DETERMINATION OF THE n-OCTANOL/WATER PARTITION COEFFICIENT 

OF DIETHYLTOLUENE DIAMINE (DETDA) BY THE SHAKE FLASK METHOD 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471C-143 

U.S. EPA Product Properties Test Guidelines 

OPPTS 830.7550, Partition Coefficient (n-Octanol/Water), Shake Flask Method 

and 

OECD Guideline for the Testing of Chemicals 

OECD 107, Partition Coefficient (n-octanol/water): Shake Flask Method 

AUTHORS: 

William R. Schutt, B.S. 

Willard B. Nixon, Ph.D. 

STUDY INITIATION DATE: February 15, 2012 

STUDY COMPLETION DATE: June 27, 2012 

SUBMITTED TO: 

Albemarle Corporation 

451 Florida Street 

Baton Rouge, LA 70801 

Wildlife International, Ltd. 

8598 Commerce Drive 

Easton, Maryland 21601 

(410) 822-8600 

Page 1 of 42 



Wildlife International, Ltd. Project Number 471C-143 

- 5 - 

TABLE OF CONTENTS 

Title/Cover Page ..................................................................................................................................... 1 

Good Laboratory Practice Compliance Statement ................................................................................. 2 

Quality Assurance Statement ................................................................................................................. 3 

Report Approval ..................................................................................................................................... 4 

Table of Contents ................................................................................................................................... 5 

Summary ................................................................................................................................................ 7 

Introduction ............................................................................................................................................ 8 

Objective ................................................................................................................................................ 8 

Experimental Design .............................................................................................................................. 8 

Materials and Methods ........................................................................................................................... 8 

 Test Substance ........................................................................................................................... 9 

 Solvents and Reagents ............................................................................................................... 9 

 Saturated Solvent Preparation ................................................................................................... 9 

 Stock and Standards Preparations ........................................................................................... 10 

 Partition Sample Preparation for Feasibility Trial ................................................................... 10 

 Partition Sample Preparation for Definitive Test .................................................................... 11 

 Analytical Methods ................................................................................................................. 11 

 Calibration Curve and Limit of Quantitation .......................................................................... 12 

 Matrix Blank and Matrix Fortification Samples ..................................................................... 13 

 Example Calculations .............................................................................................................. 13 

Results and Discussion ......................................................................................................................... 14 

 Feasibility Trial ....................................................................................................................... 14 

 Stock Confirmation and pH Confirmation .............................................................................. 14 

 Mass Balance Determination ................................................................................................... 15 

 Partition Coefficient Determination ........................................................................................ 15 

Conclusions .......................................................................................................................................... 15 

References ............................................................................................................................................ 16 
Wildlife International, Ltd. Project Number 471C-143 

- 6 - 

TABLE OF CONTENTS 

- Continued - 

TABLES

Table 1. HPLC Operational Parameters .......................................................................................... 17 

Table 2. Quality Control Samples of DETDA in n-Octanol and Reagent Water Partition Phases ... 18 

Table 3. Mass Balance Results for the n-Octanol/Water Partition Coefficient Definitive Test 

 with DETDA ..................................................................................................................... 19 

Table 4. Measured Concentrations and Partition Coefficient Results for the n-Octanol/Water 

 Partition Coefficient Definitive Test with DETDA .......................................................... 20 

FIGURES 

Figure 1. Analytical method flow-chart for the analysis of DETDA in 

 n-octanol/water partition samples ..................................................................................... 21 

Figure 2. A representative calibration curve for the analysis of DETDA by 

 HPLC/UV ......................................................................................................................... 22 

Figure 3. Representative chromatograms of low-level and high-level DETDA 

 calibration standards for HPLC/UV analyses ................................................................... 23 

Figure 4. Chromatograms of the matrix blank samples.................................................................... 24 

Figure 5. Chromatograms of the matrix fortification samples ......................................................... 25 

Figure 6. Representative chromatograms of a partition test sample (471C-143-1) .......................... 26 

APPENDICES 

Appendix 1. Protocol ...................................................................................................................... 27 

Appendix 2. Characterization Report .............................................................................................. 38 

Appendix 3. Personnel Involved in the Study ................................................................................. 42 
Wildlife International, Ltd. Project Number 471C-143 

- 7 - 

SUMMARY 

STUDY TITLE: Determination of the n-Octanol/Water Partition Coefficient of 

Diethyltoluene Diamine (DETDA) by the Shake Flask Method 

WILDLIFE INTERNATIONAL, LTD. 

PROJECT NUMBER: 471C-143 

SPONSOR: Albemarle Corporation 

451 Florida Street 

Baton Rouge, LA 70801 

TESTING FACILITY: Wildlife International, Ltd. 

