Research Journal of Chemistry and Environment___________________________________Vol.
26 (10) October (2022)
A highly sensitive and selective method for
the development and validation of potential genotoxic
impurity in Raltegravir by high-throughput
UPLC-MS/MS
Annapoorna Vadlamani1, Ravindhranath K.1,2* and Sreenivasa Rao B.3
1. Department of Engineering Chemistry, College of Engineering, Koneru Lakshmaiah Education Foundation, Vaddeswaram, AP, INDIA
2. Department of Chemistry, Koneru Lakshmaiah Education Foundation, Vaddeswaram, AP, INDIA
3. Department of Chemistry, GITAM Institute of Science, GITAM (Deemed to be University), Visakhapatnam - 530 045, AP, INDIA
*ravindhranath.kunta@gmail.com
Abstract
Raltegravir is a human immunodeficiency virus (HIV)
integrase inhibitor, a novel anti-AIDS drug. To
quantify genotoxic impurities (GTIs), conventional
analysis techniques are inadequate as a result, there is
a high need to develop critical analytical methods for
drug analysis using the hyphenated analytical
technique. A highly sensitive and selective method was
developed and validated for the estimation of genotoxic
impurity in Raltegravir Active pharmaceutical
ingredient (API) by using UPLC-MS/MS. The UPLC-
MS/MS analysis for the quantification of GTI was
performed on the symmetry C18 (150 mm×4.6
mm,3.5μm) analysis column and separation of
genotoxic impurity was achieved under a gradient
mode system using a mobile phase comprising 1%
acetic acid as mobile phase (A) and acetonitrile as
mobile phase (B) with a column temperature of 40°C
and flow rate of 0.5 mL/min. A triple mass quadrupole
detector was used to measure GTI by combining with
the negative-electrospray ionization used in multi-
response monitoring mode (MRM).
The newly developed method was validated as per ICH
guidelines and was able to measure GTI at 2.0 ppm for
about 10mg/mL solution of Raltegravir. The %RSD
obtained for GTI for six preparations was 1.74 in
method precision and 2.56 during the variation of
analyst, system and column on a different day. The
overall %RSD of 2.73 showed the ruggedness of the
method. The GTI is linear from 0.039 to 2.9ppm (LOQ
to 150%) and R= 0.9998 showed good linearity. The %
recovery obtained for genotoxic impurity was in the
range 103.0-109.6% with %RSD 1.51-3.19 from 0.42
to 2.5ppm. The %RSD in precision and accuracy at
LOQ is 1.63 and 3.19 indicating the sensitivity of the
method. The newly proposed and developed high-
sensitive method can be used to detect GTI in
Raltegravir API and formulation.
Keywords: Antiretroviral, Genotoxic Impurity, UPLC-
MS/MS, Validation, Negative ESI.
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Res. J. Chem. Environ.
Introduction
Raltegravir is an antiretroviral drug used to treat
AIDS/HIV11. It is human immunodeficiency virus-1 (HIV-1)
integrase inhibitor1,19,22 and a human immunodeficiency
enzyme that converts viral genetic material into the genetic
material of HIV-infected human cells. Glucuronidation is
the metabolic pathway of the drug16. Raltegravir is approved
by USFDA and it is sold with the brand name Isentress7 and
Isentress HD for the treatment of HIV infection in children
and adults15. It is available in chewable tablets (25 or
100mg), film coated tablet (400mg) administered orally once
or twice daily. The common side effects are allergic
reactions, skin reactions.
The IUPAC name of Raltegravir is 4-[(4-fluorophenyl)
methylcarbamoyl]-1-methyl-2-[2-[(5-methyl-1,3,4-oxadiaz
ole-2- carbonyl) amino]propan-2-yl]-6-oxopyrimidin-5-
olate. Raltegravir is member of monofluorobenzene,
secondary carboxamide, a primidone, 1,2,4-oxadiazole, a
dicarboxylic acid amide and a hydroxyl primidine.
Molecular formula is C20H21FN6O5.The molecular weight is
444.42gr/mol. The chemicals that cause harm to an organism
by damaging the genetic material (DNA) are called
genotoxic impurities (GTI) and result in causing cancer or
mutations.
The presence of these potential genotoxic impurities even at
a trace level may affect the safety and efficacy of
pharmaceutical products. In order to control these impurities,
different Pharmacopeias like British Pharmacopeia’s (BP),
United States Pharmacopeia’s (USP), Indian Pharmacopeia
(IP) and so on, have slowly incorporated allowable level of
limits in the APIs (Active pharmaceutical ingredients). The
chemical structure of Raltegravir and N’-{(1-Hydroxy-
ethylidene)-hydrazino}-oxo-acetic acid (GTI) shown in
figure 1a, b is the important key intermediate that is used in
Raltegravir synthesis, identified as GTI due to its
electrophilic functional group present in API and finished
pharmaceutical substances.
There are several analytical methods revealed in the
literature survey for determination of Raltegravir in the pure
drug, human plasma as a single analyte and in combination
with other drugs. There are few analytical methods reported
by using various analytical techniques such as
HPLC5,12,17,18,21,23,25 and LC-MS/MS4,6,9,10,13,14,20,24,26,27.
