1 SCOPE

This method is applicable to detect the use of certain azo colourants that may not be used in the
manufacture or treatment of certain commodities made of textile fibres and that are accessible to reducing
agent with and without extraction.

2 PRINCIPLE

2.1 After selection of coloured textile specimen from the textile article, the test specimen is tested
according to the method of colorant extraction for disperse dyes and/or the method of direct
reduction for the other classes of dyes
2.2 The application of the combined methods or one of the two methods is based on the nature of the
fibre(s) of the test specimen and colour treatment. When relevant, if the test specimen is not
discoloured during the application of one of the two methods, the other one is carried out.
2.3 When the method of the colourant extraction for disperse dyes is carried out, the colourant is first
extracted from the fibre in the headspace using appropriate solvents under reflux, (using
chlorobenzene). The extract is concentrated and transferred with methanol, taken up in aqueous
citrate buffer solution
2.4 If the textile specimen is not completely discoloured after chlorobenzene extraction, the specimen is
added to the reaction vessel with the methanolic solution of the disperse dye for combined reduction
2.5 The sample is treated with sodium dithionite in a citrate-buffered aqueous solution (pH = 6) at 70C in
a closed vessel. The amines released in the process are transferred to a t-butyl methyl ether phase
by means of solid phase extraction or liquid-liquid extraction. The t-butyl ethyl ether extract is then
concentrated and the residue is taken up in Acetonitrile for the detection and determination of amines
using chromatography.

2.6 The extract is subjected to GC-MS for qualitative analysis first. If the amines are detected by GC-MS,
confirmation and quantitation should be made by using HPLC-DAD. If confirmation and quantitation
with LC-DAD is necessary, freshly prepared sample extract should be used in the analysis, as
amines decompose after being exposed at room temperature for a long period.

3 REFERENCE DOCUMENTS

3.1 EN 14362-1: 2012 – Methods for determination of certain aromatic amines derived from azo
colorants. Detection of the use of certain azo colorants that are accessible with and without
extracting the fibers

4 PROCEDURE

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4.1 APPARATUS
4.1.1 Analytical balance accurate to 0.1mg
4.1.2 Reaction vessel (20mL to 50mL) of heat-resistant glass, with tight closure
4.1.3 Thermostat-controlled oven
4.1.4 Turbo evaporator/ Rotary Evaporator
4.1.5 Pipettes: 1mL, 2mL, 5mL, 10mL
4.1.6 100mL round bottom flask
4.1.7 Glass pestle
4.1.8 Nitrogen gas (Purity 99.5%)
4.1.9 Volumetric flask: 5mL, 10ml, 25mL, 100mL
4.1.10 Gas Chromatography equipped with Mass Selective Detector (MSD), Agilent 6890GC
with Agilent 5973 mass spectrometer or equivalent
4.1.11 High Performance Liquid Chromatography equipped with Photodiode Array Detector,
4.1.12 Mechanical shaker providing horizontal shaking motion
4.1.13 Ultrasonic bath
4.1.14 Extraction apparatus
4.1.15 Separating Funnel (250 or 500ml)

4.2 CHEMICALS, REAGENTS AND STANDARD SOLUTION PREPARATION

4.2.1 Methanol
4.2.2 Acetonitrile
4.2.3 Chlorobenzene
4.2.4 N-Pentane
4.2.5 t-butyl methyl ether
4.2.6 Ethyl Acetate
4.2.7 Citrate buffer solution (pH 6, pre-heated to 70 + 2C(1000ml buffer solution containing
12.526 g of citric acid and 6.320 g NaOH)
Note: Adjust the pH value of the buffer to 6 using citric acid/NaOH
4.2.8 Sodium dithionite,
4.2.9 Sodium Hydroxide
4.2.10 Sodium Chloride
4.2.11 Distilled water, Grade-3
4.2.12 Diatomaceous earth - Extrelut column, Extrelut NT refill packs or equivalent ChemElut
20ml unbuffered column, Varian, Cat No. 1219-8022 or equivalent
4.2.13 Preparation of standard solution
4.2.13.1 Stock solution of 1000mg/L of individual amine (Table 1) was prepared by
dissolving accurately 0.025g of chemical standard in methanol (4.2.1) in a 25mL
volumetric flask.