Easton, Maryland 21601 

SPONSOR’S REPRESENTATIVE: Marcia L. Hardy, DVM, Ph.D. 

LOCATION OF STUDY, RAW

DATA AND A COPY OF THE

FINAL REPORT: 

Wildlife International Ltd. 

Easton, Maryland 21601 

TEST SUBSTANCE: Diethyltoluene Diamine (DETDA) 

TEST DATES: Experimental OECD Start – February 16, 2012 

Experimental EPA Start – February 16, 2012 

Experimental Termination – February 23, 2012 

TEST SYSTEM: HPLC-grade reagent water saturated with n-octanol, and n-octanol 

saturated with HPLC-grade reagent water. 

SUMMARY: The n-octanol/water partition coefficient (Pow) of DETDA at 25oC was determined 

by the shake flask method. The partition coefficient was evaluated as a function of 

solvent volume ratio. Three n-octanol/water volume ratios were evaluated in 

duplicate. After a 15-minute partition equilibration period (via gentle mechanical 

agitation) followed by centrifugation, sub-samples from each phase of each 

partition test vessel were prepared for analysis by volumetric dilution. Aliquots 

from the n-octanol phase samples were diluted in 50:50 (v/v) acetonitrile:reagent 

water (ACN:H2O). An aliquot of each aqueous phase sample was diluted to a final 

solvent composition of 50:50 (v/v) ACN:H2O. The dilutions of both the n-octanol 

phase samples and the aqueous phase samples were analyzed by high performance 

liquid chromatography with ultraviolet absorbance detection (HPLC/UV). Mass 

balance for DETDA was obtained in all test vessels. At n-octanol/water solvent 

ratios of 1:1, 1:2 and 1:4, the mean Pow was 23.3, 24.9 and 24.0 respectively, 

corresponding to mean log Pow values of 1.37, 1.40 and 1.38, respectively. The 

overall mean log Pow was 1.38 ± 0.019 (n = 6, CV = 1.4%). 
Wildlife International, Ltd. Project Number 471C-143 

- 8 - 

INTRODUCTION 

Tests were conducted to determine the n-octanol/water partition coefficient of diethyltoluene 

diamine (DETDA) at 25C using the shake flask method. Wildlife International, Ltd. conducted this 

study for Albemarle Corporation at the Wildlife International, Ltd. analytical chemistry facility in Easton, 

Maryland. The experimental portion of this study was conducted between February 16, 2012 and 

February 23, 2012. Raw data generated by Wildlife International, Ltd. and a copy of the final report are 

filed under Project Number 471C-143 in archives located on the Wildlife International, Ltd. site. The 

original signed report will be maintained by Albemarle Corporation, Baton Rouge, Louisiana. 

OBJECTIVE 

The objective of this study was to experimentally determine the n-octanol/water partition 

coefficient of DETDA by equilibrating the test substance in a two-phase system of n-octanol/water by the 

flask-shaking method and analyzing the two phases by appropriate analytical methodology. 

EXPERIMENTAL DESIGN 

The n-octanol/water partition coefficient of DETDA was determined using the flask-shaking 

method. The method consisted of shaking the solute (DETDA) with the two immiscible solvents 

(n-octanol and HPLC-grade reagent water) at 25 ± 1°C over a period of time (15 minutes) and analyzing 

the n-octanol and aqueous phases for the solute concentration using HPLC/UV. The concentration of 

DETDA in each partition phase was maintained at less than 0.01 M. A feasibility trial was performed at 

an n-octanol/water volume ratio of 1:1 (v/v) to confirm that the shake flask experimental design was 

appropriate for the test substance and to provide an estimate of appropriate solvent ratios for the definitive 

test. The definitive portion of the test was conducted at three n-octanol/water solvent ratios. 

MATERIALS AND METHODS 

The study was conducted according to the procedures outlined in the protocol, “Determination of 

the n-Octanol/Water Partition Coefficient of Diethyltoluene Diamine (DETDA) by the Shake Flask 

Method” (Appendix 1). The study was also performed based on procedures in U.S. EPA Product 

Properties Test Guidelines, OPPTS 830.7550, Partition Coefficient (n-Octanol/Water), Shake Flask 

Method (1) and OECD Guideline for the Testing of Chemicals, OECD 107, Partition Coefficient 

(n-octanol/water): Shake Flask Method (2). 
Wildlife International, Ltd. Project Number 471C-143 

- 9 - 

Test Substance 

The test substance was received from Albemarle Corporation on September 21, 2011, and was 

assigned Wildlife International, Ltd. identification number 10232. The test substance was a liquid 

identified as DETDA; Lot number – 4500051816-011110; CAS Number 68479-98-1. A purity of 

97.27% was obtained from the characterization report provided by the Sponsor (Appendix 2). The test 

substance was stored under ambient conditions. An expiration date of March 3, 2013 and molecular 

weight of the test substance, 178.28 g/mol, were provided by the Sponsor. 

