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CHAPTER 3

SIMULTANEOUS TRACE LEVEL DETERMINATION OF

POTENTIALL GENOTOXIC HYDRAZINES AND ALKYL AMINES

IN PHARMACEUTICALS BY CAPILLARY ELECTROPHORESIS

WITH INDIRECT PHOTOMETRIC DETECTION

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3.1 Introduction to genotoxic hydrazines and alkylamines

Genotoxic impurities have the potential to induce genetic mutations,

chromosomal aberrations and rearrangements and can cause cancer

in humans. These impurities may be introduced into the drug

substances and drug products as residual starting materials and

reagents, excipients, process related by-products or through drug

substance degradation. Impurities with known genotoxicity (Muller’s

Class 1 and Class 2 type genotoxic impurities [1]) and impurities

containing characteristic functional moieties not contained in the

parent API (Muller’s Class 3 type alerting structure impurities[1]) are

capable of inducing genotoxicity, therefore the ICH Q3A(R), Q3B(R2)

and Q3C(R5) guidelines [2-4] cannot be applied for controlling these

impurities in drug substances and drug products. The Guideline on

the Limits of Genotoxic Impurities [5] issued by the Committee for

Medicinal Products (CHMP) and a more recent draft guidance

document on limits of genotoxic and carcinogenic impurities [6] issued

by the US Food and Drug Administration recommend that an

exposure level of 1.5 μg per person per day can be considered an

acceptable qualification threshold for each genotoxic impurity. The

guidelines [6-7] further clarify that for structurally similar genotoxins,

the TTC limits should be applied to the sum total of these impurities

as they are expected to have the same mode of action and the same

target. Any impurity found at a level below this threshold, generally

needs no further safety qualification for genotoxicity and

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carcinogenicity concerns. This concept of TTC when applied ensures

a lifetime risk of cancer of less than 10-5, a risk level that is thought to

pose negligible safety concerns as compared to the real time cancer

risks. A staged TTC based on the duration of exposure may also be

acceptable when the drug substance is not intended for a life time

consumption [1]. While stringent limits with a 10-fold lower values

amounting to 0.15µg/day are recommended for high potency

carcinogens such as N-nitrosoamines, aflatoxins and azoxy

compounds, more relaxed limits may be acceptable in other

circumstances such as carcinogens with established dietary exposure

and genotoxic impurities present in drug substances used for life

threatening indications such as cancer. The analyses of genotoxic

impurities remains a challenge, as their absolute concentrations

should be limited to ppm levels in the drug substances.

Hydrazine is a useful building block in organic synthesis of

pharmaceuticals and pesticides owing to its reducing properties. It

has been used as a synthetic intermediate in the manufacturing of

several drugs such as nufuroxazide, hydrazaline, isoniazid etc.

Hydrazine is a degradation impurity in isoniazid [8], hydralazine,

phenelzine and isocarboxazid [9-10]. Hydrazine and methylhydrazine

are genotoxic in nature [11-12] and therefore warrant a control in the

drug substances at appropriate levels such that the total intake of

hydrazine or its derivatives does not exceed 1.5µg per day.

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The alkylamines such as ethylamine, diethylamine and

triethylamine are used as solvents in the pharmaceutical synthesis.

Because of their basic nature, the alkylamines are frequently used to

trap acids. The primary and secondary alkylamines react with acyl

chloride and acid anhydrides to yield amides. The Mannich reaction

which involves the aminoalkylation of a carbonyl compound with a

primary or a secondary amine is used in the synthesis of several

medicinal compounds such as rolitetracycline, fluoxetine, tramadol

and tolmetin. While most of the arylamines are considered to be

genoxotic, the alkylamines are highly corrosive and create respiratory

problems and need to be controlled at acceptable levels in the drug

substances. No specific limits for these alkylamines have been

proposed in the ICH Q3 guidelines on impurities. In case of

triethylamine, based on repeated dose toxicity and reproductive

toxicity studies, the regulatory assessors recognize a level of 320 ppm

(considering a 10 g daily dose) as acceptably safe. However this limit

cannot be directly applied to other organic bases and their proposed

limits should be supported with available toxicological data.