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4.2.13.2 Four mixtures of amine standards were prepared. Please refer to Table 1 for the
detail of amines in each mixture.
4.2.13.3 Pipette 1.5mL of individual stock solution to a 10mL volumetric flask to afford a
secondary standard solution of 150mg/L.
4.2.13.4 Make up the solution to volume with methanol (4.2.1).
Standard solutions for LC-DAD analysis
4.2.13.5 Prepare solutions with methanol (4.2.1) to afford calibration standard solution of
2.5, 5, 10, 15 and 20mg/L.
4.2.13.6 Repeat procedure 4.2.6.5 with Mix B, Mix C and Mix D 150mg/L working
standard solution to afford calibration standard solution of Mix B Mix C and Mix
D, respectively.
Standard solutions for GC-MS analysis
Note: Working standard solutions Mix A, Mix B, Mix C and Mix D could be mixed in
preparing standard solutions for GC analysis. (1, 2.5, 5, 10, 15 mg/L) with internal
standard concentration of 10 mg/L in all the five calibration solutions
Internal standard solution
Anthracene(100ppm): Weigh accurately 0.01g of in 100 ml volumetric flask and make up to
volume with methanol (4.2.1) to afford a solution of 100 mg/L
2,4,5-Trichloraniline(1000ppm): Weigh accurately 0.1g in 100 ml volumetric flask and
make up to volume with methanol (4.2.1) to afford a solution of 1 g/L(1000ppm)

CAS No. Internal Standard Brand Name

636-30-6 2,4,5-Trichloroaniline Aldrich

ISO TEC
1719-06-8 Anthracene – d10

Standard solutions

4.2.14 Prepare 5 different calibration solutions for MIX A, MIX B,MIX C,MIX D (5,10,15,25,30
mg/L) with internal standard concentration of 10 mg/L in all the five calibration solutions

4.2.15 Preparation of aqueous sodium dithionite solution (reducing agent)

4.2.15.1 Weigh accurately 10.0g of sodium dithionite. (4.2.4)
4.2.15.2 Pour the sodium dithionite (4.2.7.1) into a 100ml beaker.
4.2.15.3 Dissolve the sodium dithionite (4.2.7.2) with 50ml hot distilled water.(70°C) to
afford an aqueous sodium dithionite solution of 200mg/ml. Make sure the final
solution is clear which indicates that all solid particles are dissolved completely
**********
Table 1 Detail of amines in mixed standard solutions Mix a, Mix B, Mix C and Mix D
Mix A

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CAS No. Amines Substances Purity Brand Name

95-80-7 2,4-diaminotoluene / 4-methyl-m-phenylenediamine 99.5% Dr Ehrenstorfer
95-53-4 o-toluidine 99.5% Dr Ehrenstorfer
92-87-5 Benzidine 99.0% Dr Ehrenstorfer
106-47-8 4-chloroaniline 99.5% Dr Ehrenstorfer
119-90-4 3,3'-dimethoxybenzidine 98.0% Dr Ehrenstorfer
119-93-7 o-tolidine / 3,3’-dimethylbenzidine 99.0% Dr Ehrenstorfer
Mix B
CAS No. Amines Substances Purity Brand Name
Dr Ehrenstorfer
92-67-1 4-aminobiphenyl 98.4%
2-methoxy-5-methylaniline / Dr Ehrenstorfer
120-71-8 6-methoxy-m-toluidine 99.0%
Dr Ehrenstorfer
90-04-0 o-anisidine / 2-methoxyaniline 99.5%
95-68-1 2,4-dimethylaniline / 2,4-xylidin 99.0% Dr Ehrenstorfer

101-77-9 4,4'-methylenedianiline 98.0% Dr Ehrenstorfer

4-chloro-o-methylaniline / Dr Ehrenstorfer
95-69-2 98.0%
4-chloro-o-toluidine
Mix C
CAS No. Amines Substances Purity Brand Name
4-methoxy-1,3-phenylenediamine / Dr Ehrenstorfer
615-05-4 4-methoxy-m-phenylenediamine 99.0%
4,4'-methylene bis(o-chloroaniline) / Dr Ehrenstorfer
101-14-4 2,2’-dichloro-4,4’-methylenedianiline 98.2%
137-17-7 2,4,5-trimethylaniline 99.5% Dr Ehrenstorfer

97-56-3 o-aminoazotoluene 98.0% Dr Ehrenstorfer

Dr Ehrenstorfer
60-09-3 p-phenylazoaniline / 4-aminoazobenzene 98.8%
101-80-4 4,4'-oxydianiline 99.0% Dr Ehrenstorfer

87-62-7 2,6-dimethylaniline / 2,6-xylidin 99.0% Dr Ehrenstorfer

Mix D
CAS No. Amines Substances Purity Brand Name

91-94-1 3,3'-dichlorobenzidine 99.2% Dr Ehrenstorfer

Dr Ehrenstorfer
91-59-8 b-naphthylamine / 2-naphthylamine 99.5%
3,3'-dimethyl-4,4'-diaminodipenylmethane / Dr Ehrenstorfer
838-88-0 98.8%
4,4’-methylenedi-o-toluidine
139-65-1 4,4'-thiodianiline 99.5% Dr Ehrenstorfer

