CiToxLAB Hungary Ltd.

Veszprém - Szabadságpuszta

FINAL REPORT

Validation of the Analytical Method for the 

Determination of Ethanedioic acid, hydrate (1:2) [CAS 

RN 6153-56-6] (hydrated form of ethanedioic acid [CAS 

RN 144-62-7])

Study code: 11/339-316AN

Study Director: Zsolt Sárvári, M.Sc.

21 February 2013



Study code: 11/339-316AN Final Report Page 5 of 18

STUDY TITLE: Validation of the Analytical Method for the Determination 

of Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] 

(hydrated form of ethanedioic acid [CAS RN 144-62-7])

SPONSORS: Univar USA Inc.

Address: 17425 NE Union Hill Road,

Redmond, WA 98052, USA

Phone: +1 425 889 3400

Fax: +1 425 889 3760

WEGO Chemical and Mineral Corp.

Address: 239 Great Neck Road,

Great Neck, NY 11021, USA

Phone: +1 516 487 3510

Fax: +1 516 487 3794

STUDY MONITOR: John V. Massingale

Greenwich Chemical Consulting, Inc.

Address: 3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410, USA

Phone: +1 561 578 8110

Fax: +1 561 578 8120

E-mail: john@grcci.com

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

TEST FACILITY

MANAGEMENT: Christopher Banks, DABT

Managing Director

Alyson Leyshon, M.Sc.

Senior Director of Toxicology Operations

STUDY DIRECTOR: Zsolt Sárvári M.Sc.

Phone: +36 88 545 361

E-mail: zsolt.sarvari@hu.citoxlab.com
Study code: 11/339-316AN Final Report Page 6 of 18

ASSISTANT SCIENTIST: Olga Ulbert, M.Sc.

LEAD QA: Szabolcs Gáty M.Sc.

Head of Quality Assurance Unit

Vanda Gyimesi, M.Sc.

Deputy Head of QAU

RESPONSIBLE PERSONS: Ildikó Kacz - technical assistant

Andrea Egei - technical assistant

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

"Validation of Analytical Method"
Study code: 11/339-316AN Final Report Page 7 of 18

CONTENTS

Page

1. SUMMARY 8

2. MATERIALS 9

2.1. TEST ITEM 9

2.2. REAGENTS AND MATERIALS 9

2.3. MIXTURES, SOLUTIONS 9

3. TEST METHODS AND RESULTS 10

3.1. APPARATUS 10

3.2. ANALYTICAL METHOD 10

3.3. METHOD VALIDATION 11

4. ARCHIVES 14

5. DISTRIBUTION OF THE FINAL REPORT 14

6. DEVIATION 14

APPENDIX 15

Copy of the GLP-Certificates 16

Certificate of Analysis 18

Study code: 11/339-316AN Final Report Page 9 of 18

2. MATERIALS 

2.1. TEST ITEM

Name: Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] 

(hydrated form of ethanedioic acid [CAS RN 144-62-7])

Chemical name: Oxalic acid, hydrate

CAS number: 6153-56-6

Batch/Lot number: YP2-20110704B

Molecular formula: C2H2O4×2H2O

Molecular weight: 126.1 (dihydrate form)

Appearance: White crystals

Purity: 99.71 %

Manufacture date: 04 July 2011

Expiry date: 03 July 2012

Storage conditions: Controlled room temperature (15-25o

C)

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: HPLC grade, VWR, Batch: 12Z0028

Potassium dihydrogen phosphate: reagent, Lach-Ner, Batch: PP/2010/02241

Phosphoric acid 85%: reagent, Reanal, Batch: KBM55463

2.3. MIXTURES, SOLUTIONS

Stock Solution: 1.0 mg Ethanedioic acid / 1 ml water

Calibration series: 1, 2, 5, 10, 20, 50 and 100 μg test item / ml: Stock 

Solution and 50 μg/ml working solution were properly 

diluted with mobile phase.
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Recovery Formulations: 100 mg/l: About 10 mg of test item was measured into 

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

dissolved with 100 ml matrix.

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

100 ml flask and diluted with 100 ml matrix.