Hydrazine, its methylated analogues and the aliphatic amines in

general are highly polar and basic molecules. Hydrazines and the

aliphatic amines are transparent to UV detection as they lack

chromophores and therefore cannot be analyzed by HPLC-UV in their

native form. The non-substituted hydrazines do not contain carbon

hence cannot be detected by flame ionization detection technique. As

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hydrazines are low molecular weight compounds, the trace level

analysis using MS detection can be challenging due to interfering

species. Elder et.al. [13] have recently reviewed the control and

analysis of hydrazine, hydrazides and hydrazones in APIs and drug

products. Derivatisation followed by HPLC [14] and GC [15] analysis

have been the techniques of choice for determining hydrazines. TLC,

IC and electro-chromatographic methods have also been employed for

determining hydrazines after derivatisation. The authors [13] also

quote two very recent examples of hydrophilic interaction

chromatography (HILIC) for hydrazine analysis [16-17] and predict

that this may become the favored analytical approach in future, as it

does not involve derivatisation. However CE can be a very cost

effective alternative to ion-chromatography and HILIC but

unfortunately is often underutilized. Recently M.Khan et. al [18] have

demonstrated the application of CE for determining genotoxins

dimethylsulfate and chloroacetyl chloride at trace levels in drug

substances.

The analysis of hydrazines and other trace analytes by

chromatographic techniques is often preceded by sample pretreatment

which involves removal of substrate interferences through liquid-

liquid extraction (LLE) and liquid-solid extraction (LSE) techniques.

The electromigration techniques such as CE, CEC and MEKC provide

better selectivities when compared to chromatographic techniques.

This is due to the fact that the driving forces (electrophoretic

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mobilities) that induce separation between analyte species are

intrinsically specific. Therefore, obtaining selectivity between charged

species is seldom a challenge in capillary electrophoresis. Recently

three hydrazines namely hydrazine, methylhydrazine and 1,1-

dimethylhydrazine were separated on a poly(methylmethacrylate)

microchip with contactless conductivity detection [19]. Determination

of hydrazine and its methylated analogues by non-aqueous capillary

electrophoresis with amperometric detection [20] and by

electrochemical detection using a 4-pyridyl hydroquinone self-

assembled microdisk platinum electrode [21] has been reported. In

another research, 14 amino acids and amines have been analyzed by

a CE method after derivatising with naphthalene-2,3-

dicarboxaldehyde with LED induced fluorescence detection [22].

The purpose of this research was to develop a simple and fast

generic method to determine hydrazine, methylhydrazine and other

alkyl amines in drug substances, avoiding laborious and cumbersome

steps of derivatisation. The developed CE method offers speed, high

selectivity, low reagent consumption and is environment-friendly.

A simple, fast and sensitive capillary electrophoretic method was

developed for determining two genotoxic impurities hydrazine and

methylhydrazine and eight alkyl amines at trace levels in

pharmaceutical substances using indirect photometric detection. The

method development involved a systematic screening of cationic

visualizing reagents and optimization of separation conditions to

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obtain the best resolution and sensitivities for targeted analytes. The

optimized method was validated for specificity, precision, linearity and

accuracy. Linear calibration curves (R > 0.999) were obtained for all

analytes in the range LOQ - 200% of nominal concentrations. The

developed CE method was effectively implemented for estimating the

hydrazines and alkyl amines in several active pharmaceutical

ingredients (APIs).

3.2 Experimental

3.2.1 Chemicals and Reagents

CE grade water was procured from Agilent technologies, (Waldbronn,

Germany). Pyridine, 4-aminopyridine, imidazole and benzyl amine

were procured from Merck (Darmstadt Germany), ethylamine,

triethylamine, ethylenediamine, triethanolamine, isopropylamine,

diisopropylamine, N,N-diisopropyl ethylamine and N-butylamine were

procured from Sigma Aldrich (Steinheim, Germany). Hydrazine,

methylhydrazine were procured from Spectrochem (Mumbai, India).

