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A VALIDATED RP-HPLC METHOD FOR IMPURITY PROFILING OF SODIUM NITROPRUSSIDE IN
INJECTION DOSAGE FORM
Original Article
MURALI KRISHNAM RAJU P.a,b, VENKATA NARAYANA B.b, SHYAMALA P.a*, SRINIVASU KONDRAb, HSN RAJU
DANTULURIb
aDepartment of Physical, Nuclear Chemistry and Chemical Oceanography, School of Chemistry, Andhra University, Visakhapatnam
530003, Andhra Pradesh, India, b
Received: 25 Aug 2020, Revised and Accepted: 08 Oct 2020
Aurobindo Pharma Limited, Bachupally, R. R District, Hyderabad 500090, Telangana, India
Email: shyamalapulipaka06@gmail.com
ABSTRACT
Objective: The main objective of this research work is to develop and validate a single reverse-phase high-performance liquid chromatography (RP-
HPLC) method. This method should be capable of quantifying all the known, as well as other possible degradation impurities of sodium
nitroprusside (SNP) in its injection formulation.
Methods:
Results: In the proposed method, SNP was eluted at 22.5 min. Nitrite, nitrate, and ferrocyanide were linear from 0.25 to 37 μg/ml, ferricyanide was
linear from 1.0 to 37 μg/ml, and SNP was linear from 0.75 to 37 μg/ml. The % RSD for six spiked samples (precision) was found to be less than 0.5
%. Accuracy was performed for known impurities from LOQ to 150 % for a 0.5 % specification level. The results were found to be in the acceptance
range of 90-110 %. The LOQ concentration of nitrite, nitrate, and ferrocyanide was 0.25 μg/ml each, LOQ of ferricyanide and SNP was found to be
1.0 μg/ml and 0.75 μg/ml, respectively. The SNP injection samples were exposed to different degradation conditions, and the results were found
specific in the proposed methodology.
Of all method development trails, we have observed better separations between known and degradation impurities in Inert sustain C18,
(250 x 4.6) mm, 5 µm column at 30 °C temperature. Isocratic elution was carried out by using pH 8.6 phosphate buffer and acetonitrile in the ratio of
65:35 %v/v with a flow rate of 0.8 ml/min. The detection was carried out at 220 nm, with an injection volume of 10 µl.
Conclusion: The proposed RP-HPLC method is specific, precise, accurate, linear, stable, and robust for quantification of known and other possible
degradation impurities in SNP injection formulation.
Keywords: RP-HPLC, Impurity profiling, Injection formulation, Sodium nitroprusside, Degradation
© 2021 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2021v13i1.39534. Journal homepage: https://innovareacademics.in/journals/index.php/ijap
INTRODUCTION
SNP is a sodium salt of di anionic metal complex dihydrate with
molecular formula Na2[Fe(CN) 5NO].2H2O. It is chemically named
as Ferrate (2-), pentakis(cyano-C)nitrosyl-, disodium, dihydrate [1,
2]. It is a potent rapid-acting hypotensive agent and when
administered intravenously. SNP is used to lower blood pressure
during surgical operations.
The literature survey of SNP revealed some of the reported
pharmacopeia methods for the estimation of ferricyanide and
ferrocyanide in SNP [7, 8]. The reported HPLC methods were found for
estimation of nitrates and nitrites [9, 10]. A HPLC method was proposed
for the estimation of ferrocyanide in food-grade salts [11]. A
spectrophotometric method was published for the estimation of SNP and
its photo degradants [12]. An assay by HPLC method was reported for
SNP [13], and an in vitro stability of SNP for intravenous administration
methodology was reported [14]. Based on the literature survey, it was
observed that most of the reported works concentrated on the assay of
SNP, the content of ferricyanide, and ferrocyanide in SNP. To date, no
analytical method was published to quantify all known and possible
degradation impurities of SNP simultaneously in the single liquid
chromatography (LC) method for injection formulation.
Continuous injection of SNP might cause
cyanide poisoning; therefore, intravenous administration rate (10
mcg/kg/min) is strictly controlled [3, 4]. However, there are several
problems associated with the clinical use of sodium nitroprusside,
including tolerance and the toxicity of its metabolites, cyanide, and
thiocyanate [5, 6].
Taking cues from literature, we have decided to develop and validate
a short RP-HPLC method for detecting all possible impurities of SNP
in injection formulation. During development, we have performed
several stress conditions such as hydrolysis, oxidation, photolysis
and thermal conditions in order to highlight the possible
degradation impurities in the proposed methodology [15-17]. The
developed method was checked for specificity, precision, linearity,
accuracy, robustness and stability of solutions based on ICH Q2 (R1)
[18]. Based on method development and validation results, the
proposed LC method could quantify all known and other possible
degradation impurities. The chemical structures of four known
impurities and SNP are given in fig. 1.
MATERIALS AND METHODS
Chemicals
SNP standard and injection formulation (labelled 25 mg/ml)
samples were provided by Aurobindo pharma research center-1,
Hyderabad, Telangana, India. Impurities, sodium nitrite USP
reference standard and sodium nitrate USP reference standards
were procured from the United States pharmacopeial convention,
USA. Potassium ferrocyanide trihydrate and potassium ferricyanide
procured from Sigma Aldrich, USA. Chemicals, such as Di-sodium
hydrogen phosphate (ACS), orthophosphoric acid (AR), sodium
hydroxide (AR), hydrogen peroxide (LR), and acetonitrile (HPLC)
were procured from Merck, India. HPLC grade water and
Hydrochloric acid (AR) were procured from Rankem, India. Tetra-n-
butylammonium hydroxide 40% solution in water was procured
from Alfa aesar, UK.