99-55-8 5-nitro-o-toluidine 99.0% Dr Ehrenstorfer

106-50-3 1,4-phenylenediamine 98.0% Fluka

62-53-3 Aniline 99.5% Fluka

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For m-Toluidine and P-Toluidine

CAS No. Amines Substances Purity Brand Name

106-49-0 p-Toluidine 99.0 % Fluka

m-Toluidine Fluka
108-44-1 99.5%

4.3FIBRE COMPOSITION
4.3.1 Based on the extraction of colourants, identify the nature of the textile components so that
the possible use of disperse dyestuffs can be determined
Nature of fibre Use of disperse Cases Colourant extraction
dyestuffs for disperse dyes
necessary or not
Nature fibre No A No
No B No
Man-made fibre Undetermined C Yes
Yes D Yes
Note: if the fibre is not dyed, the fibre shall not be tested

4.3.2 Case of the fibre blends:

4.3.2.1 In case when fibres of different types are mixed, refer the below table in order to decide if
the application of the colourant extraction for disperse dyes shall be applied
Colourant extraction for disperse dyes Other component of the blend
Necessary or not A B C D
A No No Yes Yes
Component of the B No No Yes Yes
Blend C Yes Yes Yes Yes
D Yes Yes Yes Yes

4.4SAMPLE PREPARATION
4.4.1 Sampling and slicing
4.4.1.1 If the textile article is semi manufactured products such as yarns, fabrics, etc., cut
out test specimens from it
4.4.1.2 If the textile article is composed of several parts of textile products, such as a
garment, cut out test specimens from all the parts of the textile article that have
direct and prolonged contact to skin or mouth.
4.4.1.3 Take sample randomly or base on client requests. For complex sample, pictures of
the sample must be taken and mark the sampling site.

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4.4.1.4 Single-coloured homogeneous sample

(a) Cut the sample into small pieces of size 5 mm x 5 mm and mix (Sampling
should be made at different parts of the sample. Provided there are
finished products to be analyzed sample parts should be collected
proportionately).
4.4.1.5 Multi-coloured samples and samples with pattern
(a) Collect samples according to the proportion of colour in the finished product.
(b) Cut the sample into 5 mm x 5 mm pieces.
Note: If the mass of some parts(eg., labels, threads, etc.,) does not reach the mass(1g) to be tested,
gather identical parts when possible. If the total mass of material is below 0.5g, this material is defined
as minor component. Below 0.2g of material, the analysis is omitted. Embroidery shall be weight with
ground fabric
4.4.1.6 Case of colour gathering: Upto 3 colours may be tested together
4.4.1.6.1 In order to gather 3 colours, the following rules shall be applied
 Select three colours from same part of the textile article
 If the three colours do not come from the same part of textile article
select these 3 colours from textile parts made of the same type of the textile
fibre
 If the 3 colurs do not come from the same part of textile article and do not
come from the same type of textile fibre, select these 3 colours from textile
parts on which the same procedure shall be applied
4.4.1.6.2 Preparation of the 3 colour test specimen
 Each colour shall have approximately the same weight in order to obtain the
total mass of 1g.