3. TEST METHODS AND RESULTS 

Start of experiment: 14 May 2012

End of experiment: 25 May 2012

3.1. APPARATUS

HPLC system: Merck-Hitachi LaChrom HPLC system

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

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

Refrigerator: Zanussi, No.: ZLKI-262

3.2. ANALYTICAL METHOD

3.2.1. HPLC Conditions:

Column: Zorbax SB-C8 250 × 4.6 mm, 5μm

Mobil Phase: 50 mM KH2PO4 (pH:2.1) : ACN 9:1

Flow: 1.0 ml/min.

Injection volume: 10 μl
Study code: 11/339-316AN Final Report Page 11 of 18

Temperature: 25 °C

Detector: UV at 220 nm 

Retention time: 2.5 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 2 and 50

μg/ml calibration standards on two different days. CV of the replicate injections 

was determined. The repeatability was found to be 1.6 % or better.

Table 2.: The injection repeatability of the method

Sample Measured 

parameter

RSD (CV) %

Analytical 

occasion 1.

Analytical 

occasion 2.

Standard 2 μg/ml

Retention time 0.2 0.0

Area 1.6 0.8

Standard 50 μg/ml

Retention time 0.0 0.2

Area 0.4 0.7

3.3.3. Linearity

Linearity of the HPLC method was examined in 1 - 100 μg/ml range. Seven

concentration levels were tested: 1, 2, 5, 10, 20, 50 and 100 μg/ml.

Five such series were prepared from separate stock solutions and analysed on five

different days. 

The detector response proved to be linear in 1 – 100 μg/ml range. The calibration

graph was calculated applying weighted linear regression using 1/concentration as 

weighting factor. 
Study code: 11/339-316AN Final Report Page 12 of 18

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

14 May 2012 -286 2771 1.000

15 May 2012 264 2617 1.000

17 May 2012 108 2900 1.000

18 May 2012 -523 2861 1.000

21 May 2012 -468 2878 1.000

0

50000

100000

150000

200000

250000

300000

0,00 20,00 40,00 60,00 80,00 100,00 120,00

Area

Concentration (μg/ml)

Figure 1: Linearity test on 21 May 2012

3.3.4. Limit of Quantification

The lowest concentration of the calibration curve, 1 μg/ml is considered to be the 

quantification limit of the HPLC method. 
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3.3.5. Recovery, Accuracy and Precision

Recovery of test item was tested from alga and daphnia test solutions and water. 

Concentration of the test solutions was 5 and 100 mg/l and the water was 75 mg/ml.

Five replicate solutions were made.

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

presented in Table 4.

Table 4: Recovery, Accuracy and Precision of the analytical method 

Matrix Nominal 

concentration

Recovery

%

Accuracy

%

Precision

%

Alga test solution

5 mg/l 105 5 2

100 mg/l 100 0 1

Daphnia test solution

5 mg/l 105 5 2

100 mg/l 101 1 3

Water 75 mg/ml 106 6 4

3.3.6. Stability

Stock solution stability

Stock solution (1 mg/ml) prepared in water proved to be stable for at least 4 days at 

5 ± 3 °C.

: 

Peak area of the 10 μg/ml solution prepared from the 4 days old stock solution was

100 % of the peak area of the fresh one.

Stability of the samples in the autosampler

Calibration samples (2 and 50 μg/ml) were reinjected after 19 hours storage period 

in the autosampler. Peak areas after the storage period were 94 and 98 % of the 

areas measured at the start.

:

Stability of the test item in test solutions

Test solutions and water sample was stored at room temperature. At the end of the 

storage period formulations were analysed.

:

The results are detailed in Table 5.
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Table 5: Results of the stability test in aqueous solutions

Matrix Nominal 

concentration

Storage 

period End/Start

Alga test solution

5 mg/l 3 day 94 %

100 mg/l 3 day 100 %

Daphnia test solution

5 mg/l 2 day 89 %

100 mg/l 2 day 100 %

Daphnia test solution

for fish test

5 mg/l 4 day 112 %

100 mg/l 4 day 115 %

Water 75 mg/ml 3 day 97 %

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.

5. DISTRIBUTION OF THE FINAL REPORT

Sponsor:

- 1 × pdf-file

Archives:

- 1 × original, bound

6. DEVIATION

The date of the Draft Report was 19 June 2012 instead of 15 June 2012, because of 

the internal scheduling. This deviation does not have any effect on study results.
Study code: 11/339-316AN Final Report Page 15 of 18

APPENDIX
Study code: 11/339-316AN 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: 11/339-316AN Final Report Page 18 of 18

Certificate of Analysis