Instrumentation
This research work was carried out on Shimadzu prominence HPLC
(LC-20AD), consisting of a quaternary solvent manager and an
online degasser unit (DGU-20A5R). This instrument is equipped
with a photodiode array detector (SPD-M20A), it operates and data
processes with waters empower 3 software. Waters Alliance e2695
separations module HPLC with a quaternary solvent delivery system
and an online degasser unit. This instrument is equipped with a
photodiode array detector (SPD-M20A), it operates and data
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ISSN- 0975-7058 Vol 13, Issue 1, 2021
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Int J App Pharm, Vol 13, Issue 1, 2021, 160-169
161
processes with waters empower 3 software. Other instruments used
in this research include Sartorius make Microbalance (SE2),
Sartorius make Analytical balance (CPA225D), Eutech make pH
meter (pH 2700), Cintex make Water bath (CIC-2BM) and Borosil
make filtration kit equipped with Millipore 230V, 50HZ vacuum
pump.
Fig. 1: Chemical structures of ferricyanide, ferrocyanide, nitrate, nitrite and SNP
Chromatographic conditions
Optimum results were obtained in isocratic mode using a mobile
phase containing a degassed mixture of disodium hydrogen
phosphate dihydrate (25 mmol), 13 ml/l of Tetra-n-butylammonium
hydroxide 40% solution in water [19], pH adjusted to 8.6 with
orthophosphoric acid and acetonitrile in the ratio of 65:35 %v/v
[19]. Other chromatographic conditions include Column:
Inertsustain C18 (250×4.6 mm, 5 μm) (make: GL Sciences) [19],
Column temperature: 30 °C, Flow rate: 0.8 ml/min [20], Injection
volume: 10 µl [20] at Detector wavelength: 220 nm. The mobile
phase, standard, and sample solution were filtered through a 0.45
μm membrane filter before injecting it into the HPLC system.
Diluent preparation
Mobile phase was used as diluent.
Preparation of standard stock-1: (1000 µg/ml)
Accurately transferred 38 mg of
Preparation of standard stock-2: (1000 µg/ml)
sodium nitrite, 41 mg of
potassium nitrate, 50 mg of potassium ferrocyanide trihydrate and
25 mg of SNP standard into a 25 ml volumetric flask containing 10
ml of diluent, dissolved and made up to the mark with diluent,
mixed well.
Accurately transferred 39 mg of potassium ferricyanide standard in
to a 25 ml volumetric flask containing 10 ml of diluent, dissolved and
made up to the mark with diluent, mixed well.
Preparation of standard solution-1: (10 µg/ml)
Accurately transferred 1 ml of standard stock-1 into a 100 ml
volumetric flask, made up to the mark with diluent and mixed well.
Preparation of standard solution-2: (10 µg/ml)
Accurately transferred 1 ml of standard stock-2 into a 100 ml
volumetric flask, made up to mark with diluent and mixed well.
Preparation of sample solution: (5000 µg/ml)
Pooled sample was prepared by mixing the contents of 3 vials of SNP
injection formulation (fill volume: 2 ml each). Accurately transferred
2 ml of pooled sample into a 10 ml volumetric flask, made up to
mark with diluent and mixed well.
RESULTS AND DISCUSSION
Method development and optimization
According to available pharmacopoeia monographs, titrimetric
methods were used for the quantification of ferricyanide and
ferrocyanide in SNP. These methods are only specific for
quantification of ferricyanide and ferrocyanide, where other
impurities cannot be quantified [21, 22]. In some articles, authors
reported ferricyanide, ferrocyanide and other possible
photodegradation impurities by using spectrophotometry in SNP
[23, 24]. By some articles, authors reported A HPLC method for
determining nitrate and nitrite levels in vegetables [25]. Till now, no
reported article was published by addressing ferricyanide,
ferrocyanide, nitrate, nitrite and other possible degradation
impurities, likely to be formed during the product shelf life all
together in a single chromatographic method.
Taking cues from the available literature, we have taken it as a
challenge to develop a short, stable, sensitive, accurate and robust,
RP-HPLC method for detecting all possible impurities of the SNP
with sensitivity less than 1 µg/ml. The SNP and their impurities are
charged species and are highly polar. Hence conventional HPLC
mobile phases and columns could not hold the polar components in
reverse phase chromatography, as it leads to poor resolutions. To
get adequate resolutions between the negatively charged
components such as ferricyanide, ferrocyanide and SNP in an
octadecylsilane bonded column (C18), basic ion pain reagent like
tetrabutylammonium hydroxide (TBAH) was introduced into the
mobile phase [26].