4.5SAMPLE EXTRACTION
4.5.1 Extraction of dyes with Chlorobenzene:-
4.5.1.1 Accurately weigh 1.0  0.01g (w) of sample cut into stripes (5mm x 40mm) and
record the weight.
4.5.1.2 Tighten the cut striped with a white or colourless yarn
4.5.1.3 Suspend the sample in the headspace of the extractor
4.5.1.4 Position vertically the sample in the headspace of the extractor and make sure that
the condensed solvent percolates through the suspended specimen.
4.5.1.5 The suspended specimen is kept in the extractor with boiling chlorobenzene until all
the dyes are extracted from the sample (The extraction process takes about 30
mins. The colour of the sample becomes white after the extraction).
4.5.1.6 Cool the extract to room temperature.
4.5.1.7 Concentrate the extract to about 1 ml with rotary vacuum evaporator/turbo vap
( Keep the temperature at 45°C to 60°C)
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4.5.1.8 Add 2ml of methanol to the extract and sonicate in ultrasonic bath for 2 minutes to disperse the ex
Transfer the extract into a reaction vessel
4.5.2 Textile dyed with disperse dyes and/or other dyes:
4.5.2.1 Remove from the extractor, the extracted textile specimen. If it contains fibers
belonging to case A or B(refer above table), remove the solvent by washing the
specimen with appropriate solvent. Eg., n-Pentane or TBME and let it dry. If
necessary cut it into small pieces for reductive cleavage. Add the extracted textile
specimen to the reaction vessel with the methanolic solution of the dispersed dyes
for combined reduction.
4.5.3 Textile dyed with dyes other than disperse dyes:
4.5.3.1 If the textile specimen contains fibers belonging only to Case A and/or B, put the test
specimen directly in a reaction vessel and add 2ml methanol.
4.6REDUCTIVE CLEAVAGE:
4.6.1 Accurately weigh 1.0  0.01g (w) cut sample and record the weight. Transfer the sample
into a reaction vessel (4.1.2)
4.6.2 Add internal standard(2,4,5-TCA)
4.6.3 Add 15mL of citrate buffer solution (4.2.7) pre-heated to 70 + 2C to the reaction vessel
loaded with sample.
4.6.4 Close the reaction vessel.
4.6.5 Shake the vessel for about 30s to ensure all sample is wetted and soaked with buffer
solution.
4.6.6 Keep the reaction vessel in an oven at 70 + 2C for 30 + 1 min.
4.6.7 Add 3.0mL of freshly prepared aqueous sodium dithionite solution for reductive cleavage
of the azo groups.
4.6.8 Close the reaction vessel.
4.6.9 Shake the vessel for about 30s to mix well the solution mixture. Make sure all sample is
soaked in solution.
4.6.10 Keep the reaction vessel in an oven at 70 + 2C for 30 + 1 min.
4.6.11 Cool the reaction mixture to room temperature (20 -25C) within 2 minutes.

4.7SEPARATION AND CONCENTRATION OF THE AMINES.

Either procedure in clause 4.7.1 or 4.7.2 or 4.7.3 or 4.7.4 is applicable.
4.7.1 Extraction with diatomaceous earth column.
4.7.1.1 Add to the reaction vessel(step 4.6)0.2ml of Sodium Hydroxide solution(10% W/W)
and shake vigorously
4.7.1.2 Squeeze the solution out of the fibres with a glass pestle.
4.7.1.3 Decant the solution on the diatomaceous earth column
4.7.1.4 Allow absorption of solution by the column for 15 min.

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4.7.1.5 Wash the fibres with 10mL of t-butyl methyl ether by shaking the mixture
manually for 30s.
4.7.1.6 Decant the t-butyl methyl ether on the diatomaceous earth column.
4.7.1.7 Collect the eluate in a 150mL round bottom flask
4.7.1.8 Repeat procedure 4.7.1.5 to 4.7.1.7 with another 10mL and 20mL of t-butyl
methyl ether.
4.7.1.9 Pour directly 60mL of t-butyl methyl ether on the column.
4.7.1.10 Collect all the eluent
Note: The whole elution process should take around 50 min. A long elution process would result in
loss of amines
4.7.1.11 Concentrate the t-butyl methyl ether extract (4.7.1.10) to about 1mL (Not to
dryness) in a turbo evaporator under nitrogen condition/rotary evaporator
4.7.1.12 Temperature of water bath of turbo evaporator/rotary evaporator should not
exceed 50C.
4.7.1.13 Remove the remaining solvent with weak flow of nitrogen carefully.
Note: Removal of solvent to dryness may lead to loss of amine under
uncontrolled conditions.
4.7.1.14 Wash the dried content in the reaction vessel with 2.0mL of Acetonitrile or
TBME
4.7.1.15 Transfer the extract to a 1.5mL vial for chromatographic analysis.

4.7.2 Liquid-liquid extraction(Screening)

4.7.2.1 Add to the reaction vessel(step 4.6) 0.5ml of Sodium Hydroxide
solution(40%W/W), 7g of sodium chloride and TBME 5ml
4.7.2.2 Shake for 15min with a horizontal mechanical shaker
4.7.2.3 For complete phase separation after shaking, it is recommended to centrifuge
the mixture
4.7.2.4 Remove the upper phase for determining the amines without a concentration
step
4.7.3 Liquid-liquid extraction(Screening) – In-house method
4.7.3.1 Add to the reaction vessel(step 4.6) 0.5ml of Sodium Hydroxide
solution(40%W/W), 7g of sodium chloride and Ethyl acetate 5ml
4.7.3.2 Shake for 15min with a horizontal mechanical shaker
4.7.3.3 For complete phase separation after shaking, it is recommended to centrifuge
the mixture
4.7.3.4 Remove the upper phase for determining the amines.
4.7.4 Liquid-liquid extraction(Quantification)
4.7.4.1 Add to the reaction vessel (step 4.6)0.2ml of Sodium Hydroxide solution (10%
W/W), 7g of sodium chloride and TBME 5ml.