All reagents were of analytical grade or highest available purity. The

drug substances were synthesized in Aptuit Laurus Private Limited

(Hyderabad, India).

3.2.2 Instrumentation and electrophoretic conditions

An Agilent Technologies Capillary Electrophoresis system equipped

with an on-column diode-array detector, an autosampler,

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autoinjector, a capillary thermostat and a power supply able to deliver

upto 30 kV was used for performing the separations. Bare fused silica

capillary of total length 80.5 cm (length to detector = 72 cm); 50-μm

id, having an extended light path with a bubble factor of 3 (Agilent

Technologies, Waldbronn, Germany) was used for enhanced

sensitivity. An alignment interface, containing an optical slit matched

to the internal diameter of 150-µm was utilized. The Agilent

ChemStation software was used for system control, data acquisition

and post-run processing.

A voltage of 30 kV was applied in positive polarity mode.

Injections were made hydrodynamically by pressure at 50 mbar for 30

s followed by injection of a BGE plug by pressure at 50 mbar for 2 s.

The capillary was thermostated at 25°C. The detector signals were

monitored at 350 nm (bandwidth 80 nm) with a reference wavelength

of 264 nm (bandwidth 20 nm).

New capillaries were washed with CE grade water for 30 min.

followed by the BGE for 50 min. Prior to every use, on each day the

capillary was conditioned by flushing with water for 10 min. and then

with the BGE for 20 min. Between analyses, the capillary was flushed

with the BGE for 3 min. To maintain a high level of reproducibility for

both migration times and peak areas the BGE was replaced after every

15 injections.

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3.2.3 Preparation of solutions

Preparation of background electrolyte

The background electrolyte (BGE) was prepared by dissolving about

47.0 mg of 4-aminopyridine in 100 mL CE grade water. The pH of this

solution was adjusted to 5.5±0.1 with 10% ortho-phosphoric acid. The

resultant BGE was filtered through 0.2 μm nylon syringe filters before

use.

Preparation of standard and sample solutions

Stock solutions of each analyte were prepared at a concentration of

1.0 mg/mL independently by dissolving appropriates amounts in

diluent (water:methanol 90:10 v/v). Each stock solution was further

diluted 10 folds (1 mL – 10 mL) with the diluent to obtain standard

stock solutions. A 3.0 mL aliquot of each of these standard stock

solutions were diluted to 100 mL in a single volumetric flask to obtain

the standard solution and filtered through 0.2 µm nylon syringe

filters.

About 0.5 g of each API was accurately weighed into a 5 mL

volumetric flask. A 0.5 mL aliquot of methanol was added to the flask

and sonicated for about 5 min. The flask was cooled to room

temperature and volume was made up to 5 mL mark with the diluent

and sonicated for 15 min. with intermittent shaking. The sample

solutions were filtered through 0.2 µm nylon syringe filters.

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3.2.4 Method validation

Specificity

Specificity was evaluated by analyzing hydrazine, methylhydrazine

and each alkylamine individually and spiked to the respective APIs

along with all other known process related impurities. The

electropherograms were examined for interferences from other

impurities or the sample matrix.

Precision

The repeatability of the method was evaluated by analyzing replicate

preparations (n = 6) of a spiked solution containing the analytes at

LOQ level, 100% (3.0 µg/mL) and 200% (6.0 µg/mL) of nominal

analyte concentrations. Six replicate spiked solutions were prepared

at these same concentrations and injected every day, on three

different days to evaluate intermediate precision. The %RSD for peak

areas and their migration times were determined to assess the

method’s precision.

Detection and Quantitation Limits

A series of dilute solutions with known concentrations of hydrazine,

methylhydrazine and the alkylamines were injected to obtain the

detector response. The detection limits and quantitation limits for

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each analyte were derived from the concentrations resulting in signal-

to-noise ratios of about 3:1 and 10:1 respectively.

Linearity and range

The linearity solutions were prepared from individual stock solutions

of hydrazine, methylhydrazine and the alkylamines at LOQ, 50, 100,

150, and 200% of analyte concentrations and analyzed. The data was

subjected to linear regression analysis with the least squares method.