Solvents and Reagents 

All solvents were HPLC-grade or higher quality and reagents were ACS reagent-grade or better. 

Solvent/Reagent Manufacturer/Lot 

Reagent Water: HPLC-Grade, Burdick & Jackson, Lot Nos. DE958-E 

(equivalent to ASTM Type II Designation D1193-06 (3)). 

Acetonitrile (ACN): HPLC-Grade, Burdick & Jackson, Lot Nos. DF154 

n-Octanol: Sigma-Aldrich, Lot No. SHBB3592V 

Saturated Solvent Preparation 

Mutually saturated partition phases were prepared. The solvents were mutually saturated by 

combining 500 milliliters of reagent water with 300 milliliters of n-octanol in a clean French-square 

bottle. The bottle was placed in an agitating water bath (Boekel 290400) maintained at 25oC and agitated 

for approximately 20 hours at 25 ± 0.2°C (temperature monitored using a min-max thermometer). After 

the saturation period, the contents of the bottle were decanted into a separatory funnel and allowed to 

partition for a minimum of 10 minutes. The mutually saturated n-octanol and aqueous phases were 

separated by draining the lower aqueous phase and upper n-octanol phase into separate French-square 

bottles. The separated n-octanol and aqueous saturated solvents were stored under ambient conditions. 

Before use in an experimental trial, the saturated solvents were equilibrated at the test temperature 

(25 ± 1oC) in a temperature-controlled incubator (Fisher Model 3724) with a data logger to continuously 

monitor temperature. Before use in the definitive test, the pH of the saturated reagent water was 

measured using the pH meter at the test temperature. 
Wildlife International, Ltd. Project Number 471C-143 

- 10 - 

Stock and Standards Preparations 

A primary stock solution of the test substance was prepared at a nominal concentration of 

1.00 mg DETDA/mL by accurately weighing 0.1029 grams of the test substance in a scintillation vial 

(weight adjusted for 97.27% purity). The test substance aliquot was then quantitatively transferred into a 

100-mL volumetric flask and was brought to final volume using ACN. The 1.00 mg DETDA/mL 

(1000 mg DETDA/L) primary stock solution, identified as 10232-001, was used to prepare a set of 

standard solutions in a final solvent composition of 50:50 (v/v) ACN: H2O, using the following dilution 

scheme: 

Stock 

Concentration 

(mg DETDA/mL) 

Aliquot 

(μL) 

Final 

Volume 

(mL) 

Standard 

Concentration 

(mg DETDA/L) 

1.00 

1.00 

1.00 

1.00 

1.00 

50.0 

100 

200 

300 

500 

10.0 

10.0 

10.0 

10.0 

10.0 

5.00 

10.0 

20.0 

30.0 

50.0 

A primary stock solution of the DETDA test substance in n-octanol saturated with reagent water 

was prepared by accurately weighing 0.4112 grams of the test substance directly into a 100-mL 

volumetric flask using an analytical balance. The volumetric flask was then brought to final volume with 

saturated n-octanol. The nominal DETDA concentration in the resulting primary stock was 4.00 mg 

DETDA/mL. This primary stock solution in n-octanol was used to fortify all partition samples and the 

n-octanol phase matrix fortification sample. 

Partition Sample Preparation for Feasibility Trial 

An initial feasibility trial was conducted with equal volumes of saturated reagent water and 

n-octanol solutions (n-octanol:reagent water volume ratio of 1:1). Duplicate samples were prepared using 

volumetric pipettes to transfer 10.0 milliliters of reagent water (saturated with n-octanol) into each labeled 

40-mL glass sample vial followed by 6.00 milliliters of n-octanol (saturated with reagent water). Each 

partition sample was then fortified with DETDA by transferring 4.00 milliliters of the 4.00-mg 

DETDA/mL primary stock solution in n-octanol saturated with reagent water. This brought the total 

volume of n-octanol saturated with reagent water in each sample to 10.0 milliliters. The nominal fortified 

mass of DETDA was 16.0 milligrams in each partition sample. After fortification, the partition samples 
Wildlife International, Ltd. Project Number 471C-143 

- 11 - 

were sealed, gently swirled or agitated by hand for approximately thirty seconds and placed into a 25 ± 

1oC agitating water bath for 15 minutes (temperature monitored using a min-max thermometer). 

