determination of nitrosodiethylamine (ndea) in phase

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Sonavane et al. World Journal of Pharmacy and Pharmaceutical Sciences

DETERMINATION OF NITROSODIETHYLAMINE (NDEA) IN PHASE

TRANSFER CATALYST BY SENSITIVE LIQUID

CHROMATOGRAPHY TECHNIQUE

Dr. Sachin Sonavane*, Vishal Solse and Samadhan Kadam

Element Chemilink Pvt Ltd (Unit-iv). PLOT No 1808, 1809/2, 1811-1815, Near Asian

Paint Chokdi, Behind Atique Bakery, GIDC Estate, Ankleshwar-393002.Gujarat-393002.

ABSTRACT

Objective: Detection and control of Nitrosodiethylamine (NDEA) in

phase transfer catalyst i.e Triethyl benzyl ammonium chloride.

Hydrochloride salt of benzyltriethylammonium which acts as a phase-

transfer catalyst in chemical reactions. Phase-transfer catalyst is

a catalyst that facilitates the migration of a reactant from

one phase into another phase where reaction occurs. Tri ethyl benzyl

ammonium chloride catalyst is easily available and inexpensive which

is used in many API molecules. Nitrosoamine is an organic compound

containing the group –NNO attached to two organic groups.

Nitrosoamines are formed by reaction of secondary or tertiary amines

with a nitrosating agent. Some of the nitrosoamines are classified as

probable human carcinogens. A sensitive High performance liquid

chromatography (HPLC) method was developed and validated for the

determination of Nitrosodiethylamine in Triethyl benzyl ammonium chloride. HPLC column

Kanak PH-HEX, wavelength 250 mm, internal diameter 4.6mm, particle size 5μm with ultra

violet detector (UV) was used. The proposed method is specific, linear, accurate, rugged and

precise. The calibration curve of Nitrosodiethylamine showed good linearity over the

concentration range of 0.125 ppm to 3.75 ppm with respect to test solution concentration and

the regression coefficient was 0.999 for analyte. Method had very low limit of detection

(LOD) and limit of quantification (LOQ) of analyte which proves that the method is sensitive

and suitable for quantification of Nitrosodiethylamine impurities at trace level.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 9, Issue 11, 1591-1603 Research Article ISSN 2278 – 4357

*Corresponding Author

Dr. Sachin Sonavane

Element Chemilink Pvt Ltd

(Unit-iv). PLOT No 1808,

1809/2, 1811-1815, Near

AsianPaint Chokdi, Behind

Atique bakery, GIDC Estate,

Ankleshwar-393002.

Gujarat-393002.

Article Received on

01 September 2020,

Revised on 22 Sept. 2020,

Accepted on 12 October 2020

DOI: 10.20959/wjpps202011-17612

https://en.wikipedia.org/wiki/Catalyst

https://en.wikipedia.org/wiki/Phase_(matter)


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KEYWORDS: Triethyl benzyl ammonium chloride (TEBAC); NDEA; method development

and validation.

INTRODUCTION

Benzyltriethylammonium chloride is a lipophilic phase-transfer catalyst that can be used as

phase-transfer catalysis (PTC) to catalyze polycondensation reactions to form high molecular

weight polymers under bi-phasic conditions. It is used in aldol condensation reactions and

base-catalysed dehydration reaction. TEBAC, having a chemical structure as shown in figure

1, formation of Nitrosodiethylamine (NDEA) may get generate and it is well proved that

nitrosoamines are classified as probable human carcinogens.[2-3,10]

ICH M7 (R1) classifies Nitrosamine impurities as Class 1, which is known to be mutagenic

and carcinogenic, based on both rodent carcinogenicity and mutagenicity data. These

Nitrosamine impurities impact the genetic material by means of mutations through

chromosomal breaks, rearrangements, covalent binding or insertion into the DNA during

replication. These changes in the genetic materials caused by the exposure to very low levels

of Nitrosamine impurities can lead to cancer.[1-3]

Thus, it is important to identify Nitrosamine

impurities in all the key chemicals used in the manufacturing of drugs at very low levels to

ensure safety to the public.

Nitrosamine impurities can get incorporated into the drug substance and drug product

basically through process formation, direct introduction, degradation or cross-contamination.