Accuracy

The API samples were spiked with hydrazine, methylhydrazine and the

alkylmines expected to be present at LOQ, 100 and 200% of the

nominal analyte concentrations covering the range. Triplicate

samples at each level were prepared and analyzed as per the method.

The recoveries of the spiked analytes were calculated against freshly

prepared standards and mean percentage recoveries were used to

assess the accuracy.

3.3 Results and Discussion

3.2.1 Development and optimization of method

Hydrazine, methylhydrazine and the alkylamines (Fig. 3.1) carry

positive charge at the working pH of 5.5 tend to migrate towards the

cathode (negative) terminal under the influence of applied electrical

field. The development and optimization trials were made by

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evaluating different cationic visualizing reagents for obtaining the

desired selectivities, maximum sensitivity and peak symmetry for the

target analytes through indirect photometric detection.

Analyte Structure Analyte Structure

Hydrazine N N

H

H

H

H

Triethanolamine N

OHOH

OH

Methylhydrazine

NH2 NH

CH3

Isopropylamine

NH2

Ethylamine NH2 Diisopropylamine

NH

Triethylamine N

N,N-diisopropyl

ethylamine N

Ethylenediamine NH2

NH2

n-butylamine NH2

Fig. 3.1 Chemical structures of Hydrazines and alkylamines

The principle of indirect photometric detection is based on the

fact that the UV-absorbing ions present in BGE are displaced by the

transparent, non-UV absorbing analyte ions [23]. This displacement

causes a negative signal in the analyte zone when compared to the

high continuous background signal. The negative signals

corresponding to the analyte peaks can be made upright by

subtracting suitable reference energy from the sample energy. The

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sensitivity of detection depends upon the molar absorptivity of the

visualizing reagent at the detection wavelength and also upon the

baseline noise, the transfer ratio and the optical path length [24]. An

extended path capillary (bubble factor = 3) was employed in the

method to maximize the detection sensitivities.

The selection of the visualizing reagent and detection

wavelength are the key parameters to obtain the highest sensitivities

in indirect photometric detection. The pH of the background

electrolyte plays a very vital role in ionizing the visualizing reagent and

the target analytes, thus controlling their electrophoretic mobilities

and selectivity of the method. In CE, the Kohlraush regulating

function determines the probe displacement by the analyte [25] and

the electrophoretic mobility of the probe ions relative to the analyte

ions influence the peak shapes due to electromigration dispersions.

BGEs containing visualizing reagents pyridine, imidazole,

benzylamine and 4-aminopyridine each at four different

concentrations namely, 2.5, 5, 7.5 and 10 mM were evaluated for

obtaining optimum separation between the analytes and sensitivity.

The structures of the visualizing reagents are provided in Fig. 3.2.

N Pyridine

NH

N

Imidazole

N

NH2

4-Aminopyridine

NH2

Benzylamine

Fig-3.2: Structures of cationic visualizing reagents

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The order of mobilities of the visualizing reagents being

evaluated was: pyridine < benzylamine < 4-aminopyridine < Imidazole.

The high electrophoretic mobility of imidazole resembles more closely

to that of the metal cations and for this reason imidazole has been the

most efficient probe for the analysis of alkali and alkaline earth metals

[26]. The molar absorptivities of the visualizing reagents at the most

appropriate wavelengths were found to be in the order pyridine <

benzylamine < imidazole < 4-aminopyridine. In accordance, the

analyte response obtained with benzylamine and pyridine was less

when compared to the other probes. Interestingly, when imidazole was

being evaluated as a suitable probe, the addition of a 2 mM citrate

counter ion to the BGE significantly improved the separation between

the late migrating diisopropyl amine, triethanol amine and diisoproyl

ethylamine. However the addition of citrate buffer to other visualizing

reagents such as 4-aminopyridine did not provide any better

separation than the BGE devoid of citrate counter ion. The pH of the

BGE played a critical role in separating the analytes especially the late

eluting amines. The separation was evaluated at pH 4.2, 5.1, 5.5 and

6.0 and the best separation was obtained at a pH of 5.5.

