Short Communications

Determination of Dimethylamine and Triethylamine in

Hydrochloride Salts of Drug Substances by Headspace
Gas Chromatography using Dimethyl Sulphoxide-
imidazole as Diluent
J. GOPALAKRISHNAN* AND S. ASHA DEVI1
Piramal Enterprises Limited, Analytical Development Laboratory, 1, Nirlon complex, Off Western expressway,
Goregaon (E), Mumbai-400 101, 1School of Biosciences and Technology, VIT University, Vellore-632 014,
India

Gopalakrishnan and Asha Devi: Determination of Amines in Drug by Headspace Gas Chromatography

A simple, rapid, accurate and precise analytical method for determination of secondary and tertiary
amines in hydrochloride salt form of drug substances in non-aqueous medium, which does not require
any derivatization, was developed by headspace gas chromatography. Dimethylamine hydrochloride and
triethylamine hydrochloride were analyzed in metformin hydrochloride. The sample was dissolved in a vial
containing dimethyl sulphoxide and imidazole. The vials were incubated at 100°, for 20 min. The syringe
temperature was maintained at 110°. DB624 column with 30 m×0.32 mm i.d., 1.8 µm film thickness was used.
The injector and detector temperature were 200° and 250°, respectively. The analytical program used was
carrier gas (nitrogen) flow rate: 1 ml/min; injected gas volume: 1.0 ml; split ratio: 1:10; oven temperature
program: 40° for 10 min, followed by an increase up to 240° at 40°/min. The analytical method was validated
with respect to precision, recovery, limit of detection and quantification and linearity.

Key words: Aliphatic amines, dimethylamine, triethylamine, metformin hydrochloride, DMSO, imidazole,
headspace gas chromatography

Organic solvents are used in the synthesis of limit for unspecified impurities is 0.10%, i.e., 1000
pharmaceutical compounds and they must be controlled ppm[4]. These amines are chemically reactive in nature
to a minimum level in the final product. Their limits and their influence in experimental condition during
are fixed based on their toxicity[1]. In general, these analysis by headspace gas chromatography was briefly
residual solvents are analyzed by headspace gas studied by Kott and Chen[5]. Various techniques of
chromatographic technique. Most of the common analyzing organic amines after derivatization using gas
organic solvents such as toluene, hexane, benzene, chromatography and headspace gas chromatography
chloroform, dichloromethane are chemically non- (GC) techniques were reported by Hiroyuki[6]. Influence
reactive and does not involve in any chemical reaction of pH of the diluent in aqueous medium during static
during analysis. Selection of suitable diluent for analysis headspace analysis of aliphatic amines was reported by
for these types of residual solvents is relatively simple. Maris et al.[7]. Analytical method for determination of
For these solvents, diluents are selected based on their aniline in water using microwave assisted headspace
higher boiling point, their solubility in the sample and solid phase micro extraction (SPME) technique was
the organic solvents to be determined. reported by Yan and Jen[8].
Aliphatic amines such as triethylamine and
dimethylamine are used in synthesis of many This is an open access article distributed under terms of the Creative
pharmaceutical products. Triethylamine should be Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
allows other the remix, tweak, and build up to the non-commercially, as
controlled to the level of maximum 320 ppm according to long as the author is credited and the new creations are licensed under
European Directorate of Quality Medicines Document the identical terms.

(EDQM)[2,3]. Limit for dimethylamine is not mentioned Accepted 03 May 2016

in any literature. However, as per ICHQ3A (R2), the Revised 20 Mar 2016
Received 29 Sep 2015
*Address for correspondence
E-mail: [email protected] Indian J Pharm Sci 2016;78(3):409-413

