CiToxLAB Hungary Ltd.

Veszprém - Szabadságpuszta

FINAL REPORT

Validation of the Analytical Method for the 

Determination of Benzene, 1-chloro-4-(trifluoromethyl)-

Study code: 12/056-316AN

Study Director: Zsolt Sárvári, M.Sc.

12 March 2013



Study code: 12/056-316AN Final Report Page 5 of 18

STUDY TITLE: Validation of the Analytical Method for the Determination 

of Benzene, 1-chloro-4-(trifluoromethyl)-

SPONSOR: PCBTF Testing Consortium

Address: c/o Greenwich Chemical Consulting, Inc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

USA

TEST FACILITY: CiToxLAB Hungary Ltd.

(Formerly known as LAB Research Ltd. before 1st September 2011)

Address: H-8200 Veszprém, Szabadságpuszta

Hungary

Phone: +36 88-545-300

Fax: +36 88-545-301

STUDY DIRECTOR: Zsolt Sárvári, M.Sc.

ASSISTANT SCIENTIST: Olga Ulbert, M.Sc.

QUALITY ASSURANCE: Vanda Gyimesi, M.Sc.

On behalf of QAU

BASIS OF STUDY: SOP of CiToxLAB Hungary Ltd.; ANE 124, 

"Validation of Analytical Method"
Study code: 12/056-316AN Final Report Page 6 of 18

CONTENTS

Page

1. SUMMARY 7

2. MATERIALS 8

2.1. TEST ITEM 8

2.2. REAGENTS AND MATERIALS 8

2.3. MIXTURES, SOLUTIONS 8

3. TEST METHODS AND RESULTS 9

3.1. APPARATUS 9

3.2. ANALYTICAL METHOD 10

3.3. METHOD VALIDATION 10

4. ARCHIVES 13

5. DISTRIBUTION OF THE FINAL REPORT 14

6. DEVIATION 14

APPENDIX 15

Copy of the GLP Certificates 16

Certificate of Analysis 18

Study code: 12/056-316AN Final Report Page 8 of 18

2. MATERIALS 

2.1. TEST ITEM

Name: Benzene, 1-chloro-4-(trifluoromethyl)-

Synonym: PCBTF

CAS Number: 98-56-6

Batch/Lot number: 20111028

Molecular formula: C7H4ClF3

Molecular weight: 180.56

Appearance: Clear colourless liquid

Purity: 99.69%

Manufacture date: 28 October 2011

Expiry date: 28 October 2013

Storage conditions: Room temperature

2.1.1. Receipt

The test item of a suitable chemical purity was supplied by the Sponsor. All safety 

instructions required in the handling and disposal of the test item were supplied by 

the Sponsor as well.

Identification of the test item was made by its name, batch, appearance and colour by 

Central Dispensary Unit of CiToxLAB Hungary Ltd.

2.2. REAGENTS AND MATERIALS

Ultra pure water (ASTM Type I): prepared by Direct-Q 3 system, Millipore

Acetonitrile (ACN): HPLC grade, VWR, Batch: 12Z3715

2.3. MIXTURES, SOLUTIONS

Stock Solution: 1.0 mg Benzene, 1-chloro-4-(trifluoromethyl)- / 1 ml 

ACN

Calibration standards: 0.1, 0.2, 0.5, 1, 2, 5, 10 and 20 μg Benzene, 1-chloro-4-

(trifluoromethyl)-/ ml: Stock Solution and 10 μg/ml 

working solution were properly diluted with ACN : 

water = 7:3.

Recovery Formulations: 20 mg/l: About 10 mg of test item was measured into 

500 ml flasks (one decimal place accuracy). It was 

dissolved with 500 ml alga test solution.

1 mg/ml: 5 ml 100 mg/ml solution measured into the 

100 ml flask and diluted with 100 ml matrix.
Study code: 12/056-316AN Final Report Page 9 of 18

3. TEST METHODS AND RESULTS 

Start of experiment: 16 October 2012

End of experiment: 26 October 2012

3.1. APPARATUS

HPLC system: Merck-Hitachi LaChrom HPLC system III

D-7000 Interface, No.: 1126-175

L-7100 HPLC pump, No. : 1388-056

L-7200 Autosampler, No.: 1380-033

L-7400 UV Detector, No.: 1374-025

L-7614 Degasser, No.: 144116A0094

L-7360 Column Oven, No.: 00118060

Merck-Hitachi LaChrom HPLC system IV

D-7000 Interface, No.: 1442-122

L-7100 HPLC pump, No. : 1516-030

L-7200 Autosampler, No.: 1406-005

L-7400 UV Detector, No.: 1502-017

L-7614 Degasser, No.: 14412YA0500

L-7360 Column Oven, No.: 00107295

Balances: BP221S Sartorius, No.: 11809117

L2200P Sartorius, No.:3810037

Ultrasonic bath: Elmasonic S300H, ELMA, No.: 010890105

Water purification system: MILLIPORE, DIRECT Q3, FOMNO 7334I

pH meter: SevenEasy S20 Mettler Toledo, No.:1231025119

Shaking incubator: Gallencamp 8B 3262 J

Spectrophotometer: Hitachi U-2910, No.: 2221-004

Refrigerator: Zanussi, No.: ZLKI-262

Climatic chamber
Study code: 12/056-316AN Final Report Page 10 of 18

3.2. ANALYTICAL METHOD

3.2.1. HPLC Conditions:

Column: Zorbax Eclipse Plus C8 150 × 4.6 mm, 5μm

Mobile Phase: ACN : water = 7:3

Flow: 1.0 ml/min.