Research Journal of Chemistry and Environment___________________________________Vol. 26 (10) October (2022)
Figure 1a: Chemical structure of Raltegravir
Figure 1b: Chemical structure of GTI
However, the above-mentioned techniques are developed to
determine Raltegravir in pure drug form in combination in
solvent mixture or in biological fluids. There are some
drawbacks in the above methods because retention times can
vary and some methods are needed to characterize the
impurities online when the impurity standards are not
available.
Therefore, for accurate determination of GTIs at trace levels,
the above-mentioned techniques are inadequate and there is
a great need to develop better analytical methods for the
analysis of such GTIs in pharmaceutical industries. As a
result, various kinds of hyphenated chromatographic
techniques and methodologies have been explored as useful
approaches. Ideally, during initial development, many
conventional analytical instruments with various
combination of instruments and detector are used in the
pharmaceutical industry for the identification and
quantification such as spectrophotometer2, HPLC/UPLC3
with UV/IR/ fluorescence and GC with flame ionization
detector.
Finally, in the present study we made an attempt to develop
a simple method with a combination of UPLC-MS/MS
method for the quantification of GTI in Raltegravir at
specified levels. The developed method was verified and
validated as per ICH guidelines8 in terms of specificity,
precision, accuracy, limit of detection, the limit of
quantification, linearity, robustness and solution stability.
The above developed and validated method can be applied
to determine GTIs in the API and formulation batches of
Raltegravir.
Material and Methods
Chemicals and reagents: In the process of analysis, the
chemicals and solvents used were: Acetonitrile (HPLC-
Grade) and acetic acid (AR) procured from Merck (Mumbai,
India). References standard for GTI as well as Raltegravir
143
Res. J. Chem. Environ.
were obtained as a gift sample from Spectrum Pharma
Research Solutions. Pure Milli-Q water was procured from
Millipore Milli-Q plus purification system, ultra-wave
sonicator (Thermo lab).
Diluent Preparation and Mobile phase: A solution of
0.1% acetic acid was prepared in water and mixed with
acetonitrile in the ratio of 65:35 used as a diluent. Solutions
A (1% Acetic acid) and B (Acetonitrile) were used as mobile
phase.
Standard stock and sample solutions preparation: The
standard stock solution of 0.015 ppm GTI was prepared by
dissolving an accurate amount in the diluent. The standard
solution of Raltegravir of 7500 ppm was prepared as a stock
solution of GTI.
Accurately weighed drug samples of Raltegravir and diluent
were added. The sample solution was sonicated in an ultra-
wave sonicator for 5minutes and diluted to get a
concentration of 7500ppm. The sample solutions prepared
were transferred to a vial for analytical quantification. A
good peak shape and desired base line with signal-to-noise
ratio (S/N) was achieved by optimizing the standard and
sample concentration. The standards and sample were
filtered through 0.22-μm membrane filters before injecting
in the UPLC for the analysis.
Chromatographic conditions
LC Parameters: The chromatographic tests were
performed on the Acquity UPLC system (Waters H-Class)
integrated with MS-MS XEVO TQ-S, Waters-USA. The
column used for analysis is Symmetry C18 (150x4.6mm, 3.5
μm). The gradient mode of analysis used solutions A (1%
Acetic acid) and B (Acetonitrile) as a mobile phase with a
flow-rate of 0.5 mL/min. and the column temperature was
maintained at 40°C. The gradient mode of analysis was set
as follows: Time (Minutes) / % Mobile phase A: 0/55, 4/55,
4.2 / 14, 10.1 / 14, 10.2 / 55, 15/55. Injected 10 µL as
injection volume. All the solutions were filtered through a
0.22 µm PVDF filter before injecting into UPLC for
analysis.
Mass spectrometer: The mass system used was MS/MS
Waters Xevo TQ–S triple quadrupole mass spectrometer
with electrospray ionization (ESI) probe used with negative
polarity using software (MassLynx). Typical followed
operating conditions are: Desolvation Gas 1000 (L/hr),
Nebulizer gas 6.0 (bar), Cone gas 140 (L/hr), Capillary 2.5
(KV), Source temperature 1500C, Desolvation temperature
5000C, Dwell time 200 msec, Scan distance 50-1000 m/z.
MRM mode (Multiple Reaction Monitoring) was measured
to use the parent ion of GTI at 144.88 m/z and daughter ion
at 72.93 m/z respectively.
Results and Discussion
Sample solution optimization: In the trace level analysis,
preparation of sample solution is an important part of drug
Research Journal of Chemistry and Environment___________________________________Vol. 26 (10) October (2022)
analysis especially for impurity solution preparation because
of the matrix effects, causing analyte instability, sensitivity
loss and recovery abnormality. The chromatographic
efficiencies were tested by using different diluents.