Maximum sensitivities for the analytes were obtained using 4-

aminopyridine as the visualizing reagent and the response was nearly

doubled when compared with other probes which can be attribute to

the high molar absorptivity of 4-aminopyridine. Lower concentrations

of the visualizing reagents (2.5 mM) resulted in broad peaks due to

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electromigration dispersion and a diminished resolution. On the other

hand higher concentrations resulted in higher noise levels (10 mM),

decreased sensitivity and an adverse effect on the linearity of detection

[27]. Representative electropherograms with 4-aminobenzoic acid

probe at 2.5 and 10 mM concentrations are presented in Fig. 3.3 and

Fig. 3.4 respectively.

min1 2 3 4 5

mAU

-5

0

5

10

15

1 2

3 4

56

7

8

9

10

Fig. 3.3: Effect of probe’s concentration (2.5 mM 4-aminopyridine)

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min1 2 3 4 5 6 7 8 9

mAU

-30

-25

-20

-15

-10

-5

0

1

2

3

4

5 6

7

8

9

10

Fig. 3.4: Effect of probe’s concentration (10 mM 4-aminopyridine)

1. Ethylene diamine, 2.Hydrazine, 3. Methyl hydrazine, 4. Ethylamine, 5. Isopropylamine, 6. n-Butyl amine, 7. Triethylamine, 8.Diisopropylamine, 9. Triethanol amine, 10.Di isopropyl ethylamine

Good peak symmetries, high sensitivity and optimum resolution

between the analytes could be achieved using 5 mM 4-aminopyridine

in the BGE system at a pH of 5.5 which was chosen as final

conditions. The sample and reference wavelengths were optimized to

achieve the highest sensitivity and minimum baseline noise. Based

on the visualizing reagent’s absorption profile, different combinations

of sample wavelength and reference wavelength were evaluated with

the features of Diode Array Detector (DAD). The method was found to

be most sensitive when the signals were acquired at a sample

wavelength of 350nm (bandwidth 80 nm) using a reference wavelength

of 264nm (bandwidth 20 nm).

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The sampling rate of the detector (measured in hertz) i.e. the

number of data points acquired by the detector per second, plays an

important role in both the sensitivity and the resolution of the analyte

peaks. The effects are more prominent when the peaks are sharp and

narrow, as is the case of peaks obtained in CE and GC. The impact of

sampling rate (as peak width) on the peak height (Table 3.2) and

baseline noise (Table-3.3) was evaluated taking the hydrazine peak as

the model peak. The graphical representation of sampling rate effects

on peak height and peak width are presented in Fig. 3.4 and Fig. 3.5

respectively.

Table-3.1: Effect of sampling rate on peak height

Sampling rate (hertz)

Peak width (min.)

Hydrazine Peak Height Mean

height Inj-1 Inj-2

200 0.005 12.68066 12.75487 12.71777

100 0.01 14.74398 12.80253 13.77326

33 0.03 13.1378 13.36097 13.24939

20 0.05 14.11922 13.69552 13.90737

10 0.1 9.10089 9.19478 9.147835

5.0 0.2 8.24145 6.272212 7.256831

2.5 0.4 5.26139 4.66382 4.962605

~1.2 0.85 2.69857 2.37105 2.53481

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Fig. 3.5: Effect of sampling rate on peak height

Table 3.2: Effect of sampling rate on baseline noise

Sampling rate

(hertz) Peak width

(min.) Baseline Noise Mean

Noise Noise Inj-1 Noise Inj-2

200 0.005 0.5140 0.4642 0.4891

100 0.01 0.3461 0.4309 0.3885

33 0.03 0.2405 0.2271 0.2338

20 0.05 0.1503 0.1290 0.1397

10 0.1 0.1815 0.1260 0.1538

5.0 0.2 0.1831 0.1189 0.1510

2.5 0.4 0.1050 0.1146 0.1098

~1.2 0.85 0.0373 0.0441 0.0407

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Fig. 3.6 Effect of sampling rate on noise

From the results presented above, it can be concluded that the

peak height and hence the sensitivity is almost constant when the

sampling rate is in the range of 200 to 20 hertz. The peak height is

drastically decreased when the sampling rate is further reduced.