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4.7.4.2 Shake for 15min with a horizontal mechanical shaker

4.7.4.3 Transfer the t-butyl methyl ether layer into a clean 100ml R.B. flask using
separating funnel.
4.7.4.4 Add another 10mL of t-butyl methyl ether to the vessel containing buffer extract.
4.7.4.5 Repeat procedures 4.7.3.2 to 4.7.3.3
4.7.4.6 Add 5mL t-butyl methyl ether to rinse the reaction vessel and transfer to
separating funnel and shake in manually for 5 min.
4.7.4.7 Combine the wash with the extract obtained in 4.7.3.3.
4.7.4.8 Wash the separating funnel with 5mL of t-butyl methyl ether.
4.7.4.9 Combine all the t-butyl methyl ether extract and wash.
4.7.4.10 Repeat procedures 4.7.1.11 to 4.7.1.15 for the preparation of chromatographic
analysis as stated in 4.9.

4.8 QUALITY CONTROL SAMPLES PREPARATION

4.8.1Prepare method blank solution by repeating clause 4.5 to 4.7 without sample.
4.8.2 Prepare laboratory control sample solution by repeating clause 4.5 to 4.7 with the addition
of 1mL of 15 mg/L standard solution in the absence of test sample.
4.8.3 Prepare sample spike solution by repeating clause 4.5 to 4.7 with the addition of 1mL of
15 mg/L standard solution in the presence of test sample.
4.8.4 Proceed to clause 4.9 for chromatographic analysis.

4.9 INSTRUMENTAL ANALYSIS BY GC-MS

4.9.1The extract is subjected to GC-MS for qualitative analysis first. If listed amines are
detected by GC-MS, confirmation and quantitation should be made by using HPLC-DAD
(4.8). Freshly prepared sample extract should then be used in confirmation and
quantitation.
4.9.2Develop a calibration curve of the target amines
4.9.3Set up the GC/MSD system with below conditions

Agilent 6890GC/7890 GC with Agilent 5973/5975 mass

GC-MS instrument
spectrometer
Acquisition Mode SCAN mode
Injection Mode Split
Split ratio 5:1
Split flow 5.0 mL/min
Total flow 8.9 mL/min
Injection Volume 1.0 l
Injector Temperature 250 C
Column DB-5MS / HP-5MS, 0.25mm x 30m x 0.25m

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(or) DB-35MS / HP-35MS, 0.25mm x 30m x 0.25m

Carrier Gas Helium
Flow Rate 1.0 ml/min
Initial Temperature 60 C
20 C/min to 250 C hold for 2 min;
Temperature Program 20 C/min to 280 C hold for 4 min;
Total run time 18 min.
Max Temperature 280 C

4.9.4 Run the calibration solution and laboratory control sample

4.9.5 Establish calibration curve of each compound using “Peak area” vs. “Conc.”. The
coefficient of linear regression should be  0.995.
4.9.6 Inject the standard check solution to the GC-MS.
4.9.7 Check the recovery of the calibration standard solution at 5ppm. The recovery should be
within 85-115%. Otherwise, investigate source of problems.
4.9.8 Inject sample blank to check for any contamination.
4.9.9 Inject QC sample to check for recovery.
4.9.10 Inject sample solution to the GC-MS.
4.9.11 Identify the presence of target analyte based on the retention time and on comparison of
the intensity ratio characteristic ions of sample mass spectrum, after background
correction, with that in a reference mass spectrum. Table 2 summarizes the characteristic
ions used in identification.
4.9.12 Compounds are identified when the following criteria are met:
4.9.12.1 Retention time of the sample component is within ± 0.1 retention time units of
the standard compounds.
4.9.12.2 The relative intensities of Qualifier 1/Target-Ion (i.e. Relative response, Q1) and
Qualifier 2/Target-Ion (i.e. Relative response, Q2) agree within ± 30% of the
relative intensities of these ions in the reference spectrum.
4.9.13 If banned amines are identified, the result should be confirmed and quantitated with LC-
DAD (4.8). Freshly prepared sample extract should be used for these purposes.
4.9.14 If aniline and / or 1,4-phenylenediamine are / is detected, perform 4-aminoazobenzene
determination in accordance with Standard Operating Procedures SGS/IN/CTS
LAB/ECO-04-4 “Verification of the use of Azo dyes which can release 4-aminoazo
benzene by § 64 LFGB BVL B 82.02.9 – 2008 and ISO/DIS 17234-2: 2010”
4.9.15 Estimate the amount of identified amines using the ions as specified in Table 2 from the
calibration curve.

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4.9.16 If the estimated amount of identified amines exceed the initial calibration range of the
GC/MS system, the sample extract must be diluted before subjected to HPLC-DAD
confirmation and quantitation (4.8).

