Substance by the Flask-Shaking Method Following OECD Guideline 107", Springborn Smithers 

Laboratories Protocol No.: 040805/OECD/Kow (Appendix 1). 

2.2 Test Substance and Standard Reagents 

2.2.1 Test Substance 

The test substance, EFKA 8530, was received on 31 October 2007 from Ciba Specialty 

Chemicals Company, Allentown, Pennsylvania. The following information was provided: 
Springborn Smithers Study No. 13658.6319 Page 9 

 Name: EFKA 8530 

 Lot No.: 15306 

 CAS No.: Not Listed 

 Purity: 93.56% 

 Recertification Date: 31 January 2009 

Upon receipt at Springborn Smithers, the test substance (SSL No. 126-09) was stored at room 

temperature in the original container in a dark ventilated cabinet. Concentrations were adjusted 

for the purity of the test substance and are presented as active ingredient (a.i.). 

Determination of stability and characterization, verification of the test substance identity, 

maintenance of records on the test substance, and archival of a sample of the test substance are 

the responsibility of the Study Sponsor. 

2.2.2 Standard Reagents 

All solutions were prepared using reagent water (meeting ASTM Type II requirements) purified 

with a Barnstead NANOpure® system. The n-octanol used was obtained from Sigma-Aldrich 

Chemical Company, Inc. All chemicals were at least reagent grade and were obtained from 

commercial sources. 

2.3 n-Octanol-Saturated Water and Water-Saturated n-Octanol Solution 

Preparation 

n-Octanol-saturated water and water-saturated n-octanol were prepared simultaneously in 

Erlenmeyer flasks by combining 700 mL of n-octanol with 700 mL of purified reagent water 

(PRW). The flasks were tightly sealed and then placed on a shaker table (Lab-Line Orbital 

Model 3590). The flasks were shaken for 24 hours at 150 rpm to saturate both phases. After 

being removed from the shaker table, the solutions were transferred into separatory funnels and 

allowed to settle until the two phases had completely separated. The two individual phases were 

transferred into separate amber bottles. This yielded n-octanol-saturated water and 

water-saturated n-octanol. 
Springborn Smithers Study No. 13658.6319 Page 10 

2.4 Stock Solution Preparation 

A 2.02 mg a.i./mL stock solution was prepared by placing 0.4312 g of EFKA 8530 (0.4034 g as 

active ingredient) in a 200-mL volumetric flask and bringing it to volume with water-saturated 

n-octanol. This stock solution was used to prepare test samples during preliminary testing. 

A 2.05 mg a.i./mL stock solution was prepared by placing 0.4372 g of EFKA 8530 (0.4090 g as 

active ingredient) in a 200-mL volumetric flask and bringing it to volume with water-saturated 

n-octanol. This stock solution was used to prepare test samples during definitive testing. 

A 5.16 mg a.i./mL stock solution was prepared by placing 0.2760 g of EFKA 8530 (0.2582 g as 

active ingredient) in a 50.0-mL volumetric flask and bringing it to volume with n-octanol. This 

stock solution was used to prepare n-octanol quality control samples. 

A 5.00 mg a.i./mL primary stock solution was prepared by placing 0.5344 g of EFKA 8530 

(0.5000 g as active ingredient) in a 100-mL volumetric flask and bringing it to volume with 

tetrahydrofuran (THF). A 1000 mg a.i./L secondary stock solution was prepared by placing 

10.0 mL of the 5.00 mg a.i./mL primary stock solution in a 50.0-mL volumetric flask and 

bringing it to volume with THF. A 100 mg a.i./L tertiary stock solution was prepared by placing 

5.00 mL of the 1000 mg a.i./L secondary stock solution in a 50.0-mL volumetric flask and 

bringing it to volume with THF. The 1000 mg a.i./L secondary stock solution and the 

100 mg a.i./L tertiary stock solution were used to prepare calibration standards. 

2.5 Preparation of Quality Control Samples 

n-Octanol quality control samples were prepared at concentrations of 206, 1032 and 

2064 mg a.i./L by fortification of water-saturated n-octanol with the 5.16 mg a.i./mL stock 

solution. 
Springborn Smithers Study No. 13658.6319 Page 11 

2.6 Preparation of Calibration Standards 

Calibration standards were prepared at concentrations of 2.00, 5.00, 10.0, 25.0, 35.0 and 

50.0 mg a.i./L by fortifying THF with the 100 mg a.i./L (2.00 and 5.00 mg a.i./L standards) and 

1000 mg a.i./L (10.0, 25.0, 35.0 and 50.0 mg a.i./L standards) stock solutions. 