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When the drug substance is present in the form of triethylamine) and dimethylamine hydrochloride (25
salt, the amines which could be present in the residual µg/ml of dimethylamine) in DMSO were used as
amount are also expected to be present in the form standard solutions. Headspace vials for standard were
of salt. Therefore, it is required to convert the salt prepared by adding imidazole to the standard solutions.
form of amine to free base, during headspace gas Similarly sample vials were prepared in headspace
chromatographic technique. Michael et al. developed vials by adding sample, imidazole and DMSO. For
analytical method for determination of triethylamine determination of triethylamine and dimethylamine,
in sarafloxacin hydrochloride on capillary gas sample size was 200 and 100 mg, respectively and
chromatography by using glass inlet liners and glass imidazole quantity was 0.5 and 1 g, respectively. Blank
wool which were treated with methanolic potassium vials were prepared by adding DMSO in to a headspace
hydroxide[9]. Raghuram et al. reported quantification vial containing imidazole.
of cyclopropylamine, diethylamine and triethylamine Analytical method validation was carried out with
in oseltamavir phosphate and candesartan cilexetil respect to system suitability, linearity, limit of detection
by headspace gas chromatography technique. They (LOD) and limit of quantification (LOQ), precision
have treated the active pharmaceutical ingredients and accuracy. Specificity of the analytical method was
in a mixture of aqueous sodium hydroxide and established by injecting individually diluent, standard
dimethyl sulphoxide and analyzed by headspace solutions of dimethylamine hydrochloride and
gas chromatography[2]. Deshpande et al. developed triethylamine hydrochloride in to the chromatograph.
analytical method for determination of diethylamine For LOD and LOQ, various concentrations of
and triethylamine in oxybutynin hydrochloride and dimethylamine and triethylamine were prepared and
trazadone hydrochloride. They have treated the active injected in to the chromatograph up to their detectable
pharmaceutical ingredient in a mixture of aqueous concentration and quantifiable concentration. Precision
sodium hydroxide solution and dimethyl sulphoxide of the method was determined by injecting six replicate
and analyzed by headspace gas chromatography[10]. preparations of sample. Accuracy of the method was
The aim of the present study is to find out a suitable carried out by spiking the analytes at three different
base which can convert the salt form of amine in to levels, i.e., 50, 100 and 150% of the specification limit.
free base in presence of the drug substance in non- The samples at each level were prepared in triplicate
aqueous medium. It was found that imidazole was able and injected in to the chromatograph. Linearity of
to convert the salt form of amine in to free base when it dimethylamine hydrochloride and triethylamine
was used in non-aqueous medium. The novelty of our hydrochloride were carried out by injecting standard
study is that imidazole has not been used as a diluent solutions of these analytes from LOQ to 150% level of
for preparation in any of the residual solvents methods specification limit. Each level was prepared in triplicate
and analyzed.
published so far. Metformin hydrochloride was used as
the drug substance for the study. Dimethylamine was Thermo focus GC was equipped with split/split less
used as a raw material in the synthesis of metformin injector, column oven, a flame ionization detector,
hydrochloride[11]. Triethylamine is in general used as headspace auto sampler (Thermo). This auto sampler
a base for many organic reactions. A simple, rapid, was equipped with a glass syringe (2.5 ml capacity)
accurate and precise headspace gas chromatographic with temperature controller and vials of standard size
technique for analyzing dimethylamine hydrochloride and volume of 20 ml capacity. The vial temperature
and triethylamine hydrochloride in metformin was controlled by using an incubator equipped with
hydrochloride without any derivatisation was automatic mixing system by micro vibration. The vials
developed. containing solutions were kept at 100°, for 20 min. Vials
were pressurized due to incubator temperature. The
Triethylamine hydrochloride was obtained from
syringe temperature was maintained at 110°. DB624
Sigma Aldrich, USA. Triethylamine and dimethyl
column with 30 m×0.32 mm i.d., 1.8 µm film thickness
sulphoxide (DMSO) were obtained from Merck,
was used. The injector and detector temperature were
India. Dimethylamine hydrochloride and imidazole
200° and 250°, respectively. The analytical program
were obtained from S.D. Fine-Chem Limited, India.
used was carrier gas (nitrogen) flow rate: 1 ml/min;
Metformin hydrochloride was procured from a
injected gas volume: 1.0 ml; split ratio: 1:10; oven
pharmaceutical company.
temperature program: 40° for 10 min, followed by an
Triethylamine hydrochloride (13 µg/ml of increase up to 240° at 40º/min.
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In general, DMSO, N-methyl pyrrolidone, 1,3 dimethyl was same and it was not altered due to further addition
2-imidazolidone and dimethyl formamide are the non- of imidazole. A similar study was carried out using
aqueous solvents which are being used as a solvent dimethylamine hydrochloride to optimize the quantity
medium for preparation of samples for residual solvents of imidazole and the results are shown in fig. 2. In
analysis. These solvents are preferred for residual order to generate the data on pH of the solutions, the
solvent analysis because of their high solubility and solutions at each stage were mixed with equal amount
high boiling point. Experiments were carried out using of water and measured. The results are tabulated in
all these solvents. It was not possible to convert the Table 1. When the pH of the solutions containing free
salt form of the amine in to free base, using these base and salt form of the amine was same, the area
solvents. Imidazole dissolved in DMSO was preferred response was also same.
because of its basic as well as buffer nature and trials Specificity of the method was confirmed as there was
were made. It was found that imidazole in DMSO was no any interference due to diluent at the retention
able to convert the salt form of amine in to free base times of the triethylamine and dimethylamine. LOD
quantitatively. Thus, DMSO-imidazole mixture was was determined by calculating the signal to noise ratio
selected as suitable diluent for this method. with respect to the corresponding noise in the blank
To optimize the quantity of imidazole required for the injection. Precision at LOQ level was carried out by
sample preparation, solutions of triethylamine and injecting six different preparations of analytes at
triethylamine hydrochloride were prepared separately their LOQ level and the percentage relative standard
in DMSO. They were transferred in to headspace vials deviation (% RSD) for area was calculated. The results
containing various amount of imidazole. At each level, are tabulated in Table 2.
solutions were prepared in duplicate and analyzed For linearity study five different concentrations of
as per the experimental condition. A graph of area triethylamine and dimethylamine in the range of 0.37-
response of triethylamine (base and hydrochloride 19.5 µg/ml and 2.8-37.5 µg/ml, respectively were
salt) against amount of imidazole used was plotted and prepared. The vials were prepared in triplicate for each
shown in fig. 1. The response of triethylamine which concentration and analyzed. A linear graph was plotted
was present in the form of triethylamine hydrochloride with respect to area response against the concentration
was very less when less amount of imidazole was used. of the analytes and their corresponding correlation
The response was increasing with respect to increase coefficient, slope and intercept values were calculated.
in amount of imidazole and reached a maximum value. The results are attached in Table 3.
When adequate amount of imidazole was used, the
response of triethylamine (both salt and free base) The sample was containing only 0.07% of