Manufacturing of drug substances involves raw materials, Catalysts, intermediates, solvents,

chemicals and reagents.[4-7]

Triethylamine (TEA), which is key starting material for the manufacturing of Triethyl benzyl

ammonium chloride (TEBAC). TEA also contains 100-300 ppm levels of diethylamine as a

residual in TEA and hence the chances of formation of NDEA increases as secondary amine

are very reactive and prone to get oxidise easily to convert to Nitroso counterpart. It is well

proved that nitrosoamines are classified as probable human carcinogens. Because of the

known carcinogenecity residual Nitrosodiethylamine in Triethyl benzyl ammonium chloride

substance must be checked or controlled at trace level. To achieve quantification of such a

trace level it is necessary to develop sensitive, accurate and robust analytical method.

https://en.wikipedia.org/wiki/Aldol_condensation


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Nitrosodiethylamine are not controlled in the official monograph by HPLC. During literature

survey several analytical methods were found reported for estimation of Nitrosodiethyllamine

by other techniques but quantification at trace level by HPLC is not reported. In most of the

literature NDEA is tested by GC. Determination of Nitrosodiethylamine by LC-MS is also

reported.[8-10]

In present research, analytical method is developed to quantify the Nitrosodiethyllamine at

trace level using HPLC-UV detector. Nitrosodiethylamine which may generates during

synthesis of Triethyl benzyl ammonium chloride. The proposed analytical method is

validated as per International Council for Harmonization of Technical Requirements for

Pharmaceuticals for Human Use (ICH) guidelines ICH Q2-R1.

Tri ethyl benzyl ammonium chloride Nitrosodiethylamine

Figure: 1.

MATERIALS AND METHODS

Instrumentation: Shimadzu high performance liquid chromatography (HPLC) system

prominence i with UV detector with Lab solutions software or its equivalent.

Chemical and reagents: HPLC column used of Kanak PH-HEX. length 250 mm, internal

diameter 4.6mm, particle size 5μm. Nitrosodiethylamine standard of purity 99.9% was

procured from Clearsynth Inspiring research, Acetonitrile HPLC gradient grade of Rankem,

Methanol HPLC grade of Rankem, Di-sodium hydrogen phosphate anhydrous Emparta grade

from merck, Triethylamine Lab grade of Rankem, Orthophosphoric acid Emparta grade of

merck and purified HPLC grade water was used in the experimentation.

Chromatographic parameters

HPLC method was performed using Kanak PH-HEX. length 250 mm, internal diameter

4.6mm, particle size 5μm. Separation and peak symmetry was achieved with the mixture of

mobile phase-A: buffer and mobile phase-B: Acetonitrile in gradient elution with timed


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programme T min/A:B: T0/80:20; T12/80:20; for standard solution and gradient elution with

timed programme for sample solution is T min/A:B: T0/80:20; T12/80:20; T15/30:70;

T27/30:70; T30/80:20 and T35/80:20 with flow rate 1.2 mL/minute. The column temperature

was maintained at 25°C and auto sampler temperature 10°C. Ultraviolet detection was

performed at wavelength 229 nm. Injection volume was 25 μL, injector rinsing solution was

Acetonitrile and run time 12.0minutes for standard solution and 35.0 minutes for sample

solution.

Preparation of buffer solution

Buffer was prepared by dissolving 1.4g of Disodium hydrogen phosphate anhydrous in

1000mL of water. 1.0mL triethylamine was added, pH 3.0 was set with Orthophosphoric

acid, Filtered the solution and degassing was performed with the aid of sonicator to remove

air bubbles.

Solution preparation

Diluent preparation: Acetonitrile was used as a diluent.

Standard stock solution A: Accurately weighed and transferred 25 mg of NDEA standard in

to 25 mL volumetric flask. add 15 mL of diluent Sonicated to dissolve and diluted up to the

mark with diluent and mixed.

Standard stock solution B: Transferred 5.0 mL of standard stock solution A in to a 50 mL

volumetric flask and diluted up to the mark with diluent and mixed.

Standard solution: Transferred 0.50 mL of standard stock solution B in to a 200 mL

volumetric flask and diluted up to the mark with diluent and mixed. (NDEA are 2.5 ppm with

respect to test solution concentration).

Test solution: Accurately weighed 1000 mg of Tri ethyl benzyl ammonium chloride test

sample in to a 10 mL volumetric flask and diluted up to the mark with diluent. Solution was

mixed and sonicated. Filtered the solution if it required, through 0.45u syringe filter and

injected the filtrate.

Note: All standard and test solutions were prepared freshly before analysis.


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Procedure

Performed blank followed by standard solution six replicate and test preparation. Run the

blank and test solution chromatograph for 35.0 minutes and standard solution for 12.0

minutes. The peak of NDEA was integrated at wavelength 229 nm at retention time 7.8

minutes. desired peak were integrated in standard and test solution at specified wavelength

and calculated by external standard addition method.