Along with TBAH, other basic ion-pair reagents are also available in
the market, include tetraethylammonium hydroxide (TEAH) and
tetramethylammonium hydroxide (TMAH) etc. As TBAH is available
at our research laboratory, the same is used for the preparation of
the mobile phase to hold the negatively charged species in the
column. TBAH in the mobile phase could hold the negatively charged
species by decreasing the charge to mass ratio. Due to the presence
of basic ion pair reagent in the mobile phase, the hydrophobic
property of negatively charged species increases, resulting in longer
retention times. Based on the literature, the mobile backup phase
was prepared at basic pH by using TBAH [26-28]. The development
work was carried out in columns which could withstand basic
mobile phase pH such as Gemini C18 (250×4.6 mm, 5 μm) mak e:
Phenomenex, Inertsustain C18 (250×4.6 mm, 5 μm) (mak e: GL
Shyamala et al.
Int J App Pharm, Vol 13, Issue 1, 2021, 160-169
162
Sciences), X-Bridge C18 (250×4.6 mm, 5 μm) (Make: Waters), Zorbax
extend C18 (250 ×4.6 mm, 5 μm) (mak e: Agilent). Of all the
mentioned columns, the optimum separation between the
impurities was achieved in the Inertsustain C18 column with
dimensions (250×4.6 mm, 5 μm) (mak e: GL Sciences). Along with
columns, different mobile phase pH, compositions, column
temperatures and flow rates were also evaluated. Some of the
method development trials for mobile phase composition, pH and
column oven temperature by keeping other chromatographic
conditions constant were given in table 1.
Table 1: Method development trials to improve the resolution between SNP related impurities
Buffer pH
Composition*
Temp** ( °C)
Observation
6.0
58:42
40 °C
Poor resolution between ferricyanide and SNP
7.1
70:30
40 °C
Poor resolution between ferrocyanide and unknown impurity.
8.0
62:38
40 °C
Resolution between ferricyanide and SNP was not satisfactory.
8.0
65:35
40 °C
Resolution between ferricyanide and SNP was not satisfactory.
8.5
58:42
35 °C
Resolution between ferrocyanide and unknown impurity was not satisfactory.
8.5
58:42
40 °C
Resolution between ferrocyanide and unknown impurity was not satisfactory.
8.5
62:38
35 °C
Resolution between ferricyanide and SNP was not satisfactory.
8.5
60:40
35 °C
Resolution between ferrocyanide and unknown impurity was not satisfactory.
8.5
60:40
40 °C
Resolution between ferrocyanide and unknown impurity was not satisfactory.
8.6
65:35
30 °C
Peak shapes and resolutions between all degradants found satisfactory.
8.8
65:35
30 °C
Peak shapes and resolutions between all degradants found satisfactory.
*Mobile phase composition (Buffer: Acetonitrile) % v/v, **Column oven temperature
Response factor calculation for both ferricyanide and
ferrocyanide
The percentage of known impurities were calculated against their
corresponding standard solutions. Out of four known impurities,
ferrocyanide and ferricyanide are readily interconvertible [Fe
(CN)6]3−+e− ⇌ Fe (CN)6]4− [29]. During the stress study, it was
observed that the presence of peroxides in diluent is facilitating the
conversion of ferricyanide to ferrocyanide. Ferricyanide and
ferrocyanide redox reaction could be controlled by purging the diluent
with inert gas (Nitrogen/Helium) from the bottom of the flask to
remove the residual oxygen. Even after taking such precautions,
ferricyanide is slightly converting to ferrocyanide based on time. In
addressing the issue of quantifying the known impurities and
degradation impurities, two different standards were proposed. The first
standard consists of nitrite, nitrate, ferrocyanide and SNP, whereas the
second standard consists of ferricyanide. Actual areas of ferrocyanide
and ferricyanide from both the standards even after slight
interconversion were determined by using the response factor (RF).
Ferricyanide easily converts to ferrocyanide in the presence of
peroxides. Hence, RF for ferrocyanide and ferricyanide redox
conversion was determined using area lost to the area gain by using
2% hydrogen peroxide to facilitate the redox reaction. RF
determination for ferrocyanide and ferricyanide redox conversion
was given in table 2.
Table 2: Determination of response factor for redox couple by using 2% hydrogen peroxide
Degradation
time*
Area from peroxide stress sample
(h)
Response factor (RF)
Ferricyanide
Ferrocyanide
Area of ferricyanide
lost (A)
Area of ferrocyanide
gained (B)
Ferricyanide RF
(B/A)
Ferrocyanide RF
(A/B)
0
5509424
458792
NA
NA
NA
NA
1
5468329
595608
41095
136816
3.33
0.30
2
5417119
777561
92305
318769
3.45
0.29
5
4987023
2065594
522401
1606802
3.08
0.33
8
4392412
4234070
1117012
3775278
3.38
0.30
12
3976131
5566206
1533293
5107414
3.33
0.30
Average response factor
3.3
0.3
*Ferricyanide degradation time after addition of 2% hydrogen peroxide, Actual area of ferrocyanide and ferricyanide from both the standard
solutions were calculated with the help of RF. The details of the calculation were given in table 3.