Fig. 1: Triethylamine and Triethylamine hydrochloride response with respect to imidazole quantity.
-♦- TEAHCl; -■- TEA. There was no response observed when triethylamine hydrochloride was analysed at lower pH. At higher pH
values, the response of triethylamine and triethylamine hydrochloride by headspace GC was found to be high and it remained same.

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Fig. 2: Dimethylamine hydrochloride response in headspace GC in DMSO-imidazole with respect to imidazole quantity.
The quantity of imidazole required to completely convert the salt form of dimethylamine to free base by headspace GC was
determined using this experiment.

TABLE 1: pH OF TRIETHYLAMINE AND TRIETHYLAMINE HYDROCHLORIDE IN DMSO WITH VARIOUS

QUANTITIES OF IMIDAZOLE
Diluent composition TEA HCl TEA DMA HCl
pH (50:50) in water
DMSO (Blank) 7.2
DMSO+1000 mg imidazole (Blank) 10.1
0 mg imidazole+DMSO 7.5 10.3 7.2
25 mg imidazole+DMSO 9.1 10.3 9.0
50 mg imidazole+DMSO 9.3 10.2 9.2
100 mg imidazole+DMSO 9.6 10.1 9.5
250 mg imidazole+DMSO 9.8 10.1 9.8
500 mg imidazole+DMSO 9.9 10.0 9.9
750 mg imidazole+DMSO 10.0 10.0 10.1
1 g imidazole+DMSO 10.1 10.1 10.1
TEA HCl: triethylamine hydrochloride, TEA: triethylamine; DMA HCl: dimethylamine hydrochloride. pH was checked after adding equal
amount of water in to each vial. pH of the solution was more than 10 when triethylamine was used and it remained same with respect to
different quantities of imidazole added to the solution.

TABLE 2: LOD AND LOQ FOR DIMETHYLAMINE AND TRIETHYLAMINE

Analyte LOD (µg/ml) S/N Ratio for LOD LOQ (µg/ml) Precision at LOQ (% RSD)
Dimethylamine 0.93 5 2.8 2.4
Triethylamine 0.12 3 0.37 4.7
Precision at limit of detection (LOD) and limit of quantification (LOQ) was performed in six replicates.

dimethylamine and triethylamine was absent. Accuracy and 96.6% for dimethylamine and triethylamine,
of the experiment was carried out by spiking 50, 100 respectively. Overall % RSD of recovery was found to
and 150 µg of dimethylamine in 100 mg of metformin be 2.4% and 4.0% for triethylamine and dimethylamine,
hydrochloride. For triethylamine, accuracy was carried respectively.
out by spiking 32, 64 and 96 µg of triethylamine in The proposed method for determination of
200 mg of metformin hydrochloride. 92.5-99.4% of dimethylamine hydrochloride and triethylamine
recovery was obtained for triethylamine and 93.1- hydrochloride in metformin hydrochloride is a direct
104.3% of recovery was obtained for dimethylamine. method and it does not involve in any derivatisation.
Overall recovery at all levels was found to be 97.6 The possible interaction between the analyte, drug
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TABLE 3: LINEARITY STUDY RESULTS FOR REFERENCES
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413 Indian Journal of Pharmaceutical Sciences May - June 2016