Partition Sample Preparation for Definitive Test 

Based on the findings of the feasibility trial, partition samples in the definitive tests were 

prepared at n-octanol/water volume ratios of 1:1, 1:2 and 1:4. The partition samples were prepared in 

duplicate at each volume ratio in 40-mL glass vials. Volumetric pipettes were used to transfer appropriate 

volumes of reagent water (saturated with n-octanol) to the sample vessels, followed by appropriate 

volumes of n-octanol (saturated with reagent water). The samples were each fortified using a volumetric 

pipette to transfer 4.00 or 2.00 milliliters of the 4.00-mg DETDA/mL stock solution in n-octanol 

(saturated with reagent water). The nominal fortified mass of DETDA was 16.0, 16.0, and 

8.00 milligrams for the 1:1, 1:2, and 1:4 partition samples, respectively. After fortification, the samples 

were sealed and gently agitated or swirled by hand for approximately 30 seconds. The samples were 

placed into a 25 ± 1oC agitating water bath for 15 minutes (temperature was monitored using a min-max 

thermometer). 

Analytical Methods 

The sample preparation and instrumental determination procedures used for the analysis of 

DETDA in the n-octanol and aqueous phases of the partition samples were developed at Wildlife 

International, Ltd. A method flow-chart for the analysis of DETDA in the definitive test partition 

samples is presented in Figure 1. 

The samples were centrifuged (Thermo Sorvall Legend XF) at a setting of 1500 rpm for 

10 minutes to promote complete phase separation. A portion of the top (n-octanol) phase of each 

partition sample was transferred into a labeled glass vial. Using gas-tight syringes and volumetric flasks, 

a 0.100-mL aliquot of each n-octanol phase sample was diluted to a 10.0-mL final volume with 50:50 

(v/v) ACN:H2O dilution solvent into the calibration range of the HPLC/UV methodology. An aliquot of 

each diluted n-octanol phase sample was transferred to an autosampler vial and submitted for HPLC/UV 

analysis. Following n-octanol phase sampling, the aqueous phase was collected. Using a disposable 

syringe fitted with a detachable needle, the needle was inserted through the remaining n-octanol phase in 

the 40-mL partition sample vial while gently and continuously expelling air. Once the tip of the needle 
Wildlife International, Ltd. Project Number 471C-143 

- 12 - 

was in the aqueous phase, a portion of the aqueous phase was drawn into the syringe. The syringe was 

removed from the sample and the needle was immediately detached. The contents of the syringe were 

expelled into a labeled glass vial. Using gas-tight syringes, a 1.00-mL aliquot of each aqueous phase 

sample was transferred into separate 5.00-mL volumetric flask preloaded with 2.50-mL of ACN. The 

volumetric flasks were brought to final volume with reagent water to yield a final sample composition of 

50:50 (v/v) ACN:H2O. An aliquot of each diluted aqueous phase sample was transferred to an 

autosampler vial and submitted for HPLC/UV analysis. The aqueous phase of one partition sample from 

the definitive test was selected and its pH was measured using a calibrated pH meter. 

To confirm the DETDA concentration of the 4.00-mg DETDA/mL n-octanol stock solution used 

to fortify the partition samples, triplicate aliquots were volumetrically diluted with 50:50 (v/v) ACN:H2O 

dilution solvent (overall dilution factor of 250) using gas-tight syringes and volumetric flasks and an 

aliquot of each diluted n-octanol stock sample was submitted for HPLC/UV analysis concurrently with 

the feasibility trial samples. 

Concentrations of DETDA were determined by Agilent Series 1100/1200 HPLC systems 

equipped with an Agilent Series 1100 and 1200 Variable Wavelength Detector (VWD) set at 216 nm for 

the feasibility trial and definitive test, respectively. Chromatographic separations were achieved using a 

YMC-Pack ODS-AM analytical column (150 mm x 4.6 mm, 3-m particle size). The instrument 

parameters for analysis of DETDA in the definitive test are summarized in Table 1. 

Calibration Curve and Limit of Quantitation 

Five calibration standards of DETDA in 50:50 (v/v) ACN: H2O, ranging in concentration from 

5.00 to 50.0 mg DETDA/L, were analyzed concurrently with each set of n-octanol and aqueous phase 

samples. The calibration set was injected at the beginning and end of the analytical sequence with one 

calibration standard injected after a maximum of every five test samples. Linear regression equations 

were generated for the analytical sequence using the summed/total peak area response for retention times 

between approximately 7.4 and 7.5 minutes versus the respective DETDA concentrations of the 

calibration standards. The concentration of DETDA in the samples was determined by substituting the 

total peak area responses for retention times between approximately 7.4 and 7.5 minutes of the samples 

into the applicable linear regression equation. The calibration curve for DETDA is presented in Figure 2. 
Wildlife International, Ltd. Project Number 471C-143 

- 13 - 

 Representative chromatograms of low- and high-level DETDA calibration standards are presented in 

Figure 3. 