Table 3: Area calculations
Calculation for determination of actual ferrocyanide area from standard-1
A
B
1
RF
1
1
C
(3.3)
1 = B 1 x RF
C
1
1+A1
Ferrocyanide
area from standard-1
Ferricyanide area
from standard-1
Response factor [To convert area of
ferricyanide (B1) to ferrocyanide (C1
Area of
ferrocyanide lost
)
Actual area of ferrocyanide
from standard-1
Calculation for determination of actual ferricyanide area from standard-2
A
B
2
RF
2
2
C
(0.3)
2 = B2 x RF
C
2
2+A2
Ferricyanide
area from standard-2
Ferrocyanide area
from standard-2
Response factor (To convert area of
ferrocyanide (B2) to ferricyanide (C2
Area of ferricyanide
lost
)
Actual area of ferricyanide
from standard-2
System suitability
The finalized related substances method was capable of quantifying
all known and other possible degradation impurities in SNP
injection formulation. As a part of the system suitability, two
different standard solutions are adopted to find the exact areas of
ferrocyanide and ferricyanide.
Standard-1 contains 10 µg/ml of each nitrite, nitrate,
ferrocyanide and SNP and Standard-2 contains 10 µg/ml of
ferricyanide. Chromatograms of both standards were given in fig.
2 and 3.
System suitability results were calculated from both the standards to
check the ability of the analytical instrument and method. The system
suitability results from both the standards were given in table 4.
System suitability results show the peak asymmetry, USP plate
count, resolution between the closely eluting impurities nitrite and
nitrate are meeting the acceptance criteria.
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Int J App Pharm, Vol 13, Issue 1, 2021, 160-169
163
Fig. 2: Representative chromatogram of standard-1 containing nitrite, nitrate, ferrocyanide and SNP
Fig. 3: Representative chromatogram of standard-2 containing ferricyanide and its redox counterpart ferrocyanide
Table 4: System suitability results obtained from both standard-1 and 2
S. No.
Components
RT*
RRT**
Area
Resolution
Peak asymmetry
#
USP plate count
1
Nitrite
3.92
0.17
598085
---
1.2
11172
2
Nitrate
4.71
0.21
414971
5.0
1.2
13163
3
Ferrocyanide
8.76
0.39
668677
---
1.1
9739
4
Ferricyanide
18.74
0.83
172022
---
1.2
15500
5
SNP
22.49
1.00
237932
---
1.1
18059
*Retention time (min), **Relative retention time (RT of impurity/RT of SNP), #
Resolution between closely eluting impurities nitrite and nitrate from
standard-1
Method validation
The proposed LC method for quantification of known and other
possible degradation impurities obtained during stress studies of
SNP was validated as per ICH guidelines Q2 (R1).
Specificity
Specificity indicates the analytical method’s ability to distinguish
accurately and specifically each analyte of interest in the presence of
other components such as blank, placebo matrix and other possible
impurity peaks related to SNP.
The specificity experiment was conducted by injecting blank,
individual impurities, and known impurity spiked samples in LC
equipped with a photodiode array (PDA) detector to check the peak
purity. The SNP injection formulation does not have any placebo
matrix; hence placebo is not injected. Peak purity was calculated for
known impurities and SNP from the spiked sample, and the data
were given in table 5. Typical chromatograms of the blank, control
sample and spiked sample were given in fig. 4-6
Selectivity
Selectivity indicates the ability of an analytical method to distinguish
all possible degradation impurities that are going to be generated in
any sample over a period of time. To make sure the method
selectivity SNP injection was exposed to different stress conditions
such as hydrolysis, thermal, oxidation and photolytic [30-32].
Degraded samples were chromatographed by using HPLC equipped
with PDA detector to establish method selectivity. Chromatograms
of these stress conditions were given in fig. 7-11. From these stress
conditions, peak purity of the SNP was measured to make sure the
co-elution of any degradation impurity. Peak purity results of the
SNP from degraded samples were given in table 6.
Acid degradation
Into a 10 ml volumetric flask containing 2 ml of SNP injection, 1 ml of 1
mol hydrochloric acid was added and the solution was kept at room
temperature for 3 h. The degraded sample was neutralized with 1 ml of
1 mol sodium hydroxide, made up to 10 ml mark with diluent and
chromatographed. The resultant chromatogram was given in fig. 7.
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Int J App Pharm, Vol 13, Issue 1, 2021, 160-169
164
Fig. 4: Blank chromatogram
Fig. 5: Control sample chromatogram
Fig. 6: Chromatogram of spiked sample with known impurities at 0.5% level (25 µg/ml)
Table 5: Peak purity results of known impurities and SNP from spiked sample
Component
RT
RRT
Peak purity*
Purity angle
Purity threshold
Nitrite
3.915
0.19
0.069
0.246
Nitrate
4.689
0.23
0.051
0.265
Ferrocyanide
8.701
0.43
0.055
0.243
Ferricyanide
18.417
0.91
0.228
0.478
SNP
20.268
1.00
0.069
0.292
*To consider any peak is pure, purity angle should be less than purity threshold
Base degradation
Into a 10 ml volumetric flask containing 2 ml of SNP injection, 1 ml of 1
mol sodium hydroxide was added and the solution was kept at room
temperature for 1 h. The degraded sample was neutralized with 1 ml
of 1 mol hydrochloric acid, made up to 10 ml mark with diluent and
chromatographed. The resultant chromatogram was given in fig. 8.