2.7 Test Apparatus 

The test apparatus consisted of 50-mL glass centrifuge tubes with Teflon®-lined screw caps. All 

testing was conducted in a temperature-controlled environmental chamber designed to maintain a 

temperature of 20 ± 1 oC. The temperature extremes were measured with a calibrated minimummaximum thermometer. 

2.8 Test Solution Preparation 

2.8.1 Preliminary Test 

Test solutions were prepared in 50-mL glass centrifuge tubes using 20.0 mL of the 

2.02 mg a.i./mL stock solution in water-saturated n-octanol along with 20.0 mL of 

n-octanol-saturated water. 

2.8.2 Definitive Test 

During the definitive experiment, three test solutions were prepared in duplicate using the 

volume ratios of 1:2, 1:1 and 2:1 n-octanol/water. In the volume ratio of 1:2 test, 10 mL of the 

2.05 mg a.i./mL stock solution in water-saturated n-octanol along with 20 mL of corresponding 

n-octanol-saturated water was prepared. In the volume ratio of 1:1 test, 15 mL of the stock 

solution in water-saturated n-octanol along with 15 mL of corresponding n-octanol-saturated 

water was prepared. In the volume ratio of 2:1 test, 20 mL of the stock solution in watersaturated n-octanol along with 10 mL of corresponding n-octanol-saturated water was prepared. 
Springborn Smithers Study No. 13658.6319 Page 12 

2.9 Test Procedure 

2.9.1 Preliminary Test 

The centrifuge tubes containing the test solutions were sealed with Teflon®-lined screw caps and 

hand rotated through 180 degrees about their transverse axes for approximately 100 rotations in 

five minutes to establish equilibrium. The samples were then centrifuged at approximately 

1000 rpm for 20 minutes in a Beckman Model GS-6R centrifuge. The temperature was 

maintained at 18.7 oC during this period. 

A portion of the n-octanol phase was sampled by pipet and transferred to a vial. The remainder 

of the n-octanol phase and interfacial layer was removed and discarded and the water phase was 

sampled. The pH of the aqueous phase of each sample was determined using pH indicator strips 

after sampling. 

After the n-octanol and water phases were separated, the n-octanol portion of each sample 

(approximately 10 mL) was then analyzed by gel permeation chromatography with evaporative 

light scattering detection (GPC/ELSD). 

2.9.2 Definitive Test 

The centrifuge tubes containing the test solutions were sealed with Teflon®-lined caps and hand 

rotated through 180 degrees about their transverse axes for approximately 100 rotations in 

five minutes to establish equilibrium. The samples were then centrifuged for 20 minutes at 

approximately 1000 rpm in a Beckman Model GS-6R centrifuge. Test samples were allowed to 

re-equilibrate at the test temperature for approximately one hour. The temperature ranged from 

25.8 to 26.6oC throughout the definitive test. 

A portion of the n-octanol phase was removed by pipet and transferred to a vial. The remainder 

of the n-octanol phase and interfacial layer was removed and discarded. A portion of the water 

phase was sampled for pH measurements. Finally, the n-octanol samples were analyzed by 
Springborn Smithers Study No. 13658.6319 Page 13 

GPC/ELSD. Both water and n-octanol test samples were prepared for analysis in the same 

manner as described for the preliminary test. 

2.10 Chemical Analysis 

Blank water-saturated n-octanol samples and aliquots of the water-saturated n-octanol from the 

test samples were prepared and analyzed to determine EFKA 8530 concentration. For each test 

sample, one aliquot was removed from only the n-octanol phase. The octanol phase of the 

samples was diluted into the standard range with THF prior to analysis. In addition, a sample of 

the stock solution (2.02 mg a.i./mL preliminary test, 2.05 mg a.i./mL definitive test) was 

analyzed. 

Calibration standards were analyzed with the test samples, one set prior to analysis of the test 

samples and the other set after the test samples. Three QC samples were prepared and analyzed 

with each of the test samples. Analysis of the QC samples was used to judge the precision and 

instrumental control maintained during the analytical process. 