Raltegravir and genotoxic impurity solubility were good in
the diluent mixture. A proper peak shape and good response
were obtained for the genotoxic impurity and Raltegravir
when diluted with the appropriate ratio of diluent. Sample
recoveries were also observed for GTI when diluted with
diluent. Therefore, the ratio of diluent mixture solution was
optimized and used throughout the analysis as diluent.
LC parameters optimization: The current analytical
method developed was done by examining various
stationary phases to get better separation of impurity and
Raltegravir peak. It is important to achieve the appropriate
separation between impurity and Raltegravir due to the
similarity in the chemical structure of GTI and Raltegravir.
For the selection, separation and optimizing analysis time,
various analysis columns such as Kromasil C-
18(150mm×4.6 mm, 3.5 µm), Hypersil BDS C-18(150 mm
x 4.6 mm, 3.5 µm), Zorbax C-18 (150 mm × 4.6 mm, 3.5
μm) and X-Bridge C-18 column (150mm × 4.6mm, 3.5μm)
columns were tested under similar conditions. The impurity
response was very less and the Raltegravir peak split was
observed in the Kromasil column; with Hypersil, both the
peaks were merged. The impurity peak was not eluted with
Zorbax while using ACE C-18 column S/N ratio of impurity
observed on the lower side.
In the symmetry C-18 column (150mm×4.6mm, 3.5μm), the
separation and impurity responses with Raltegravir were
found to be more with a good S/N ratio of impurity. In
symmetry column, both the analytes are maintained and
separated well from each other. This distinction was
achieved with the most basic analytes of silanol surface of
silica residual. This reduced the silanol function of
symmetry column greatly with high resolution and improved
shape. Triethylamine and diethylamine exhibited high
background noise. Different compositions of the mobile-
phase with acetic acid and acetonitrile were tested; finally,
positive differences and reactions were observed with
varying degrees of acetic acid-acetonitrile. Both, isocratic
mode and the gradient elution methods were tested.
Gradient method of elution was found to be very effective
while achieving the separation completely of GTI and
Raltegravir drug peak. The column is heated with a
Table 1
System suitability of Genotoxic impurity
Standard solution of GTI (n=6)
System suitability
(% RSD )
Precision 2.20
Recovery 2.84
Linearity 3.55
Solution stability 3.31
Robustness 3.72
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Res. J. Chem. Environ.
thermostat at 40°C. The RT of GTI and Raltegravir was
observed at 2.5 minutes and 7.5 minutes respectively.
MS-MS parameters optimization: The choice of selecting
method of detection for trace level of impurities is also a very
important step in the analysis of impurities in pharmaceutical
analysis. We tested using HPLC-UV. However, in these
strategies, sufficient sensitivity to GTIs and compliance
level analysis were not achieved. UPLC-MS-MS, specific
and sensitive with MRM mode was tested to evaluate the
GTI of Raltegravir drug substance. The electrospray-
ionization source (ESI) with negative ion mode of detection
was explored during initial development phase. The strength
of signal in the positive(+ve) mode was much lower than in
the negative(-ve) mode. In addition, development of method
was done with an ESI-source that operates in the negative
mode of polarity. The ion source parameters have been
developed in obtaining the correct and proper response. The
representative mass spectra of Raltegravir and GTI are
shown in figure 2a, b.
Method validation: The newly developed method was
demonstrated for its feasibility; validation was performed.
Specificity, precision, accuracy, LOQ, LOD, linearity and
stability of the solution were verified by following
guidelines as per FDA and ICH. System suitability details
are summarized in table 1.
Specificity: The specificity of the analytical method is
shown by the elution of any peak at the RT of Raltegravir
and genotoxic impurity. The UPLC chromatogram showed
very clearly that there is no elution of other peak at specified
RT. This indicates the method is highly specific in the
separation of the Raltegravir and the GTI shown in figure 3.
The retention time of GTI was 2.5 minutes and Raltegravir
was 7.5 minutes, both the peaks were well separated from
each other. Hence the method was specific.
Precision: The %RSD of the GTI peak area obtained for six
replicate injections of standard system suitability solution
was 1.55% (Table 2) indicating system suitability for
analysis. The six sample preparation of method precision
showed %RSD as1.74 and the developed method was
proven to be precise highly for the detection of impurity. The
overall %RSD value of MP and IP is less than 10% as shown
in table 3. This implies that the developed method is highly
rugged as shown in figure 4.
Standard solution of GTI
(S/N)
63.21
78.31
103.45
81.57
58.26
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Res. J. Chem. Environ.