Acceptance criteria for system suitability

% RSD of peak area of NDEA in six replicate standard injections was found less than 10.0

RESULTS AND DISCUSSION

Nitrosodiethylamine (NDEA) CAS No. 55-18-5, molecular formula C4H10N2O, molecular

weight 102.13. Refer figure 1 for the structure of Nitrosodiethylamine (NDEA). During

literature survey it was found that most of the methods for determination of

Nitrosodiethylamine (NDEA) are by GC with FID or LC mass detector. Reverse phase high

performance liquid chromatography (RP-HPLC) was chosen as a starting point for method

development. compound are soluble in organic polar solvents. On basis of chemical

properties of the compound and to achieve detection of impurity with reverse phase high

performance liquid chromatography (RP-HPLC) was chosen. In RP-HPLC with UV detector

on C18 column using mobile phase a mixture of Phosphate buffer of pH 3.0 and acetonitrile

were used but the distorted peak was observed. In order to improve peak shape trials were

taken with different pH of the mobile phase by adding triethylamine after improving peak

shapes, interference were observed. The desired peak shape of impurity without any blank or

matrix interference and symmetrical peak shape was achieved with Phenyl-Hexyl column and

the proposed mobile phase combination of buffer and acetonitrile.

ANALYTICAL METHOD VALIDATION

Selectivity

Selectivity is the ability of the method to determine accurately and specifically the analyte of

interest in the presence of other components in a sample matrix that may be expected to be

present in the sample matrix under the stated conditions.

Selectivity of the method was evidenced by comparing chromatograms of blank,

Nitrosodiethylamine triethylamine, toluene. And Isopropyl alcohol separate injection. There

is no interfering peak at the retention time of Nitrosodiethylamine was observed. The


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interference with in the peak was checked using UV detector at 229nm wavelength. Tri ethyl

benzyl ammonium chloride test sample injected in the chromatograph. The peak purity index

of Nitrosodiethylamine was found 0.999 in presence of all other specified and unspecified

impurities of the test sample. It proves that no interference at the retention time of analyte

under given chromatographic parameters. The proposed method is selective and specific and

therefore suitable for quantification of analyte.

Solution stability

Drug stability in active pharmaceutical ingredient is a function of storage conditions and

chemical properties of the drug and its impurities. The solution stability till twelve hours of

Nitrosodiethylamine had been checked by injecting standard solution. Nitrosodiethylamine

standard solution was prepared fresh before injection and same solution was injected after

twelve hour interval. The sample cooler temperature was kept 25⁰C. The peak area of

Nitrosodiethylamine of freshly prepared standard solution was observed 24229 and after

twelve hours it was 24306. The solution stability till twelve hours of test solution had been

checked by injecting test solution. Test solution was prepared fresh before injection and

immediately injected and same solution was injected after twelve hour interval. The sample

cooler temperature was kept 10⁰C. The peak area of Nitrosodiethylamine of freshly prepared

Test solution was observed 2865 and after twelve hours it was 2849. On basis of solution

stability data it is recommended that the sample is stable upto twelve hours.

LOD and LOQ

The limit of detection (LOD) is the lowest concentration of the analyte in a sample that can

be detected but not necessary quantified. The obtained LOD values of Nitrosodiethylamine is

discussed.

LOD = 3.3 × σ / S

The limit of quantitation (LOQ) is the lowest concentration or amount of the analyte that can

be determined quantitatively within an acceptable level of repeatability precision and

trueness.

LOQ = 10.0 × σ / S

Where, σ = the standard deviation of the response and S= slope of the calibration curve

LOD and LOQ concentration is reported in parts per million (ppm) with respect to Triethyl

benzyl ammonium chloride test concentration. LOD was found 0.038 ppm of


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Nitrosodiethylamine with respect to test solution. LOQ was found 0.125 ppm of

Nitrosodiethylamine with respect to test solution. Precision of LOQ is checked by injecting

six replicate injections of Nitrosodiethylamine at their LOQ level. Relative standard deviation

(RSD) of peak area of Nitrosodiethylamine at LOQ level is observed 2.70% which proves

consistency and reproducibility of the method at trace level.

Linearity

Under the experimental conditions, the peak area vs. concentration plot for the proposed

method was found to be linear over the range of LOQ level, 25%, 50%, 100%, 120% and

150% of the specified limit, with a regression coefficient of Nitrosodiethylamine is 0.999 of

analyte. The linearity of Nitrosodiethylamine is plotted. The horizontal x-axis or abscissa is

typically chosen to represent the independent variable which is concentration of the analyte.