**********

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Table 2 Mass fragments information of amine standards

Mix A
m/z Target m/z m/z
CAS No. Amines Substances
ion Qualifier 1 Qualifier 2
95-80-7 2,4-diaminotoluene 122.10 121.00 94.00
95-53-4 o-toluidine 106.00 107.00 77.00
92-87-5 benzidine 184.00 185.00 156.00
106-47-8 4-chloroaniline 127.00 129.00 92.00
119-90-4 3,3'-dimethoxybenzidine 244.00 201.10 229.10
119-93-7 o-tolidine 212.00 180.00 106.00
Mix B
m/z Target m/z m/z
CAS No. Amines Substances
ion Qualifier 1 Qualifier 2

92-67-1 4-aminobiphenyl 169.00 168.00 141.00

90-04-0 o-anisidine 108.00 80.00 123.00

2-methoxy-5-
122.00 94.00 137.00
120-71-8 methylaniline
101-77-9 4,4'-methylenedianiline 198.10 197.10 106.10

95-69-2 4-chloro-o-toluidine 141.10 106.10 140.10

95-68-1 2,4-dimethylaniline 121.10 120.10 106.10

Mix C
m/z Target m/z m/z
CAS No. Amines Substances
ion Qualifier 1 Qualifier 2
97-56-3 o-aminoazotoluene 106.10 225.10 91.10

137-17-7 2,4,5-trimethylaniline 120.10 135.10 134.10

4,4'-methylene bis(o-
231.10 266.00 268.10
101-14-4 chloroaniline)

60-09-3 p-phenylazoaniline 92.00 197.10 120.10

4-methoxy-1,3-
123.00 138.00 95.00
615-05-4 phenylenediamine
101-80-4 4,4'-oxydianiline 200.10 108.10 171.10

87-62-7 2,6-dimethylaniline 121.10 106.10 120.10

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Mix D

m/z Target m/z m/z

CAS No. Amines Substances
ion Qualifier 1 Qualifier 2
3,3'-dimethyl-4,4'-
838-88-0 226.20 211.10 120.00
diaminodipenylmethane
99-55-8 5-nitro-o-toluidine 152.10 77.10 106.10
91-59-8 b-naphthylamine 143.00 115.00 116.00
91-94-1 3,3'-dichlorobenzidine 252.00 254.00 253.00

139-65-1 4,4'-thiodianiline 216.10 184.10 80.00

62-53-3 Aniline 93.10 66.10 65.10

106-50-3 1,4-phenylenediamine 108.10 80.10 107.10

m/z Target m/z m/z

CAS No. Internal Standard
ion Qualifier 1 Qualifier 2
636-30-6 2,4,5-Trichloroaniline 195 197 99
1719-06-8 Anthracene – d10 188 / /

For m-Toluidine and p-Toluidine

m/z Target m/z m/z
CAS No. Amines Substances
ion Qualifier 1 Qualifier 2
106-49-0 p-Toluidine 106.00 107.00 77.00
108-44-1 m-Toluidine 106.00 107.00 77.00

4.10 INSTRUMENTAL ANALYSIS BY HPLC-DAD – Confirmation and Quantitation

4.10.1 For identified amines, confirmation and quantitation of amines made in HPLC-DAD
instrument as below. Freshly prepared sample extract should be used. Dilution could be
made based on the result obtained from GC-MS in section 4.9.
4.10.2 For chromatogram and UV spectra of amines, please refer to Appendix A
4.10.3 Operation conditions of HPLC-DAD

Waters Alliance (2695 module and 2996 photodiode array

HPLC-DAD instrument
detector or Agilent(1200)
LiChrospher 60 RP-select B, 4 x 250 mm, 5 micron particle
Analytical column
(or) Zorbax Eclipse XDB C18(3.4micron); (150X4.6)mm
Temperature 25 oC
Solvent gradient program Buffer – 0.575 g ammonium dihydrogen phosphate and
0.70 g disodium hydrogen phosphate is dissolved in 1 L of
MilliQ water. The pH value of phosphate buffer is 6.9(or
use only water instead of buffer)
Time ACN Buffer Flow rate
min % % mL/min
0 23 77 0.7

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20 34 66 0.7
21 34 66 0.7
30 60 40 0.7
34 70 30 0.7
37 90 10 0.7
38 23 77 0.7
Detector DAD
Wavelength 200 to 400 nm
Injection Volume 20 l