All aqueous test and QC samples were analyzed for EFKA 8530 concentration by gel permeation 

chromatography with evaporative light scattering detection (GPC/ELSD) based on methodology 

validated at Springborn Smithers. The method validation study was conducted prior to the 

initiation of the definitive test and established an average recovery of EFKA 8530 of 

91.8% ± 2.10% from n-octanol (Appendix 2). Acceptance criteria for the recovery of QC 

samples were set at 80 to 120% of the prepared concentration. Conditions and procedures used 

throughout the analysis of the test and QC samples during this study were the same as those 

described in the method validation study. All octanol test and QC samples were analyzed using 

the same standards and instrumental conditions as in the method validation following dilution 

into the calibration standard range with THF. 
Springborn Smithers Study No. 13658.6319 Page 14 

3.0 CALCULATIONS 

The partition coefficient (Pow) for EFKA 8530 was calculated using the following equation: 

P

C

C

OW

n - octanol

water

=

Where: 

Cn-octanol = the concentration as moles/L of EFKA 8530 in the n-octanol phase 

at equilibrium 

Cwater = the calculated concentration as moles/L of EFKA 8530 in the water 

phase (based upon total measured mass minus the measured mass in 

the octanol fraction divided by the volume of PRW) 

4.0 RESULTS 

4.1 Preliminary Results 

The temperature in the environmental chamber during the preliminary test was maintained at 

18.7 oC. Results of the preliminary test are presented in Table 1. The mean partition coefficient 

of the test substance was determined to be 22.0 (log Pow = 1.30).

4.2 Definitive Results 

The temperature in the environmental chamber during definitive testing ranged from 25.8 to 

26.6 oC. The partition coefficient for definitive test samples is summarized in Table 2. The

mean partition coefficient of the test substance was 24.9 (log Pow = 1.31). A representative 

chromatogram for a water-saturated n-octanol phase sample is presented in Figure 1. 

The results of quality control sample analysis performed in n-octanol during preliminary and 

definitive testing and stock solution analysis are summarized in Table 3. Water-saturated noctanol quality control samples resulted in measurements ranging from 96.0% to 104% during 
Springborn Smithers Study No. 13658.6319 Page 15 

preliminary and definitive testing. Based on these results, it was established that the appropriate 

quality control was maintained during the analyses. Results of the stock solution analysis during 

the preliminary test and definitive test resulted in a recovery of 106% and 99.5%, respectively, of 

the nominal concentration (2020 and 2050 mg a.i./L, respectively). Representative 

chromatograms from the analysis of a quality control sample and a calibration standard are 

presented in Figure 2 and Figure 3, respectively. 
Springborn Smithers Study No. 13658.6319 Page 16 

PROTOCOL DEVIATION 

The protocol states that preliminary testing will be conducted in an environmental chamber in the 

range of 20 to 25 oC, held constant at ± 1 oC. During the preliminary test, the thermometer on 

one side of the chamber was 20 oC. The minimum/maximum thermometer used for the study 

had a temperature of 18.7 oC throughout the entire preliminary test. Due to the short period of 

time required to invert samples to 180o for 100 inversions, this temperature deviation did not 

have a negative impact on the results or interpretation of the study. 
Springborn Smithers Study No. 13658.6319 Page 17 

REFERENCES 

Dean, J.A., Ed. 1985. Lange’s Handbook of Chemistry. pp. 5; 102-103, 13th ed., McGraw-Hill 

Book Company, New York. 

OECD, 1995. OECD Guideline for Testing of Chemicals. Partition Coefficient 

(n-octanol/water): Shake Flask Method. Guideline #107. Adopted 27 July 1995. 

OECD, 1998. OECD Series on Principles of Good Laboratory Practice and Compliance 

Monitoring. Number 1. OECD Principles on Good Laboratory Practice (as revised in 

1997). Environment Directorate Chemicals Group and management Committee. 

ENV/MC/CHEM(98)17. OECD Paris. France. 41 pp. 
Springborn Smithers Study No. 13658.6319 Page 18 

Table 1. Results of the preliminary n-octanol/water partition coefficient test 

with EFKA 8530. 

 Measured Concentration 

Ratio Replicate 

Number 

n-Octanol 

Phase 

(moles/L) 

Calculated 

Water Phase 

(moles/L) 

Pow Log Pow

 

1:1a 1 3.49E-03 2.82E-04 12.4 1.09 

 2 3.65E-03 1.15E-04 31.7 1.50 

 

 Mean: 22.0 1.30 

 a

 pH = 5 
Springborn Smithers Study No. 13658.6319 Page 19 

Table 2. Results of the definitive n-octanol/water partition coefficient test 

with EFKA 8530. 