However from the noise curve (Fig. 3.3), it can be deduced that the

baseline noise decreases significantly when the sampling rate is

decreased from 200 hertz to 20 hertz. There was no further decrease

in the noise levels when the sampling rate was further reduced. Since

the highest sensitivity is achieved with the maximum peak height and

minimum baseline noise, the sampling rate was chosen as 20 hertz.

The separation was evaluated at 20, 25 and 30 kV and the

migration times decreased with the increase in the applied voltage.

The highest available voltage on the equipment (30 kV) was chosen as

it provided highest theoretical plates and resolution since the

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separation proceeds rapidly minimizing the effects of diffusion and

peak broadening.

3.3.2 Method Validation

Specificity

Specificity is the ability of the method to measure the analyte

response in the presence of potential impurities and interferences. No

interferences were observed at the migration times of the analytes of

interest in the spiked API samples. A specimen electropherogram of

the spiked standards is presented in Fig. 3.6.

min2 2.5 3 3.5 4 4.5 5 5.5 6 6.5

mAU

-30

-25

-20

-15

-10

-5

0

5

1 2 3

4

5 6

7

8

9

10

Fig. 3.7: Electropherogram of spiked standards in optimized method

1. Ethylene diamine, 2.Hydrazine, 3. Methyl hydrazine, 4. Ethylamine,

5. Isopropylamine, 6. n-Butyl amine, 7. Triethylamine,

8.Diisopropylamine, 9. Triethanol amine, 10.Di isopropyl ethylamine

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Precision

The repeatability and inter-day precision of the method was

determined in terms of %RSD for migration times and peak area

response of the analytes. The overall RSD of migration times and peak

areas was not more than 2.1% and not more than 3.4% respectively

for any of the analyte confirming the precision of the method.

Detection and quantitation limits

The quantitation and detection limits of the analytes are presented in

Table-3.4 and the corresponding electropherograms are presented in

Fig. 3.7 and 3.8 respectively. The results emphasize that the method

is sensitive enough for determining hydrazine, methylhydrazine at

ppm levels in real time samples considering the permissible levels of

these toxic impurities. Trace levels of the alkylamines can also be

determined using this method exemplified by the low detection and

quantitation limits.

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min1 2 3 4 5 6 7

mAU

-14

-12

-10

-8

-6

-4

-2

0

1

2

34 5

67

89

10

Fig. 3.8 Electropherogram of Analytes at Limit of Quantification

min1 2 3 4 5 6 7

mAU

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

1 2 34

56

7

8

910

Fig. 3.9 Electropherogram of Analytes at Limit of Detection

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Linearity and range

The detector response linearity to varying analyte concentrations was

established by analyzing standard solutions at seven different

concentrations ranging from LOQ to 300% of nominal analyte

concentration. Linearity curves (Area vs Conc.) were plotted for each

analyte and the data was subjected to regression analysis. The

linearity experiments conclude that the peak responses are directly

proportional to their concentrations. The linearity results are

presented in Table-3.3. The range of a method is the interval in which

it has a suitable level of precision, accuracy and linearity. From the

results of validation tests that were performed, the range for this

method was LOQ to 200% of the nominal analyte concentration.

Table-3.3 Linearity and Sensitivity Results of Hydrazines and

Alkylamines

Analyte Equation R2 LOQ

(µg/mL)

LOD (µg/mL)