4.10.4 Please refer to Table 3 for the wavelength to be used in confirming and quantitating each
amine with HPLC-DAD.
4.10.5 Run the calibration solution and laboratory control sample.
4.10.6 Establish calibration curve of each compound using “Peak area” vs “Conc”. The
coefficient of linear regression should be  0.995.
4.10.7 Inject the standard check solution of 10ppm to the HPLC-DAD.
4.10.8 Inject sample blank to check for any contamination.
4.10.9 Inject QC sample to check for recovery.
4.10.10 Inject sample solution to the HPLC-DAD.
4.10.11 Identify the presence of target analyte based on the retention time and on comparison of
the maxima and minima in PDA spectrum of sample mass spectrum, after background
correction, with that in a reference mass spectrum.
4.10.12 Compounds are identified when the following criteria are met:
4.10.12.1Retention time of the sample component is within ± 0.2 retention time units of
the standard compounds.
4.10.12.2Peak maxima /minima of the sample component are within ± 1 nm of that in the
reference spectrum.
4.10.13 Estimate the amount of identified amines using the wavelengths as specified in Table 3
from the calibration curve.
4.10.14 If the estimated amount of identified amines exceed the initial calibration range of the
HPLC-DAD system, the sample extract must be diluted before subjected to quantitation.

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Table 3 Wavelength used in confirmation and quantitation of amines with HPLC-DAD

Mix A
UV-VIS maxima for Wavelength for
CAS No. Amines Substances
identification (nm) quantitation (nm)
95-80-7 2,4-diaminotoluene / 4-methyl-m-phenylenediamine 238.5, 295.2 240
95-53-4 o-toluidine 232.6, 283.4 240
92-87-5 benzidine 282.2 280
106-47-8 4-chloroaniline 239.7, 294.0 240
119-90-4 3,3'-dimethoxybenzidine 282.2, 305.9 305
119-93-7 o-tolidine / 3,3’-dimethylbenzidine 282.2 280
Mix B
UV-VIS maxima for Wavelength for
CAS No. Amines Substances
identification (nm) quantitation (nm)
92-67-1 4-aminobiphenyl 275.1 280
90-04-0 o-anisidine / 2-methoxyaniline 233.8, 284.5 240
2-methoxy-5-methylaniline / 236.1, 289.3
240
120-71-8 6-methoxy-m-toluidine
101-77-9 4,4'-methylenedianiline 243.2, 288.1 240

95-68-1 2,4-dimethylaniline / 2,4-xylidin 233.8, 288.1 240

4-chloro-o-methylaniline / 240.8, 292.8
95-69-2 240
4-chloro-o-toluidine
Mix C
UV-VIS maxima for
CAS No. Amines Substances Wavelength (nm)
identification (nm)
137-17-7 2,4,5-trimethylaniline 233.8, 289.3 240
4-methoxy-1,3-phenylenediamine / 242.0, 302.3
240
615-05-4 4-methoxy-m-phenylenediamine
60-09-3 p-phenylazoaniline / 4-aminoazobenzene 247.9, 382.0 240
4,4'-methylene bis(o-chloroaniline) / 245.6, 295.2
240
101-14-4 2,2’-dichloro-4,4’-methylenedianiline
97-56-3 o-aminoazotoluene 255.0, 383.2 240

101-80-4 4,4'-oxydianiline 244.4, 297.6 240

87-62-7 2,6-dimethylaniline / 2,6-xylidin 232.6, 281.0 240

MIX D
UV-VIS maxima for
CAS No. Amines Substances Wavelength (nm)
identification (nm)
99-55-8 5-nitro-o-toluidine 250.3, 296.4 240

139-65-1 4,4'-thiodianiline 263.2 240

91-59-8 b-naphthylamine / 2-naphthylamine 236.1, 281.0 240
242.0, 289.3
3,3'-dimethyl-4,4'-diaminodipenylmethane/
838-88-0 240
4,4’-methylenedi-o-toluidine

91-94-1 3,3'-dichlorobenzidine 285.7 280

106-50-3 1,4-phenylenediamine 240.8, 305.9 240

62-53-3 Aniline 232.6, 283.4 240

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For m-Toluidine and p-Toluidine

UV-VIS maxima for
CAS No. Amines Substances Wavelength (nm)
identification (nm)
106-49-0 p-Toluidine 233.2, 287.1 240