For the definitive test, the method limits of quantitation (LOQ) for the determination of DETDA 

in the n-octanol and aqueous phases were 500 mg DETDA/L and 25.0 mg DETDA/L, respectively. Each 

LOQ was calculated as the product of the concentration of the low-level calibration standard and the 

dilution factor (DF) of the concurrently analyzed matrix blank samples. 

Matrix Blank and Matrix Fortification Samples 

Matrix blank and matrix fortification samples were prepared and analyzed in each of the partition 

solvent phases. These quality control samples were processed in the same fashion as the definitive 

samples to determine possible interferences and to assess method efficiency. No chromatographic 

interferences were observed in either the n-octanol or aqueous phase matrix blank samples. 

Chromatograms of the n-octanol and aqueous phase matrix blank samples are presented in Figure 4. The 

matrix fortification samples yielded procedural recoveries of 105% and 98.8% of the nominal 

concentrations in n-octanol (1600 mg DETDA/L) and reagent water (60.0 mg DETDA/L), respectively 

(Table 2). Chromatograms of the n-octanol and reagent water matrix fortification samples are presented 

in Figure 5. 

Example Calculations 

The analytical result for the concentration of DETDA in the n-octanol partition phase of sample 

number 471C-143-1 was calculated using the following equations. 

Test Substance (mg DETDA/L) in sample = Peak Area - (Y-intercept)

Slope x Dilution Factor 

Total Peak area = 13782.57471 Initial Volume (Vi) = 0.100 mL 

Y-intercept = -306.20822 Final Volume (Vf) = 10.0 mL 

Slope = 880.11029 Dilution Factor (Vf/Vi) = 100 

Molecular Weight DETDA = 178.28 g/mol Unit Conversion = 0.001 g/mg 

Test Substance (mg DETDA/L) in sample = 13782.57471 - (-306.20822)

 880.11029 x 100 
Wildlife International, Ltd. Project Number 471C-143 

- 14 - 

Test Substance (mg DETDA/L) in sample = 1600 

DETDA (M) in sample = Test Substance in sample (mg DETDA/L) x Unit Conversion 

Molecular Weight DETDA 

DETDA (M) in sample = 1600 mg DETDA/L x 0.001 g/mg 

178.28 g/mol

DETDA (M) in sample = 8.98 x 10-3

The molar concentration for the aqueous phase of this partition sample was calculated in the same 

manner and using the same equations as the molar concentration calculation for the n-octanol phase. The 

partition coefficient for this sample (sample number 471C-143-1) was calculated from the n-octanol and 

aqueous phase molar concentrations using the following equation: 

Partition Coefficient = DETDA in n-octanol phase (M) 

 DETDA in aqueous phase (M) 

Pow = 

8.98 x 10-3 M 

3.87 x 10-4 M 

Pow = 23.2 

Log (Pow) = 1.37 

Calculations performed using Microsoft® Excel 2000 in the full precision mode. Manual calculations may differ slightly.

RESULTS AND DISCUSSION 

Feasibility Trial 

The feasibility trial at a 1:1 n-octanol/water volume ratio yielded a mean estimated partition 

coefficient of 21.8 (log Pow = 1.34). Based on these results, it was concluded that the n-octanol to water 

volume ratio of 1:1would be appropriate for use in the definitive test. 

Stock Confirmation and pH Confirmation 

The stock solution of DETDA in saturated n-octanol used to fortify the partition samples had a 

mean measured concentration of 3.95 mg DETDA/mL (n = 3), which corresponded to 98.8% of the 

nominal DETDA concentration. 
Wildlife International, Ltd. Project Number 471C-143 

- 15 - 

Prior to the definitive test, the measured pH of the reagent water (saturated with n-octanol) was 

8.57. The measured pH value of the aqueous phase from a selected partition sample was 8.53. 

Mass Balance Determination 

The total mass recovered was calculated for each of the six definitive test vessels. Mass balance 

was obtained for DETDA in all vessels. The overall mean percent recovery for DETDA for the six 

partition test vessels was 101 ± 2.4% (Table 3). 

Partition Coefficient Determination 

Partition coefficients were calculated from the measured concentrations of DETDA in each phase 

for each of six samples. Overall mean n-octanol/water partition coefficients and overall mean of the 

logarithm (base 10) of the n-octanol/water partition coefficients were calculated for each of the six 

samples. 