Oxidative degradation
Into a 10 ml volumetric flask containing 2 ml of SNP injection, 1 ml
of 30% hydrogen peroxide was added and the solution was kept at
room temperature for 2 h. The degraded sample was made up to 10
ml mark with diluent and chromatographed. The resultant
chromatogram is given in fig. 9.
Thermal degradation
The SNP injection sample is kept in a hot air oven at 85 °C for 72 h.
After attaining the room, temperature sample was prepared as per
the test method by taking 2 ml of thermal degradation sample and
chromatographed. The resultant chromatogram is given in fig. 10.
Photolytic degradation
The SNP injection sample was kept in the photolytic chamber and
exposed to white fluorescent light, 1.2 million Lux hours and UV
light 200-watt hours/square meter. After attaining room,
temperature the sample was prepared as per the test method by
taking 2 ml of photolytic degradation sample and chromatographed.
The resultant chromatogram was given in fig. 11.
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Int J App Pharm, Vol 13, Issue 1, 2021, 160-169
165
Fig. 7: Acid degradation chromatogram of SNP injection formulation
Fig. 8: Base degradation chromatogram of SNP injection formulation
Fig. 9: Oxidative degradation chromatogram of SNP injection formulation
Fig. 10: Thermal degradation chromatogram of SNP injection formulation
Fig. 11: Photolytic degradation chromatogram of SNP injection formulation
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166
Table 6: Peak purity results of SNP from degradation samples
Degradation
Area of SNP
% of degradation
Peak purity of SNP
Purity angle
Purity threshold
Control sample
35895014
NA
0.106
0.303
Acid degradation
35932047
Nil
0.022
0.303
Base degradation
30010842
16.4
0.027
0.284
Peroxide degradation
35471819
1.2
0.027
0.314
Thermal degradation
34400144
4.2
0.105
0.393
Photolytic degradation
32324418
10.5
0.075
0.282
Precision
The precision of an analytical method expresses the closeness of
agreement between a series of injections prepared from a single
homogenized stock solution. It indicates the reproducible capability
of an analytical method.
Method precision was evaluated by preparing six different spiked
samples on the same day, and intermediate precision was evaluated by
preparing six different spiked samples on a different day. Each precision
sample was prepared by spiking known impurities at a 0.5% level to SNP
injection formulation and chromatographed as per the proposed
method. The obtained precision results were given in table 7.
Table 7: Results from both method precision and intermediate precision.
Preparation
Nitrite
Nitrate
Ferrocyanide
Ferricyanide
Method precision (n=6)*
Mean (%w/v)
0.505
0.493
0.495
0.525
SD
0.0008
0.0008
0.0015
0.0008
%RSD
0.2
0.2
0.4
0.2
Intermediate precision (n=6)*
Mean (%w/v)
0.495
0.491
0.493
0.512
SD
0.0021
0.0025
0.0021
0.0015
%RSD
0.04
0.4
0.4
0.2
Cumulative precision (n=12)
Mean (%w/v)
#
0.500
0.492
0.494
0.519
SD
0.0056
0.0020
0.0023
0.0069
%RSD
1.1
0.4
0.5
1.3
*Number of experiments done (n):6, #
Number of experiments done (n):12 (cumulative results of both precision and intermediate precision)
Linearity and sensitivity
The linearity of an analytical method indicates its ability to obtain
test results which are directly proportional to the concentration in a
specific range. Linearity was performed for all four known
impurities and SNP from 0.05 µg/ml to 38 µg/ml. Linearity solutions
were evaluated by linear regression analysis and calculated by the
least square method and studied. Limit of detection (LOD) and Limit
of quantification (LOQ) results for four known impurities and the
SNP were calculated from linearity solutions by using the slope of
the calibration curve. Based on the sensitivity results in six different
preparations of known impurities and SNP were prepared at their
LOD and LOQ to check the precision. Precision results at their lowest
detection and quantification level were given in table 8.
LOD = 3.3 x Standard deviation of the response/Slope of the
calibration curve
LOQ = 10 x Standard deviation of the response/Slope of the
calibration curve
Table 8: Linearity and sensitivity results of known impurities and SNP
Parameter
Nitrite
Nitrate
Ferrocyanide
Ferricyanide
SNP
Range (µg/ml)
0.25-37.68
0.25-37.53
0.25-37.50
1.00-37.74
0.75-37.27
Regression equation*
69738x+1266.5
50744x+2176.5
78543x-4930.4
23284x-5515.8
29661x-1238.2
Correlation**
0.9999
0.9999
0.9999
0.9999
0.9999
LOD (µg/ml)
0.084
0.083
0.083
0.332
0.248
LOQ (µg/ml)
0.253
0.252
0.252
1.005
0.752
LOD precision (n=6)#
Mean area
7132
4334
6150
6781
7424
%RSD
1.5
0.8
1.6
2.7
2.2
LOQ precision (n=6)
#
Mean area
18925
13423
19325
21378
22294
%RSD
0.4
0.1
0.8
0.7
1.2
*Y is the peak area and X is the concentration injected, **Correlation coefficient, #
Number of experiments done (n):6
Accuracy
The accuracy of an analytical method indicates the closeness of
agreement between an actual value and obtained value at a
particular range. Accuracy for all known impurities was evaluated at
levels LOQ, 50%, 100%, and 150% by spiking to the SNP injection
formulation. At each level, triplicate solutions were prepared by
spiking individual impurities from their respective stock solutions
and chromatographed. Accuracy results for known impurities were
given in table 9 and 10.
