Banned Amines

TEXTILE & GARMENT INDUSTRY RESEARCH &
DEVELOPMENT CENTER
Quality Standard and Testing Laboratory Research
Desk
SOP for Banned Amines Analysis as per ISO14362-1 Revision No
Issue Dt
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 fiber(s) of the test specimen and color treatment. When relevant,
if the test specimen is not discolored during the application of one of the two
methods, the other one is carried out.
2.3 When the method of the colorant extraction for disperse dyes is carried out, the
colorant is first extracted from the fiber in the headspace using appropriate
solvents under reflux, (using chloro benzene). The extract is concentrated and
transferred with methanol, taken up in aqueous citrate buffer solution
2.4 If the textile specimen is not completely discolored after chloro benzene 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
Initiated By Reviewed By Approved By Document No

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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 is
accessible with and without extracting the fibers
4 ROCEDURE
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 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

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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 Chloro benzene
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 un-buffered 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

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methanol (4.2.1) in a 25mL volumetric flask.
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

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1719-06-8 Anthracene – d10 ISO TEC
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
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Table 1 Detail of amines in mixed standard solutions Mix a, Mix B, Mix C and Mix D
Mix A
CAS No. Amines Substances Purity Brand Name
2,4-diaminotoluene / 4-methyl-m-
95-80-7 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
92-67-1 4-aminobiphenyl 98.4% Dr Ehrenstorfer
2-methoxy-5-methylaniline / Dr Ehrenstorfer
120-71-8 6-methoxy-m-toluidine 99.0%
90-04-0 o-anisidine / 2-methoxyaniline 99.5% Dr Ehrenstorfer
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

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Mix C
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
p-phenylazoaniline / 4- Dr Ehrenstorfer
60-09-3 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
91-94-1 3,3'-dichlorobenzidine 99.2% Dr Ehrenstorfer
91-59-8 b-naphthylamine / 2-naphthylamine 99.5% Dr Ehrenstorfer
3,3'-dimethyl-4,4'- Dr Ehrenstorfer
838-88-0 diaminodipenylmethane / 4,4’- 98.8%
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
Brand
CAS No. Amines Substances Purity
Name
106-49-0 p-Toluidine 99.0 % Fluka
108-44-1 m-Toluidine 99.5% Fluka
4.3FIBRE COMPOSITION
4.3.1 Based on the extraction of colorants, identify the nature of the textile components so that
The possible use of disperse dyestuffs can be determined
Nature of fiber Use of disperse Cases Colorant extraction

dyestuffs for disperse dyes
necessary or not
Nature fiber No A No
No B No
Man-made fiber Undetermined C Yes
Yes D Yes
Note: if the fiber is not dyed, the fiber shall not be tested
4.3.2 Case of the fiber blends:
4.3.2.1 In case when fibers of different types are mixed, refer the below table in ord
to decide if

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The application of the colorant extraction for disperse dyes shall be applied
Colorant extraction for disperse dyesOther 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.
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).

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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 color gathering: Up to 3 colors may be tested together
4.4.1.6.1 In order to gather 3 colors, the following rules shall be applied
 Select three colors from same part of the textile article
 If the three colors do not come from the same part of textile
article
Select these 3 colors from textile parts made of the same type
of the textile fiber
 If the 3 colors do not come from the same part of textile article
and do not come from the same type of textile fiber, select
these 3 colors from textile parts on which the same procedure
shall be applied
4.4.1.6.2 Preparation of the 3 color test specimen
 Each color shall have approximately the same weight in order

to obtain the total mass of 1g.
4.5 SAMPLE EXTRACTION

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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)
4.5.1.8 Add 2ml of methanol to the extract and sonicate in ultrasonic bath for 2
minutes to disperse the extract. 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.

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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.
4.6 REDUCTIVE 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.
4.7 SEPARATION AND CONCENTRATION OF THE AMINES.

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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.
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 elute 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.

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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(Quantification)
4.7.3.1 Add to the reaction vessel (step 4.6)0.2ml of Sodium Hydroxide

solution (10% W/W), 7g of sodium chloride and TBME 5ml.
4.7.3.2 Shake for 15min with a horizontal mechanical shaker
4.7.3.3 Transfer the t-butyl methyl ether layer into a clean 100ml R.B.
flask using separating funnel.
4.7.3.4 Add another 10mL of t-butyl methyl ether to the vessel containing
buffer extract.
4.7.3.5 Repeat procedures 4.7.3.2 to 4.7.3.3

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4.7.3.6 Add 5mL t-butyl methyl ether to rinse the reaction vessel and
transfer to separating funnel and shake in manually for 5 min.
4.7.3.7 Combine the wash with the extract obtained in 4.7.3.3.
4.7.3.8 Wash the separating funnel with 5mL of t-butyl methyl ether.
4.7.3.9 Combine all the t-butyl methyl ether extract and wash.
4.7.3.10 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
4.8.1 Prepare 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
4.9.1 The 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.

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4.9.2 Develop a calibration curve of the target amines
4.9.3 Set up the GC/MSD system with below conditions
Agilent 6890GC/7890 GC with Agilent

GC-MS instrument
5973/5975 mass 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
DB-5MS / HP-5MS, 0.25mm x 30m x 0.25m

Column (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

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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.