Injection volume: 10 μl

Temperature: 25 °C

Detector: UV at 220 nm 

Retention time: 4.6 min ± 10%

3.3. METHOD VALIDATION

The following characteristics were determined:

3.3.1. Selectivity

For the selectivity test the aqueous control matrices were analysed.

No interfering peak was observed on the blank chromatograms.

3.3.2. Repeatability

Re-injection repeatability was tested with 7 consecutive injections of the 0.2 and 10 

μg/ml calibration standards. CV of the replicate injections was determined. The 

repeatability was found to be 2.5 % or better.

Table 2.: The injection repeatability of the HPLC method

Sample Measured parameter RSD (CV) %

Standard 0.2 μg/ml

Retention time 0.0

Area 1.8

Standard 10 μg/ml

Retention time 0.0

Area 2.5
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3.3.3. Linearity

Linearity of the HPLC method was examined in 0.1 - 20 μg/ml range. Eight

concentration levels were tested: 0.1, 0.2, 0.5, 1, 2, 5, 10 and 20 μg/ml.

Five such series of standards were prepared from separate stock solutions and 

analysed on five different days. 

The detector response proved to be linear in 0.1 – 20 μg/ml range. The calibration 

graph was calculated applying linear regression. 

Deviation of the back-calculated values was less than 15 %.

Parameters of the curves are presented in Table 3; plot of the calibration graph can 

be seen in Figure 1.

Table 3: Summary of Standard Curve Parameters

Analytical occasion Intercept Slope Correlation Coefficient

16 October 2012 687 22055 1.000

18 October 2012 -305 23253 1.000

19 October 2012 -197 21834 1.000

24 October 2012 368 21182 1.000

25 October 2012 -435 22240 1.000

Figure 1: Linearity test on 19 October 2012
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3.3.4. Limit of Quantification

The lowest concentration of the calibration curve, 0.1 μg/ml is considered to be the 

quantification limit of the HPLC method. 

3.3.5. Recovery, Accuracy and Precision

Recovery of test item was tested from alga test solutions. Concentrations of the test 

solutions were 1 and 20 mg/l.

Five replicate solutions were made.

Recovery is the measured value expressed in percentage of the nominal 

concentration: 

T

M ∗100

where:

M mean of measurements

T nominal concentration

Precision is the standard deviation of replicates expressed as percentage of the 

mean.

( )

M

n

m M

RSD

n

i

i

1

1

2

−

−

=

∑=

where:

RSD relative standard deviation 

mi-M individual deviations from the mean

n number of replicates

The recovery samples were analysed by the above HPLC method. The results are 

presented in Table 4.

Table 4: Recovery and Precision of the analytical method 

Matrix Nominal 

concentration

Measured 

concentration

Recovery

%

Precision

%

Alga test solution 1 mg/l BQL - -

20 mg/l 3.35 mg/l 15 22

BQL: below quantitation limit

The low recovery values are due to poor water solubility of the test item.
Study code: 12/056-316AN Final Report Page 13 of 18

3.3.6. Stability

Stock solution stability

Stock solution (1 mg/ml) prepared in water proved to be stable for at least 7 days at 

5 ± 3 °C.

: 

Peak area of the 10 μg/ml solution prepared from the 7 days old stock solution was

103 % of the peak area of the fresh one.

Stability of the samples in the autosampler

Calibration samples (0.2 and 10 μg/ml) were reinjected after 20 hours storage 

period in the autosampler. Peak areas after the storage period were 98 and 85 % of 

the areas measured at the start.

:

Stability of the test item in alga test solution

Test solutions were stored at room temperature. After one day formulations were 

analysed.

:

Since the test item is volatile and the test solutions were in an open vessel after one day 

the concentration of the 20 μg/ml solution decreased below the quantitation level.

Presumably, this decrease was due to evaporation, and not to decomposition of the 

test item.

4. ARCHIVES

The study documents:

- study plan, amendment

- all raw data

- sample of the test item

- study report and any amendment(s)

- correspondence

are stored in the archives of CiToxLAB Hungary Ltd., Hungary 8200 Veszprém, 

Szabadságpuszta according to the Hungarian GLP and our SOP-s.
Study code: 12/056-316AN Final Report Page 14 of 18

5. DISTRIBUTION OF THE FINAL REPORT

Sponsor:

- 1 × pdf-file

Archives:

- 1 × original, bound

6. DEVIATION

The date of the Draft Report was 13 December 2012 instead of 16 November 2012, 

because of the internal scheduling. This deviation does not have any effect on study 

results.

There were a typo errors in the description of the test item. The correct molecular 

formula is C7H4ClF3, and the correct molecular weight is 180.56.
Study code: 12/056-316AN Final Report Page 15 of 18

APPENDIX
Study code: 12/056-316AN Final Report Page 16 of 18

Copy of the GLP Certificates

The facility name until 1st September 2011 was LAB Research Ltd. From that date, the registered name has been CiToxLAB Hungary Ltd.,

this information has been transmitted to the GLP competent authority. The above GLP certificate is valid for this facility (now known as

CiToxLAB Hungary Ltd) until the certificate expires (16 December 2012).
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Study code: 12/056-316AN Final Report Page 18 of 18

Certificate of Analysis