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167
Table 9: Accuracy results of known impurities at their minimum quantification level (LOQ)
Component
Amount added (%w/w)
Amount found (%w/w)
% recovery
Mean recovery
SD*
% RSD
n=3#
0.0051
0.0058
113.7
Nitrite
0.0051
0.0058
113.7
113.7
0.000
0.0
0.0051
0.0058
113.7
0.0051
0.0052
102.0
Nitrate
0.0051
0.0053
103.9
102.6
1.096
1.1
0.0051
0.0052
102.0
0.0051
0.0051
100.0
Ferrocyanide
0.0051
0.0050
98.0
98.7
1.154
1.1
0.0051
0.0050
98.0
0.0198
0.0199
100.5
Ferricyanide
0.0198
0.0197
99.5
100.2
0.577
0.6
0.0198
0.0199
100.5
*Standard deviation, #
Number of experiments done (n):3
Table 10: Accuracy results of known impurities
% of
specification
Amount added
(%w/w)
Amount found
(%w/w)
% of
recovery
% RSD±SD
% of recovery
% RSD±SD
n=3
n=9
#
#
Nitrite
50%
0.253
0.256
101.2
0.0±0.000
100%
0.506
0.505
99.7
0.1±0.115
100.5
0.6±0.638
150%
0.759
0.763
100.5
0.1±0.057
Nitrate
50%
0.251
0.251
100.0
0.0±0.000
100%
0.503
0.493
97.9
0.1±0.115
98.7
1.0±0.961
150%
0.754
0.741
98.3
0.2±0.173
Ferrocyanide
50%
0.251
0.249
99.2
0.0±0.000
100%
0.503
0.495
98.4
0.2±0.200
99.5
1.1±1.048
150%
0.754
0.760
100.8
0.1±0.058
Ferricyanide
50%
0.253
0.266
105.0
0.6±0.611
100%
0.506
0.525
103.7
0.1±0115
104.4
0.6±0.621
150%
0.758
0.792
104.5
0.1±0.058
#
Number of experiments done at each level (n):3, cumulative recovery for all three levels (n):9
Robustness
Robustness of the analytical method was evaluated by deliberately
altering some of the critical method parameters to check the method
capability on system suitability results and provides an indication of
its reliability during normal usage. Robustness was checked for flow
rate (±10%), column oven temperature (±5 °C), pH of the buffer
(±0.2 units), organic composition in the mobile phase (±2%
absolute), and wavelength (±5 nm). The obtained robustness results
were given in table 11 to 15.
Table 11: Robustness impact on nitrite peak from system suitability solution
Parameter
Variation
RRT
USP tailing
USP plate count
Control
-
0.19
1.2
11172
Flow rate (±10%)
0.72 ml/min
0.19
1.2
11686
0.88 ml/min
0.19
1.2
10654
Column oven temperature(±5 °C)
25 °C
0.18
1.2
10593
35 °C
0.21
1.2
11425
Buffer pH (±0.2 units)
8.4
0.18
1.2
11346
8.8
0.18
1.2
11397
Mobile phase composition (±2%)
67:33
0.15
1.2
11637
63:37
0.22
1.2
10726
Wave length (±5 nm)
215 nm
0.19
1.2
11168
225 nm
0.19
1.2
11185
Table 12: Robustness impact on nitrate peak from system suitability solution
Parameter
Variation
RRT
Resolution*
USP Tailing
USP plate count
Control
-
0.23
5.0
1.2
13163
Flow rate (±10%)
0.72 ml/min
0.23
5.0
1.2
13258
0.88 ml/min
0.23
4.8
1.2
12415
Column oven
temperature(±5 °C)
25 °C
0.21
5.2
1.2
11759
35 °C
0.25
4.6
1.2
12966
Buffer pH (±0.2 units)
8.4
0.22
4.9
1.2
10949
8.8
0.22
4.9
1.2
11513
Mobile phase composition
(±2%)
67:33
0.18
5.6
1.2
13876
63:37
0.26
4.1
1.3
7933
Wave length (±5 nm)
215 nm
0.23
4.9
1.2
13094
225 nm
0.23
4.9
1.2
13151
*Resolution between nitrite and nitrate
Shyamala et al.