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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.
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
m/z m/z
CAS No. Amines Substances Qualifier
Target ion Qualifier 1
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 m/z108.00

Target m/z
80.00 m/z
123.00
2-methoxy-5-
122.00 94.00 137.00
120-71-8
97-56-3 methylaniline
o-aminoazotoluene 106.10 225.10 91.10
101-77-9
137-17-7 4,4'-methylenedianiline
2,4,5-trimethylaniline 198.10
120.10 197.10
135.10 106.10
134.10
95-69-2 4-chloro-o-toluidine
4,4'-methylene bis(o- 141.10 106.10 140.10
231.10 266.00 268.10
101-14-4
95-68-1 chloroaniline)
2,4-dimethylaniline 121.10 120.10 106.10
Mix C 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
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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

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
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

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

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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
HPLC-DAD Waters Alliance (2695 module and 2996

instrument photodiode array detector or Agilent(1200)
LiChrospher 60 RP-select B, 4 x 250 mm, 5

Analytical column micron particle(or) Zorbax Eclipse XDB
C18(3.4micron); (150X4.6)mm
Temperature 25 oC
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
Solvent gradient
0 23 77 0.7
program
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

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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:
4.10.12.1 Retention time of the sample component is within ± 0.2

retention time units of the standard compounds.
4.10.12.2 Peak 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.

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4.10.14If 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 max for Wavelength for

CAS No. Amines Substances identification quantitation
(nm) (nm)
95-80-7 2,4-diaminotoluene 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 max for Wavelength for

CAS No. Amines Substances identification quantitation
(nm) (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
95-69-2 4-chloro-o-methylaniline / 240.8, 292.8 240

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Mix C
UV maxima for
Wavelength
CAS No. Amines Substances identification
(nm)
(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
p-phenylazoaniline / 4- 247.9, 382.0

240
60-09-3 aminoazobenzene
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 max for
CAS No. Amines Substances identification Wavelength (nm)
(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
838-88-0 242.0, 289.3 240

3,3'-dimethyl-4,4'-

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diaminodipenylmethane/
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
For m-Toluidine and p-Toluidine
UV-Max
CAS No. Amines Substances identification Wavelength (nm)
(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
5.3 Amine 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.

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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 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
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
95-68-1 2,4-dimethylaniline / 2,4-xylidin #

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Mix C
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
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
838-88-0 3,3'-dimethyl-4,4'-diaminodipenylmethane 70
62-53-3 Aniline #
106-50-3 1,4-phenylenediamine #

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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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7 Flow Chart Sample
If Polyester ,polyester If no polyester present

blend
Chloro Benzene / Cut the sample into 5mm

Xylene extraction in and transfer to reaction
25 ml vial
Concentrate to 1 ml
By rotary evaporator
Preheat the citrate buffer

at 70°C
Add 8/17 ml buffer as per the

method to reaction vial
Heat for 30 min
Add Di Thionite Soln to RV
Continue Heat for 30 min
Take out from bath and cool

down to RT
Add ether /salt/ NaOH Soln &

shake for 15 min
Collect the layer and inject in GC/ if

Conc is less do reduce the volume

Page 33 of 33

Banned Amines

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TEXTILE & GARMENT INDUSTRY RESEARCH &

Initiated By Reviewed By Approved By Document No

detected by GC-MS, confirmation and quantitation should be made by using

3.1 EN 14362-1: 2012 – Methods for determination of certain aromatic amines

4.1.1 Analytical balance accurate to 0.1mg

4.1.3 Thermostat-controlled oven

4.1.4 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),

4.1.11 High Performance Liquid Chromatography equipped with Photodiode

4.1.12 Mechanical shaker providing horizontal shaking motion

Initiated By Reviewed By Approved By Document No

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

4.2.3 Chloro benzene

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

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

4.2.13 Preparation of standard solution

4.2.13.1 Stock solution of 1000mg/L of individual amine (Table 1) was

Initiated By Reviewed By Approved By Document No

methanol (4.2.1) in a 25mL volumetric flask.

4.2.13.2 Four mixtures of amine standards were prepared. Please refer

4.2.13.3 Pipette 1.5mL of individual stock solution to a 10mL volumetric

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

4.2.13.6 Repeat procedure 4.2.6.5 with Mix B, Mix C and Mix D

Standard solutions for GC-MS analysis

Internal standard solution

Anthracene (100ppm): Weigh accurately 0.01g of in 100 ml volumetric

2,4,5-Trichloraniline(1000ppm): Weigh accurately 0.1g in 100 ml volumetric flask

CAS No. Internal Standard Brand Name

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

Initiated By Reviewed By Approved By Document No

1719-06-8 Anthracene – d10 ISO TEC

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.

Initiated By Reviewed By Approved By Document No

CAS No. Amines Substances Purity Brand Name

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

CAS No. Amines Substances Purity Brand Name

92-67-1 4-aminobiphenyl 98.4% Dr Ehrenstorfer

120-71-8 6-methoxy-m-toluidine 99.0%

90-04-0 o-anisidine / 2-methoxyaniline 99.5% Dr Ehrenstorfer

95-68-1 2,4-dimethylaniline / 2,4-xylidin 99.0% Dr Ehrenstorfer

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