Hydrazine y = 16.5739x – 0.3257 0.9989 0.7 0.2

Methylhydrazine y = 16.3909x – 1.8519 0.9985 0.6 0.2

Ethylamine y = 16.8983x + 0.1676 0.9965 0.4 0.1

Triethylamine y = 10.6926x + 0.7780 0.9993 0.7 0.2

Ethylenediamine y = 15.5253x – 1.2747 0.9939 0.8 0.3

Triethanolamine y = 10.2680x – 0.0942 0.9998 1.0 0.3

Isopropylamine y = 21.3953x + 0.4730 0.9999 0.3 0.1

Diisopropylamine y = 10.8095x + 1.1990 0.9984 0.7 0.1

N,N-diisopropyl ethylamine y = 6.3849x + 1.3981 0.9910 1.0 0.3

n-butylamine y = 12.6857x + 0.1630 0.9974 0.5 0.2

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Accuracy

The accuracy of a method expresses the closeness between the

theoretical value and the determined value. The accuracy results

calculated as mean recoveries for hydrazine, methyl hydrazine and the

alkyl amines at LOQ level, 50, 100 and 200% of the nominal analyte

concentrations are presented in Table-3.4. These findings suggest that

the described method represents a valuable and accurate tool for the

analysis of hydrazine, methyl hydrazine and alkyl amines in

pharmaceutical drug substances.

To separate out the systematic variability due to the sample

preparation, injection and integration and demonstrate linear

accuracy, a one-way hierarchical analysis of variation (ANOVA) within

the range was performed. The recoveries were found to be independent

of the spiked concentrations for all the analytes (ANOVA, p> 0.05).

Table-3.4 Accuracy results of Hydrazines and Alkyl amines

Analyte % Recovery ± Std. dev.

LOQ 50% 100% 200%

Hydrazine 94.6 ± 2.6 98.0 ± 1.8 96.8 ± 1.7 96.7 ± 2.1

Methylhydrazine 92.2 ± 1.9 98.3 ± 2.0 94.5 ± 2.3 95.5 ± 2.5

Ethylamine 94.9 ± 1.3 100.2 ± 1.4 99.8 ± 1.4 98.6 ± 2.7

Triethylamine 97.5 ± 2.3 97.5 ± 2.6 96.6 ± 2.1 99.8 ± 0.7

Ethylenediamine 96.8 ± 1.8 95.8 ± 1.8 99.6 ± 1.9 98.3 ± 3.1

Triethanolamine 97.4 ± 2.5 97.7 ± 1.0 99.5 ± 1.1 99.5 ± 1.4

Isopropylamine 101.6 ± 2.2 102.9 ± 1.7 100.6 ± 2.6 96.8 ± 1.0

Diisopropylamine 93.4 ± 2.2 96.3 ± 1.4 94.4 ± 2.5 96.1 ± 3.1

N,N-diisopropyl ethylamine 102.8 ± 2.1 98.9 ± 1.9 103.6 ± 0.8 100.5 ± 2.9

N-butylamine 93.0 ± 1.6 96.4 ± 2.9 100.1 ± 2.5 95.3 ± 1.8

* n = 3 determinations

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3.2.3 Real time sample analysis

The developed method was applied for determining hydrazine and

several aliphatic alkylamines used in the manufacturing process in

different APIs. The results of hydrazine content were successfully

defended to address regulatory queries. Similarly the method was

used for in-process and release control of alkylamines such as triethyl

amine in several other APIs.

3.4 Conclusion

A simple fast generic CE method was developed for determining the

genotoxic impurities hydrazine, methylhydrazine and aliphatic

alkylamines in drug substances through indirect photometric

detection. The method development involved a systematic selection of

the right visualizing reagent, the correct pH, detection and reference

wavelengths, sampling rate, capillary temperature, applied voltage and

the probe’s concentration. This method was validated in full terms to

comply with the needs for a routine application for estimating the

targeted analytes. The method was applied to demonstrate that the

two genotoxic impurities hydrazine and methyl hydrazine were within

the safe and acceptable levels in the drug substances to address

regulatory queries. The control of alkylamines such as triethyl amine,

diethyl amine, ethanol amine, isopropyl amine etc. in several other

drug substances was also shown through the carryover studies using

the same CE method.

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The most common technique for determining hydrazines and alkyl

amines is by Ion chromatography which is quite expensive and

requires frequent replacement of the ion-exchange columns. The CE

technique is a very cost-effective and reliable substitute and can be

effectively used for determining the hydrazines and alkyl amines.

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3.5 References

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