108-44-1 m-Toluidine 234.1, 283.1 240

5 RESULT CALCULATION AND EVALUATION

5.3Amine levels are calculated from the peak areas of the individual amine components as obtained in
HPLC-DAD. The amine level is calculated as mass portion w in mg/kg of the specimen according
to the following equation:

w (mg/kg) =  x V x D.F.
m
where
.
 Concentration of amine
V Final volume in mL.
m Weight of the textile specimen, in g.
D.F. Dilution factor (If no dilution is made, D.F. = 1)
5.2 Report the result to the nearest 1 mg/kg and at most 2 significant figures.
5.3 Lowest reporting limit: 5 mg/kg.
5.4 Quality Control Practice and Data Acceptance Criteria
5.4.1 Before commission of sample analysis
5.4.1.1 The laboratory should verify the method detection limit and quantitation limit of the
method in accordance with the Standard Operating Procedures RSTS-SL-002
“General Guidelines on Quality Control Practice”
5.4.2 Routine analysis
5.4.2.1 The results obtained in 5.4.2.2 to 5.4.2.8 should be recorded. In case of non-
compliance is observed, investigation and corrective action should be made before
further sample analysis.
5.4.2.2 Establish a five-point calibration curve for each amine standard. The regression
coefficient (r) of each curve should be equal to or greater than 0.995. (Frequency;
Once in a month) (3 points calibration – Daily)
5.4.2.3 Calibration Curve Standard Check – 15 ppm – Recovery within 85 – 115%.
5.4.2.4 Reagent blank - no compound above quantitation limit should be detected.
5.4.2.5 Method blank - no compound above quantitation limit should be detected.
5.4.2.6 Recovery criteria of QC sample for amines ranged from 20% to 70% of the
theoretical value. Please refer to Table 4 for the detail of recovery of amines.

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5.4.2.7 Recovery criteria of sample spike for amines ranged from 20% to 70% of the
theoretical value. Please refer to Table 4 for the detail of recovery of amines.
5.4.2.8 Duplicate Sample Check – Relative deviation within 30%.

6 TEST REPORT
The test report shall refer to the official method and contain at least the following information:
6.1 Reference to the method(s) specified in the SOP
6.2 Kind, origin and designation of the specimen (partial specimen, if applicable)
6.3 Date of receipt and date of analysis
6.4 Sampling procedure
6.5 Detection method and quantitation method
6.6 Results reported as level and detection limit per amine in mg/kg
6.7 Any departure by agreement or otherwise from the test procedure specified.
6.8 Any unusual features observed during the determination.

**********

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Table 4 Requirements of recoveries of amines.

Mix A
CAS No. Amines Substances Recovery (%)
95-80-7 2,4-diaminotoluene / 4-methyl-m-phenylenediamine 50
95-53-4 o-toluidine 50
92-87-5 benzidine 70
106-47-8 4-chloroaniline 70
119-90-4 3,3'-dimethoxybenzidine 70
119-93-7 o-tolidine / 3,3’-dimethylbenzidine 70
Mix B
CAS No. Amines Substances Recovery (%)
2-methoxy-5-methylaniline /
70
120-71-8 6-methoxy-m-toluidine
90-04-0 o-anisidine / 2-methoxyaniline 70
92-67-1 4-aminobiphenyl 70
101-77-9 4,4'-methylenedianiline 70
4-chloro-o-methylaniline /
95-69-2 70
4-chloro-o-toluidine
95-68-1 2,4-dimethylaniline / 2,4-xylidin #
Mix C
CAS No. Amines Substances Recovery (%)
4,4'-methylene bis(o-chloroaniline) /
70
101-14-4 2,2’-dichloro-4,4’-methylenedianiline
4-methoxy-1,3-phenylenediamine /
20
615-05-4 4-methoxy-m-phenylenediamine
137-17-7 2,4,5-trimethylaniline 70
101-80-4 4,4'-oxydianiline 70
87-62-7 2,6-dimethylaniline / 2,6-xylidin #
97-56-3 o-aminoazotoluene *
60-09-3 p-phenylazoaniline / 4-aminoazobenzene **

Mix D
CAS No. Amines Substances Recovery (%)
91-59-8 b-naphthylamine / 2-naphthylamine 70
99-55-8 5-nitro-o-toluidine *
139-65-1 4,4'-thiodianiline 70
91-94-1 3,3'-dichlorobenzidine 70
3,3'-dimethyl-4,4'-diaminodipenylmethane /
838-88-0 70
4,4’-methylenedi-o-toluidine
62-53-3 Aniline #
106-50-3 1,4-phenylenediamine #

Remarks
* o-aminoazotoluen (CAS 97-56-3) and 5-nitro-o-toluidine (99-55-8) are further reduced to o-
toluidine (CAS 95-53-4) and 2,4-diaminotoluene / 4-methyl-m-phenylenediamine (CAS 95-80-7)

** Azo colourants that are able to form 4-aminoazobenzene, generate under the condition of
this method aniline and 1,4-phenylenediamine. Therefore the test method of 64 LFGB B
82.02.9 was employed to verify the presence of 4-aminoazo benzene.

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# These amines are added to the list voluntarily. The percentage recovery of these amines
could attain 70%
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==================
END OF PROCEDURE
==================

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