The mean Pow values for DETDA at n-octanol/water solvent ratios of 1:1, 1:2 and 1:4 were 

determined to be 23.3, 24.9 and 24.0, respectively (Table 4). The corresponding mean log Pow values 

were 1.37, 1.40 and 1.38, respectively. The overall mean log Pow was 1.38 ± 0.019 (n = 6). 

Representative chromatograms of the n-octanol and aqueous phase samples are presented in Figure 6. 

CONCLUSIONS 

The n-octanol/water partition coefficient of DETDA at 25oC, expressed as the logarithm (base 

10), was determined to be 1.38 ± 0.019 (n = 6, CV = 1.4%). 
Wildlife International, Ltd. Project Number 471C-143 

- 16 - 

REFERENCES 

1. U.S. EPA Product Properties Test Guidelines. 1996. Product Properties Test Guidelines 

OPPTS 830.7550. Partition Coefficient (n-Octanol/Water), Shake Flask Method.

2. Organisation for Economic Cooperation and Development. 1995. Guideline for the Testing of 

Chemicals, 107: Partition Coefficient (n-octanol/water): Shake Flask Method.

3. American Society for Testing and Materials. 2006. Standard Specification for Reagent Water. 

D1193-06, ASTM Section II Water and Environmental Technology, Vol. 11.01: 45-47. 
Wildlife International, Ltd. Project Number 471C-143 

- 17 - 

Table 1 

HPLC Operational Parameters 

INSTRUMENT: Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC) 

with an Agilent Series 1100 or 1200 Variable Wavelength Detector 

ANALYTICAL COLUMN: YMC-Pack ODS-AM (150 mm x 4.6 mm, 3-m particle size) 

STOP TIME: 13.00 minutes 

FLOW RATE: 1.000 mL/minute 

OVEN TEMPERATURE: 40.0°C 

MOBILE PHASE: Solvent A (80.0%): HPLC-grade Reagent Water 

Solvent B (20.0%): Acetonitrile 

GRADIENT ELUTION 

PROFILE: TIME (min.) SOLVENT A (%) SOLVENT B (%) FLOW 

(mL/min.) 

 0.01 80.0 20.0 1.000 

 2.00 80.0 20.0 1.000 

 8.00 5.0 95.0 1.000 

 10.00 5.0 95.0 1.000 

 10.01 80.0 20.0 1.000 

 13.00 80.0 20.0 1.000 

 

INJECTION VOLUME: 100.00 μL 

DETDA RETENTION TIME 

RANGE: Peak area 1: 7.4 minutes 

Peak area 2: 7.5 minutes 

UV WAVELENGTH: 216 nm 
Wildlife International, Ltd. Project Number 471C-143 

- 18 - 

Table 2 

Quality Control Samples of DETDA in n-Octanol and Reagent Water Partition Phases 

Sample 

Identification 

(471C-143-) 

Sample 

Type 

Partition 

Phase 

Concentration of DETDA Percent 

 Recovery Fortified 3

(mg DETDA/L)

Measured 3

(mg DETDA/L) 

 

MAB-1 

MAS-1 

MAB-2 

MAS-2 

Matrix Blank 

Matrix Fortification 

Matrix Blank 

Matrix Fortification 

n-octanol 

n-octanol 

aqueous 

aqueous 

0.0 

1600 

0.0 

60.0 

< LOQ 1

1680 

< LOQ 2

59.3 

-- 

105 

-- 

98.8 

1

 The LOQ for the n-octanol phase samples was 500 mg DETDA/L, calculated as the product of the 

concentration of the lowest level calibration standard (5.00 mg DETDA/L) for n-octanol phase analysis 

and the dilution factor of the n-octanol matrix blank sample (100). 2

 The LOQ for the aqueous phase samples was 25.0 mg DETDA/L, calculated as the product of the 

concentration of the lowest level calibration standard (5.00 mg DETDA/L) for aqueous phase analysis and 

the dilution factor of the aqueous matrix blank sample (5.00). 3

 Results were generated using Microsoft® Excel 2000 in full precision mode. Manual calculations may 

differ slightly. 
Wildlife International, Ltd. Project Number 471C-143 

- 19 - 

Table 3 

Mass Balance Results for the n-Octanol/Water Partition Coefficient Definitive Test with DETDA 

Sample 

Identification 

(471C-143-) 1

Mass 

Fortified 

(mg DETDA) 

Measured 

Concentration 

 (mg DETDA/L) 2 

Phase 

Volume 

(mL) 

