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110
Chapter-3
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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Chapter-3
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Medicinal Products (CHMP) and a more recent draft guidance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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Chapter-3
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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Chapter-3
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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Chapter-3
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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Chapter-3
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
116
Chapter-3
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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Chapter-3
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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Chapter-3
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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Chapter-3
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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Chapter-3
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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Chapter-3
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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Chapter-3
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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Chapter-3
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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Chapter-3
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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Chapter-3
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).
126
Chapter-3
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
127
Chapter-3
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
128
Chapter-3
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
129
Chapter-3
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
130
Chapter-3
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.
131
Chapter-3
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
132
Chapter-3
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
133
Chapter-3
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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Chapter-3
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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Chapter-3
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.
136
Chapter-3
3.5 References
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Recommended