 Measured Concentration 

Ratio 

(n-octanol:water) 

Replicate 

Number 

n-Octanol 

Phase 

(moles/L) 

Calculated 

Water Phase 

(moles/L) 

Pow Log Pow

 

1:1a 1 3.47E-03 1.17E-04 29.7 1.47 

 2 3.51E-03 7.59E-05 46.3 1.67 

 

2:1a 1 3.41E-03 9.18E-05 37.1 1.57 

 2 3.25E-03 1.68E-04 19.3 1.29 

1:2a 1 3.39E-03 3.92E-04 8.65 0.937 

 2 3.38E-03 4.11E-04 8.24 0.916 

 Mean: 24.9 1.31 

 Std. Dev.: 15.5 0.321 

 % CV: 62.3 24.5 

 

a

 pH = 4 
Springborn Smithers Study No. 13658.6319 Page 20 

Table 3. Results of the analysis of the EFKA 8530 n-octanol stock solution 

and quality control samples. 

Preliminary Test 

Sample Prepared Concentration 

(mg a.i./L) 

Measured Concentration

(mg a.i./L) 

Percent 

Recovery 

(%) 

 

n-Octanol 

QC 1 206 207 100 

QC 2 1032 991 96.0 

QC 3 2064 2000 96.9 

 

Stock 

126-09E 2020 2140 106 

Definitive Test 

Sample Prepared Concentration 

(mg a.i./L) 

Measured Concentration 

(mg a.i./L) 

Percent 

Recovery 

(%) 

 

n-Octanol 

QC 1 206 214 104 

QC 2 1032 1019 98.8 

QC 3 2064 2075 101 

 

Stock 

126-09F 2050 2039 99.5 
Springborn Smithers Study No. 13658.6319 Page 21 

Figure 1. HPLC chromatogram of an EFKA 8530 water-saturated n-octanol 

phase test solution from the determination of the partition 

coefficient. 
Springborn Smithers Study No. 13658.6319 Page 22 

Figure 2. HPLC chromatogram of a quality control sample (1032 mg a.i./L) 

from the determination of the partition coefficient for EFKA 8530. 
Springborn Smithers Study No. 13658.6319 Page 23 

Figure 3. HPLC chromatogram of a calibration standard (25.0 mg a.i./L) from 

the determination of the partition coefficient for EFKA 8530. 
Springborn Smithers Study No. 13658.6319 Page 24 

APPENDIX 1 - STUDY PROTOCOL 
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APPENDIX 2 - ANALYTICAL METHODOLOGY 
Springborn Smithers Study No. 13658.6319 Page 35 

SUMMARY 

Methodology was validated (15 January 2008) to quantify the amount of EFKA 8530 present in 

recovery samples prepared in n-octanol. Recovery samples were diluted with tetrahydrofuran 

and analyzed by automated injection on a gel permeation chromatography system equipped with 

evaporative light scattering detection (GPC/ELSD). 

This method was validated by fortification of n-octanol with EFKA 8530 at concentrations of 

206, 1032 and 2064 mg a.i./L. Recoveries averaged 91.8% ± 2.10% and a limit of quantitation 

(LOQ) of 29.3 mg a.i./L. Defined limits for acceptance of quality control sample performance in 

subsequent studies were set at 80 to 120%. 

EXPERIMENTAL

Equipment

1. Instrument: Hewlett-Packard 1050 isocratic pump equipped with a 

Hewlett-Packard 1050 autosampler, Polymer 

Laboratories PL-ELS1000 evaporative light scattering 

detector, Hewlett-Packard 35900E A/D interface and a 

Hewlett Packard ChemStation Version A.06.03 for data 

acquisition 

 2. Balance: Orion 0960, Mettler Toledo AB204 

 3. Laboratory equipment: syringes, volumetric flasks, volumetric pipets, Pasteur 

pipets, glass centrifuge tubes, autosampler vials and 

amber glass bottles with Teflon®-lined caps 

Reagents

 1. n-Octanol: Sigma-Aldrich, reagent grade 

 2. Tetrahydrofuran (THF): Burdick & Jackson, reagent grade 

Test Substance 

The test substance, EFKA 8530, was received on 31 October 2007 from Ciba Specialty 

Chemicals Company, Allentown, Pennsylvania. The following information was provided: 
Springborn Smithers Study No. 13658.6319 Page 36 

 Name: EFKA 8530 

 Lot No.: 15306 

 CAS No.: Not Listed 

 Purity: 93.56% 

 Recertification Date: 31 January 2009 

Upon receipt at Springborn Smithers, the test substance (SSL No. 126-09) was stored at room 

temperature in the original container in a dark ventilated cabinet. Concentrations were adjusted 

for the purity of the test substance and are presented as active ingredient (a.i.).