Mass 

Recovered 

 (mg DETDA) 2 

Total Mass 

Recovered 

 (mg DETDA) 2 

Percent 

Recovery 

 (%) 2 

1-oct 

1-H2O 

2-oct 

2-H2O 

3-oct 

3-H2O 

4-oct 

4-H2O 

5-oct 

5-H2O 

6-oct 

6-H2O 

16.0 

16.0 

16.0 

16.0 

8.00 

8.00 

1.60 x 103 

68.9 

1.59 x 103

67.7 

1.49 x 103

63.1 

1.49 x 103

56.9 

1.35 x 103

56.4 

1.35 x 103

56.5 

10.0 

10.0 

10.0 

10.0 

10.0 

20.0 

10.0 

20.0 

5.00 

20.0 

5.00 

20.0 

16.0 

0.689 

15.9 

0.677 

14.9 

1.26 

14.9 

1.14 

6.77 

1.13 

6.75 

1.13 

16.7 

16.5 

16.1 

16.0 

7.90 

7.88 

104 

103 

101 

100 

98.8 

98.5 

Overall Mean: 

Standard Deviation: 

CV: 

101 

2.4 

 2.4% 

1

 The sample identification suffix “oct” = n-octanol phase and “H2O” = aqueous phase of a given test system. 2

 Results were generated using Microsoft® Excel 2000 in full precision mode. Manual calculations may differ slightly.
Wildlife International, Ltd. Project Number 471C-143 

- 20 - 

Table 4 

Measured Concentrations and Partition Coefficient Results for the n-Octanol/Water 

Partition Coefficient Definitive Test with DETDA 

Sample 

Identification 

 (471C-143-) 1

Solvent 

 Ratio 2

Measured 

Concentration 

 (M) 3 Pow 3 

Mean 

Pow 3 Log Pow 3 

Mean 

Log Pow 3 

1-oct 

1-H2O 

2-oct 

2-H2O 

1:1 

1:1 

8.98 x 10-3

3.87 x 10-4

8.90 x 10-3

3.80 x 10-4

23.2 

23.4 

23.3 1.37 

1.37 

1.37 

3-oct 

3-H2O 

4-oct 

4-H2O 

1:2 

1:2 

8.34 x 10-3

3.54 x 10-4

8.36 x 10-3

3.19 x 10-4

23.6 

26.2 

24.9 1.37 

1.42 

1.40 

5-oct 

5-H2O 

6-oct 

6-H2O 

1:4 

1:4 

7.60 x 10-3

3.16 x 10-4

7.58 x 10-3

3.17 x 10-4

24.0 

23.9 

24.0 1.38 

1.38 

1.38 

Overall Mean:

Standard Deviation:

CV (%):

24.1 

1.1 

4.5 

 1.38 

0.019 

1.4 

1

 The sample identification suffix “oct” = n-octanol phase and “H2O” = aqueous phase of a given test system. 2

 Expressed as the volume ratio of n-octanol to water. 3

 Results were generated using Microsoft® Excel 2000 in full precision mode. Manual calculations may differ slightly.
Wildlife International, Ltd. Project Number 471C-143 

- 21 - 

Octanol Phase Analysis: 

Transfer an aliquot from the top (octanol) phase of each sample into a clean glass vial and label the vial, 

appending the sample ID with '-oct'. Prepare a matrix blank and a matrix fortification sample in n-octanol 

saturated with water. 



Dilute an appropriate aliquot of each n-octanol sample in 50:50 ACN:H2O (v/v). Transfer an aliquot of the 

dilution of each n-octanol sample to an autosampler vial and submit for HPLC/UV analysis. Place any 

remaining n-octanol samples and n-octanol sample dilutions in refrigerated storage. 



Water Phase Analysis: 

Using a disposable plastic syringe with a detachable needle, insert the needle through the remaining n-octanol 

phase in the original sample vessel while gently expelling air. Once into the water phase, draw a sufficient 

volume of water into the syringe. 



Remove the syringe from the sample and immediately detach the needle. Expel the contents of the syringe into 

a clean glass vial and label the vial, appending the sample ID with '-H2O'. Prepare a matrix blank and a matrix 

fortification sample in water saturated with n-octanol. 



Transfer an appropriate amount of each water sample into the appropriate number of 5.00-mL volumetric flasks 

preloaded with an appropriate amount of acetonitrile (ACN) and bring volumetric flasks to a final volume by 

adding an appropriate volume of reagent water to each volumetric flask to yield a final sample composition of 

50:50 ACN:H2O (v/v). Transfer an aliquot of the dilution of each water sample to an autosampler vial and 

submit for HPLC/UV analysis. Place any remaining water samples and water sample dilutions in refrigerated 

storage. 