Int J App Pharm, Vol 13, Issue 1, 2021, 160-169
168
Table 13: Robustness impact on ferrocyanide peak from system suitability solution
Parameter
Variation
RRT
USP Tailing
USP plate count
Control
-
0.43
1.1
9739
Flow rate (±10%)
0.72 ml/min
0.43
1.1
10401
0.88 ml/min
0.43
1.1
9136
Column oven temperature(±5 °C)
25 °C
0.39
1.1
8977
35 °C
0.47
1.1
10392
Buffer pH (±0.2 units)
8.4
0.43
1.1
10122
8.8
0.43
1.1
9972
Mobile phase composition (±2%)
67:33
0.42
1.1
10980
63:37
0.44
1.1
9105
Wave length (±5 nm)
215 nm
0.43
1.1
9667
225 nm
0.43
1.1
9761
Table 14: Robustness impact on ferricyanide peak from system suitability solution
Parameter
Variation
RRT
USP Tailing
USP plate count
Control
-
0.91
0.2
15500
Flow rate (±10%)
0.72 ml/min
0.91
0.2
15611
0.88 ml/min
0.91
0.2
14532
Column oven temperature (±5 °C)
25 °C
0.88
0.2
14482
35 °C
0.93
0.2
15477
Buffer pH (±0.2 units)
8.4
0.93
0.2
14932
8.8
0.92
0.2
15568
Mobile phase composition (±2%)
67:33
0.94
0.2
15664
63:37
0.91
0.2
14484
Wave length (±5 nm)
215 nm
0.91
0.2
15060
225 nm
0.91
0.2
15048
Table 15: Robustness impact on SNP peak from system suitability solution
Parameter
Variation
RRT
USP Tailing
USP plate count
Control
-
1.0
1.1
18059
Flow rate (±10%)
0.72 ml/min
1.0
1.2
18262
0.88 ml/min
1.0
1.2
17037
Column oven temperature(±5 °C)
25 °C
1.0
1.2
16900
35 °C
1.0
1.1
18174
Buffer pH (±0.2 units)
8.4
1.0
1.1
17438
8.8
1.0
1.1
17433
Mobile phase composition (±2%)
67:33
1.0
1.2
17974
63:37
1.0
1.1
17589
Wave length (±5 nm)
215 nm
1.0
1.1
17358
225 nm
1.0
1.1
18311
Solution stability
Solution stability is necessary to assess the stability of standard and
sample solutions during product development and stability data
evaluation. Sample solution stability was evaluated by spiking all
known impurities on SNP injection formulation and injected at
different time intervals by keeping the solution at controlled room
temperature (~25 °C). Similarly, standard solutions were injected
periodically. The % difference in an area of each impurity and SNP
was calculated, and the results were given in table 16, 17.
Table 16: Standard solution stability results from standard-1 and 2
Component 0 h 2 h 6 h 12 h 24 h 48 h
Nitrite
NA
0.0
0.1
0.3
0.5
1.0
Nitrate
NA
0.4
0.1
0.1
0.6
0.9
Ferrocyanide
NA
0.1
0.3
0.3
0.6
0.2
Ferricyanide
NA
1.5
2.0
2.2
2.4
1.9
SNP
NA
0.4
0.2
0.6
1.0
1.7
Table 17: Spiked sample solution stability results
Component
0 h
2 h
6 h
12 h
24 h
48 h
Nitrite
NA
0.2
0.1
0.3
0.8
1.2
Nitrate
NA
0.2
0.1
0.3
0.9
1.4
Ferrocyanide
NA
0.6
1.2
1.6
1.3
1.3
Ferricyanide
NA
0.6
1.8
2.9
4.3
4.1
SNP
NA
0.1
0.1
0.1
0.7
1.0
Shyamala et al.
Int J App Pharm, Vol 13, Issue 1, 2021, 160-169
169
CONCLUSION
A simple RP-HPLC method for quantification of known impurities
and other possible degradation impurities in the SNP injectable
formulation was successfully developed and validated. The
proposed methodology is specific, precise, rugged, linear, accurate,
stable, and robust for the quantification of related impurities of SNP
injection. Moreover, the developed method has the lowest detectable
and quantification capability. The degradation data shows, the SNP
is sensitive to photolytic and base hydrolysis, moderately sensitive
to thermal oxidation, and stable in acid hydrolysis. The degradation
impurities formed during stress study are well resolved; hence any
degradation impurity formed during the product life cycle above the
identification threshold could be quantified. Based on the above
advantages, the proposed method could be useful for monitoring
possible degradation impurities of SNP in its injectable formulation
in quality control departments of manufacturing units.
ACKNOWLEDGEMENT
The authors are thankful to the management of the APL Research
Centre (A division of Aurobindo Pharma Limited, Hyderabad) for
providing chemicals, standards and all other essential facilities to do
the research work and the school of chemistry, Andhra University,
Visakhapatnam for their guidance.
FUNDING
Nil
AUTHORS CONTRIBUTIONS
Mr. Murali Krishnam Raju has generated the research activity and
prepared the manuscript. Dr. Venkata Narayana, Dr. Shyamala have
given guidance and supervision to carry out this research work. Mr.
Kondra Srinivasu and Mr. HSN Raju Dantuluri supported in
acquiring validation data and its compilation.
CONFLICT OF INTERESTS
The authors confirm that this article content has no conflict of
interest.
REFERENCES
1. Sodium nitroprusside, USP Monographs, official (USP42-NF37),
4045; 2020.
2. Sodium Nitroprusside-European Pharmacopoeia 10.3,
(01/2016:0565). Available from: https://
www.pharmacopoeia.com/bp-2020/monographs/sodium-
nitroprusside [Last accessed on 15 Jul 2020].
3. NITROPRESS®
4. Taylor HT, Styles M, Lamming JA. Sodium nitroprusside as a
hypotensive agent in general anesthesia. Br J Anuesth
1970;42:859-64.
(sodium nitroprusside injection) product
information leaflet, Hospira, Inc., Lake Forest, IL 60045 USA,
Document Name: QEN-3406v4. qxp, Reference ID: 3442071;
2013.