PROCEDURES

Preparation of Stock Solutions

A 5.16 mg a.i./mL primary stock solution was prepared by placing 0.2760 g (0.2582 g as active 

ingredient) in a 50.0 mL volumetric flask and bringing it to volume with n-Octanol. This stock 

solution was used to fortify recovery samples. This stock was stored refrigerated (2 to 8 oC) in 

an amber glass bottle fitted with 20 mm septa. 

A 5.00 mg a.i./mL primary stock solution was prepared by placing 0.5344 g (0.5000 g as active 

ingredient) of the test substance in a 100-mL volumetric flask and bringing it to volume with 

tetrahydrofuran (THF). A 1000 mg a.i./L secondary stock solution was prepared by placing 

10.0 mL of the 5.00 mg a.i./mL stock solution in a 50.0-mL volumetric flask and bringing it to 

volume with THF. A 100 mg a.i./L tertiary stock solution was prepared by placing 5.00 mL of 

the 1000 mg a.i./L stock solution in a 50.0-mL flask and bringing it to volume with THF. The 

1000 mg a.i./L and 100 mg a.i./L stock solutions were used to fortify the calibration standards 

used during the study. All stock solutions were stored refrigerated (2 to 8 o

C) in amber glass 

bottles fitted with Teflon®-lined caps. 

Preparation of Calibration Standards

Calibration standards were prepared in THF by fortifying with the 1000 mg a.i./L and the 

100 mg a.i./L stock solutions to yield concentrations of 2.00, 5.00, 10.0, 25.0, 35.0 and 

50.0 mg a.i./L. 
Springborn Smithers Study No. 13658.6319 Page 37 

Sample Fortification and Preparation

A set of recovery samples was prepared by fortifying n-octanol at concentrations of 206, 1032 

and 2064 mg a.i./L. Sample volumes were 5.00 mL. Each concentration was prepared in 

triplicate. Recovery samples were fortified using the 5.16 mg a.i./mL stock solution. In addition 

three samples were left unfortified to serve as controls. All samples were diluted into the 

calibration standard range with THF prior to analysis. 

ANALYSIS

Instrumental Conditions

The GPC/ELSD analysis was conducted utilizing the following instrumental conditions: 

Column: TOSOH Bioscience TSKGel G1000Hxl, 5 μm, 

300 x 7.8 mm 

 Mobile Phase: 100% THF 

 Flow Rate: 1.0 mL/minute 

Injection Volume: 500 μL 

 Retention Time: approximately 5.7 minutes 

Detector Parameters 

 Temperature: Evaporator: 100 oC 

 Nebulizer: 50 oC 

 Transfer Line Temperature: 50 oC 

 Gas Flow Rate: 1.0 L/minute (Nitrogen) 

Preparation of Standard Curve 

Two sets of calibration standards were analyzed with each recovery sample set; one set prior to 

analysis of the recovery samples, and the second set immediately following the analysis of the 

recovery samples. Injection of recovery samples and calibration standards onto the 

chromatographic system was performed by programmed automated injection. 

CALCULATIONS

A polynomial calibration curve was constructed by plotting the natural logarithm (ln) of the 

analyte concentration (mg a.i./L) in the calibration standards against the natural logarithm (ln) of 

the peak area of the calibration standards. The equation of the line (equation 1) was 
Springborn Smithers Study No. 13658.6319 Page 38 

algebraically manipulated to give equation 2. The concentration of the test substance within 

each recovery sample was determined using the regression coefficients from the quadratic 

equation, the natural logarithm (ln) of the peak area of the recovery sample and the dilution 

factor. Equations 2, 3 and 4 were then used to calculate measured concentrations and analytical 

results. 