Figure 1. Analytical method flow-chart for the analysis of DETDA in n-octanol/water partition 

samples. 
Wildlife International, Ltd. Project Number 471C-143 

- 22 - 

y = 880.11029x - 306.20822

R2 = 0.98935

0

5000

10000

15000

20000

25000

30000

0.0 5.0 10.0 15.0 20.0 25.0 30.0

Concentration (mg a.i./L)

Total Peak Area (216 nm)

Figure 2. A representative calibration curve for the analysis of DETDA by HPLC/UV 
Wildlife International, Ltd. Project Number 471C-143 

- 23 - 

0 2 4 6 8 10 12 min

mAU 

0

500

1000

1500

2000

2500

3000

3500

 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\002-0201.D) 7.377 7.543

(A) 

0 2 4 6 8 10 12 min

mAU 

0

500

1000

1500

2000

2500

3000

3500

 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\005-0501.D) 7.375 7.540

 (B) 

Figure 3. Representative chromatograms of low-level and high-level DETDA calibration standards for 

HPLC/UV analyses. 

(A): Low-level calibration standard (5.00 mg DETDA/L) 

(B): High-level calibration standard (30.0 mg DETDA/L) 
Wildlife International, Ltd. Project Number 471C-143 

- 24 - 

0 2 4 6 8 10 12 min

mAU 

0

500

1000

1500

2000

2500

3000

3500

 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\007-0801.D)

(A) 

0 2 4 6 8 10 12 min

mAU 

0

500

1000

1500

2000

2500

3000

3500

 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\015-2301.D)

(B) 

Figure 4. Chromatograms of the matrix blank samples 

(A): n-Octanol phase (Sample ID: 471C-143-MAB-1), dilution factor (DF) = 100 

(B): Aqueous phase (Sample ID: 471C-143-MAB-2), DF = 5.00 

The arrows indicate the approximate retention times of the peaks for DETDA. 
Wildlife International, Ltd. Project Number 471C-143 

- 25 - 

0 2 4 6 8 10 12 min

mAU 

0

500

1000

1500

2000

2500

3000

3500

 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\008-0901.D) 7.371 7.537

(A) 

0 2 4 6 8 10 12 min

mAU 

0

500

1000

1500

2000

2500

3000

3500

 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\016-2401.D) 7.372 7.536

 (B) 

Figure 5. Chromatograms of the matrix fortification samples 

(A): n-Octanol phase (Sample ID: 471C-143-MAS-1), nominal conc. = 1600 mg DETDA/L, 

 DF = 100 

(B): Aqueous phase (Sample ID: 471C-143-MAS-2), nominal conc. = 60.0 mg DETDA/L, 

 DF = 5.00 
Wildlife International, Ltd. Project Number 471C-143 

- 26 - 

0 2 4 6 8 10 12 min

mAU 

0

500

1000

1500

2000

2500

3000

3500

 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\009-1101.D) 7.368 7.534

(A) 

0 2 4 6 8 10 12 min

mAU 

0

500

1000

1500

2000

2500

3000

3500

 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\017-2601.D) 7.374 7.539

(B) 

Figure 6. Representative chromatograms of a partition test sample (471C-143-1) 

(A): n-Octanol phase (-oct), DF = 100 

(B): Aqueous phase (-H2O), DF = 5.00 
Wildlife International, Ltd. Project Number 471C-143 

- 27 - 

Appendix 1 

Protocol 
Wildlife International, Ltd. Project Number 471C-143 

- 28 - 
Wildlife International, Ltd. Project Number 471C-143 

- 29 - 
Wildlife International, Ltd. Project Number 471C-143 

- 30 - 
Wildlife International, Ltd. Project Number 471C-143 

- 31 - 
Wildlife International, Ltd. Project Number 471C-143 

- 32 - 
Wildlife International, Ltd. Project Number 471C-143 

- 33 - 
Wildlife International, Ltd. Project Number 471C-143 

- 34 - 
Wildlife International, Ltd. Project Number 471C-143 

- 35 - 
Wildlife International, Ltd. Project Number 471C-143 

- 36 - 
Wildlife International, Ltd. Project Number 471C-143 

- 37 - 
Wildlife International, Ltd. Project Number 471C-143 

- 38 - 

Appendix 2 

Characterization Report 
Wildlife International, Ltd. Project Number 471C-143 

- 39 - 
Wildlife International, Ltd. Project Number 471C-143 

- 40 - 
Wildlife International, Ltd. Project Number 471C-143 

- 41 - 
Wildlife International, Ltd. Project Number 471C-143 

- 42 - 

Appendix 3 

Personnel Involved in the Study 

The following key Wildlife International, Ltd. personnel were involved in the conduct or 

management of this study: 

 1. Willard B. Nixon, Ph.D., Director of Chemistry 

2. Raymond L. Van Hoven, Ph.D., Scientist 

3. William R. Schutt, B.S., Chemist 

4. John R. Murrell, B.S., Senior Chemist