5. Moraca PP, Bitte ME, Hale ED, Wasmuth EC, Poutasse FE.
Clinical evaluation of sodium nitroprusside as a hypotensive
agent. Anesthesiology 1962;23:193-9.
6. Cottrell EJ, Casthely P, Brodie DJ, Patel K, Klein A, Turndorf H.
Prevention of Nitroprusside-induced cyanide toxicity with
hydroxocobalamin. N Engl J Med 1978;298:809-11.
7. Sodium Nitroprusside, USP Monographs, official (USP42-
NF37), 4045; 2020.
8. Sodium Nitroprusside-European Pharmacopoeia 10.3,
(01/2016:0565). Available from: https://www.
pharmacopoeia.com/bp-2020/monographs/sodium-
nitroprusside [Last accessed on 15 Jul 2020].
9. Gerritse GR. Rapid simultaneous determination of nitrate and
nitrite by high-performance liquid chromatography using
ultraviolet detection. J Chromatogr A 1979;171:527-9.
10. Chou SS, Chung JC, Hwang DF. A High-performance liquid
chromatography method for determining nitrate and nitrite
levels in vegetables. J Food Drug Anal 2003;11:233-8.
11. Lim HS, Hwang JY, Choi EA, Lee G, Yoon SS, Kim MK.
Development and validation of HPLC method for the
determination of ferrocyanide ion in food-grade salts. Food
Chem 2018;239:
12. Frank JM, Johnsonx BJ, Rubin HS. Spectrophotometric
determination of sodium nitroprusside and its
photodegradation products. J Pharm Sci 1976;65:45-8.
1167-74.
13. David MB, Marilyn DS, Nancy K, James EC. High-performance
liquid chromatographic determination of sodium
nitroprusside. J Chromatogr A 1981;212:339-46.
14. Mahony C, Brown EJ, Stargel WW, Verghese PC, Bjornsson DT.
In vitro stability of sodium nitroprusside solutions for
intravenous administration. J Pharm Sci 1984;73:838-9.
15. Iram F, Iram H, Iqbal A, Husain A. Forced degradation studies. J
Anal Pharm Res 2016;3:1-5.
16. Blessy M, Ruchi DP, Prajesh NP, Agrawal YK. Development of
forced degradation and stability indicating studies of drugs-a
review. J Pharm Anal 2014;4:159-65.
17. Reynolds WD, Facchine LK, Mullaney FJ. Available guidance and
best practices for conducting forced degradation
studies.
18. International Conference on Harmonization (ICH) Harmonized
Tripartite Guidelines, Validation of Analytical Procedures, Text,
and Methodology, Q2 (R1), Parent Guidelines on Methodology
Dated; 1996. p. 6.
Pharm Technol 2002;26:48-56.
19. Inert Search TM for LC, Inertsil ® Applications, Analysis of
Potassium ferricyanide and Potassium ferrocyanide, Data No.
LB018-0811; 2020.
20. Chou SS, Chung JC, Hwang DF. A high-performance liquid
chromatography method for determining nitrate and nitrite
levels in vegetables. J Food Drug Anal 2003;11:233-8.
21. Sodium nitroprusside, USP Monographs, official (USP42-NF37),
4045; 2020.
22. Sodium Nitroprusside-European Pharmacopoeia 10.3,
(01/2016:0565). Available from: https://www.
pharmacopoeia.com/bp-2020/monographs/sodium-
nitroprusside [Last accessed on 15 Jul 2020].
23. Loenen CVA, Kemper HW. Stability and degradation of sodium
nitroprusside. Pharm Weekbl 1979;1:424-36.
24. Frank JM, Johnsonx BJ, Rubin HS. Spectrophotometric
determination of sodium nitroprusside and its
photodegradation products. J Pharm Sci 1976;65:45-8.
25. Chou SS, Chung JC, Hwang DF. A high-performance liquid
chromatography method for determining nitrate and nitrite
levels in vegetables. J Food Drug Anal 2003;11:233-8.
26. David MB, Marilyn DS, Nancy K, James EC. High-performance
liquid chromatographic determination of sodium
nitroprusside. J Chromatogr A 1981;212:339-46.
27. InertSearch TM for LC, Inertsil ® Applications, Analysis of
Potassium ferricyanide and Potassium ferrocyanide, Data No.
LB018-0811. Ion pair reagents for HPLC (Article No: A1084E
20190913
28. ); 2020.
https://www.tcichemicals.com/assets/brochure-
pdfs/Brochure_A1084_E.pdf
29. Huang HT, Salter G, Kahn LS, Gindt MY. Redox titration of
ferricyanide to ferrocyanide with ascorbic acid: illustrating the
nernst equation and beer–lambert law. J Chem Educ
2007;84:1461-63.
[Last accessed on 15 Jul 2020]
30. Jahanvee KT, Chirag JP, Patel MM. RP-HPLC method
development and validation of macitentan with its known and
unknown degradation impurities in its tablet dosage form. Int J
Appl Pharm 2018;10:81-9.
31. Adison F, Sanjay Pai PN. A validated stability-indicating rp-hplc
method for estimation of tolfenamic acid in the presence of its
pharmacopoeial impurities. Int J Appl Pharm 2019;11:264-70.
32. International Conference on Harmonization (ICH), Stability
testing of new drug substances and products, Q1A (R2); 2003.