The vertical y-axis or ordinate, is chosen to represent the dependent variable i.e. peak area,

which changes as the independent variable is manipulated. The regression coefficient (r2) is

more than 0.99 is generally considered as evidence of acceptable fit of the data to the

regression line. Refer figure -2, 3,4,5,6,7 and 8 of Standard Blank, 100% Standard solution,

Sample Blank, Sample solution, Spike solution. LOQ Level and Six replicates standard

solutions.

Figure 2: Standard Blank.


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Figure 3: 100 % Standard solution.

Figure 4: sample blank.


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Figure 5: sample solution.

Figure 6: Spike solution.


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Figure 7: LOQ Level.

Figure 8: Six replicates standard solutions.


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Accuracy

Accuracy can be defined as the closeness of agreement between a test result and the accepted

reference value. Accuracy of the method was determined by recovery study. Analytical

method may be considered validated in terms of accuracy if the mean value is within ± 20%

of the actual value. During recovery study Triethyl benzyl ammonium chloride batch was

analyzed and then Nitrosodiethylamine is spiked in the Triethyl benzyl ammonium chloride

at LOQ level, 50%, 100% and 150% with respect to the limit of Nitrosodiethylamine. The

recovery of Nitrosodiethylamine is found 98.8%, 99.2%, 97.3% and 96.8% respectively It

proves that the method is capable to quantify the analyte accurately and results will be

reliable even at trace level.

Ruggedness study

The (intra-laboratory tested) behavior of an analytical process when small changes in

environment and/or operating condition are made. The ruggedness of the method was

evaluated by estimating % RSD of Nitrosodiethylamine standard solution tested by two

different analysts on different days. % RSD of area of Nitrosodiethylamine peak in standard

solutions of both analysts should not be more than 10%. Six replicates of diluted standard

solution were injected by each analyst. Relative standard deviation of area of

Nitrosodiethylamine was found 1.17% and 1.03% on two different days. This shows that

there is no variation in day to day analysis. Method validation summary is given in table-1.

Ruggedness was checked using spiked solutions by two different analysts with six different

preparations. Finally RSD of total twelve preparations were calculated and found 1.14% of

Nitrosodiethylamine.

Robustness study

Robustness is a measure of the capacity of the analytical procedure to remain unaffected by

small but deliberate variations in method performance parameters, which provides an

indications of its reliability during normal usage. Robustness of the method was determined

by analyzing the standard solution and batch analysis with deliberate change in the

parameters like (a) flow rate of the mobile phase ±0.2 mL/min (b) column temperature ±2°C.

System suitability criteria were passing each time and result variation is not observed during

batch analysis. Method validation results are summarized.


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COMPLIANCE WITH ETHICAL STANDARDS

Conflict of Interest: Author Sachin sonawane, Vishal Solse, Samadhan kadam, declare that

they have no conflict of interest.

Ethical approval: This article does not contain any studies with human participants or

animals performed by any of the authors.

Table 1: Analytical method validation summary. S. NO.

Parameter Acceptance criteria Results

1 Selectivity

Nitrosodiethylamine peak should be well

separated from all known and unknown

peaks of Tetrabutyl ammonium bromide.

Complies.

Method is selective.

Peak purity passing

2 Solution stability Report Result Solutions are stable up to

12 hours

3 Limit of detection

(LOD) Report Result

Nitrosodiethylamine=0.038

ppm wrt test concentration

4 Limit of quantification

(LOQ) Report Result

Nitrosodiethylamine=0.125ppm

wrt test concentration

5 LOQ precision %RSD for six replicates of LOQ level

standard solutions is NMT:10.0%

RSD of Nitrosodiethylamine =

2.70%

6 Linearity Correlation: NLT 0.99 Nitrosodiethylamine = 0.999

7 Accuracy Recovery should be between

80% to 120% complies

8 Method precision %RSD for results of six standard

preparations is NMT:10%


1.17%

9 Ruggedness

Intermediate precision

%RSD for results of twelve spike solution

preparations (Method precision and

Intermediate precision) is NMT:10%


1.14%

Validated method can be used for routine analysis of quantification of Nitrosodiethylamine in

quality control laboratories in the pharmaceutical industry.

ACKNOWLEDGEMENT

Thanks to the management of Element Chemilink Pvt ltd for constant support.

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determination of nitrosodiethylamine (ndea) in phase

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