( ) DF x DCA 4

(x) DC(3) x)inverse(ln

2a

4aC 2bb lnx (2)

cb(lnx) 2 a(lnx)lny (1)

=

=

−+− =

++=

where: 

y = detector response (peak area) for analyte 

a, b, c = regression constants 

x = detected concentration (mg a.i./L) in the sample 

C = constant c minus the natural log of the peak area; C = (c - lny) 

DC (x) = detected concentration in the sample 

DF = dilution factor (the final sample volume divided by the original 

sample volume) 

A = concentration of the analyte in the original sample 

RESULTS AND DISCUSSION 

The mean recovery was 91.8% with a standard deviation of 2.10%. The limit of quantitation was 

set at 29.3 mg a.i./L. Defined limits for acceptance of quality control sample performance in 

subsequent studies were set at 80 to 120%. 

Analytical results for the recovery of EFKA 8530 from n-octanol are presented in Table 1A. 

Representative chromatograms from the analysis of a calibration standard, recovery sample and a 

control are presented in Figure 1A, Figure 2A and Figure 3A, respectively. A typical regression 

analysis for EFKA 8530 is presented in Figure 4A. 
Springborn Smithers Study No. 13658.6319 Page 39 

Table 1A. Analytical results for the recovery of EFKA 8530 from n-octanol 

during the method validation study. 

Sample 

ID 

Fortified Concentration 

(mg a.i./L) 

Dilution 

Factor 

Analytical Result 

(mg a.i./L) 

Percent 

of Fortified 

(%) 

Control A 0.00 20.0 < 29.3 a NA b

Control B 0.00 20.0 < 29.3 NA 

Control C 0.00 20.0 < 29.3 NA 

 

Low A 206 20.0 188 91.4 

Low B 206 20.0 193 93.9 

Low C 206 20.0 194 94.2 

 

Mid A 1032 33.3 945 91.6 

Mid B 1032 33.3 950 92.0 

Mid C 1032 33.3 964 93.4 

High A 2064 66.7 1800 87.3 

High B 2064 66.7 1900 91.8 

High C 2064 66.7 1870 90.4 

 

 Mean: 91.8 

SD: 2.10 

RSD: 2.29 

N: 9 

a Concentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each 

analysis is dependent upon the linear regression, the area of the low standards and the dilution factor of the 

controls. The limit of quantitation for the method validation was 29.3 mg a.i./L. 

b NA = Not Applicable. 

Note: Results were calculated using the actual analytical (unrounded) results and not the rounded values 

presented in this table.
Springborn Smithers Study No. 13658.6319 Page 40 

Figure 1A. Representative chromatogram of a 10.0 mg a.i./L calibration 

standard used for quantification during the method validation study 

with EFKA 8530. 
Springborn Smithers Study No. 13658.6319 Page 41 

Figure 2A. Representative chromatogram of a 1032 mg a.i./L recovery sample 

fortified with EFKA 8530. 
Springborn Smithers Study No. 13658.6319 Page 42 

Figure 3A. Representative chromatogram of a control sample during the 

method validation study with EFKA 8530. 

Note: No peak equivalent to retention time for EFKA 8530. EFKA 8530 is ordinarily detected at a retention time 

of approximately 5.7 minutes. 
Springborn Smithers Study No. 13658.6319 Page 43 

Figure 4A. A typical regression analysis for the calibration standards used to 

quantitate EFKA 8530. 

Polynomial Regression Analysis

y = -6.1918E-02x2 + 2.2171E+00x + 5.0862E+00

R2 = 9.9961E-01

0

2

4

6

8

10

12

14

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

Concentration

Detector Response

Polynomial Regression Analysis 

y = -0.061918x2

 + 2.2171x + 5.0862 

R2

 = 0.99961 

ln Standard Standard ln standard Standard 

Concentration Concentration Response Response (b^2-4aC) 

(mg a.i./L) (mg a.i./L) Area Area D 

0.693 2.00 6.6574 778.54065 4.52638 

1.609 5.00 8.4155 4516.70215 4.09095 

2.303 10.0 9.8889 19710.8 3.72603 

3.219 25.0 11.5905 108063 3.30461 

3.555 35.0 12.1870 196212 3.15688 

3.912 50.0 12.7738 352841 3.01154 

0.693 2.00 6.5736 715.91473 4.54715 

1.609 5.00 8.4353 4606.87646 4.08605 

2.303 10.0 9.9361 20662.1 3.71436 

3.219 25.0 11.5971 108780 3.30297 

3.555 35.0 12.1759 194046 3.15962 

3.912 50.0 12.8291 372920 2.99783