Figure 2a: MS/MS Fragmentation of Raltegravir

Figure 2b: MS/MS Fragmentation of GTI

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Res. J. Chem. Environ.
Table 2
System precision
Injection No. Peak area of GTI
Injection1 3842
Injection2 3695
Injection3 3842
Injection4 3799
Injection5 3805
Injection6 3855
Average 3806.33
%RSD 1.55

Table 3
Ruggedness
Preparation No. Peak area of GTI
MP IP
Preparation 1 2.095 2.153
Preparation 2 2.124 2.134
Preparation 3 2.135 2.182
Preparation 4 2.088 2.166
Preparation 5 2.186 2.254
Preparation 6 2.097 2.271
Average 2.121 2.193
%RSD 1.74 2.56
Overall mean 2.157083
Overall %RSD 2.73

(a)

(b)
Figure 3: Specificity Chromatogram of a) GTI and b) Raltegravir

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(a)
(b)
(c)
(d)
Figure 4: Representative Chromatograms of a) Blank b) Standard c) Unspiked sample and d) 100%Spiked sample
Linearity: The linearity of the method was prepared by
diluting the stock solutions of different concentrations from
LOQ to 150% of the specification level. The calibration
curves were plotted by drawing the plot against the peak area
of the GTI and against the corresponding solution
concentration. The calibration curve for GTI in the range of
0.374 ppm to 2.810 ppm was plotted with peak area on X-
axis and concentration of analyte on Y-axis. The intercept,
slope, correlation coefficient and R² value are given in
table 4 and figure 5. R² value was found to be more than 0.99
indicating that the method is linear between the peak area
and concentration.
Accuracy: Recovery studies were conducted by spiking
GTI; high accuracy of the method was found for the GTI in
the range of 103.0%–109.6% as shown in table 5. The results
demonstrated that the method is accurate and precise and
acceptable as per ICH guidelines (Figure 4c, d).
Limit of detection and limit of quantification: Detection
limit and quantification Limit for GTI were determined by
147
Res. J. Chem. Environ.
injecting different solutions to obtain S/N (Signal to Noise
ratio) greater than or equal to 3:1 and 10:1 respectively. The
signal to noise ratio for detection limit and quantification
Limit were found to be 0.15 and 0.42 ppm respectively
(Figure 6). Precision at LOQ values is represented in table 6.
Robustness: As part of robustness study, deliberate changes
to the mobile phase flow (± 10% of 0.5mL/min) and column
temperature (±2°C of 40°C) were carried out. RSD (%)
values obtained from both mobile phase flow and column
temperature studies were found to be less than 5%
demonstrating robustness of the method as reported in table
7.
Solution stability: Solution stability for standard solution,
unspiked and spiked sample (Raltegravir with GTI) was
conducted at room temperature (24 ± 2 °C) and 2–8 °C upto
24 hours. Difference of less than 3% was observed from
initial and 24 hours samples when stored at room
temperature (24 ± 2 °C) and 2–8 °C. The data is presented in
table 8.
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Res. J. Chem. Environ.

Linearity of GTI
6000
y = 1938.8x + 111.59
5000 R² = 0.9985

4000

3000

2000

1000

0
0.000000E+00 5.000000E-01 1.000000E+00 1.500000E+00 2.000000E+00 2.500000E+00 3.000000E+00

Figure 5: Linearity of GTI

Table 4
Linearity
% Level Concentration in PPM Peak area of GTI
LOQ 3.747230E-01 738
25 4.684038E-01 1027
50 9.368075E-01 2019
75 1.405211E+00 2894
100 1.873615E+00 3762
120 2.342019E+00 4563
150 2.810423E+00 5576
Slope 1938.8412
Intercept 111.5859
CC 0.9992
R.Square 0.9985

Table 5
Accuracy
Amount Added Amount Found
% Level % Recovered %RSD
(ppm) (ppm)
4.226820E-01 4.354290E-01 103.0
LOQ 4.124920E-01 4.442930E-01 107.7 3.19
4.003540E-01 4.389420E-01 109.6
9.631450E-01 1.009445E+00 104.8
50 9.835410E-01 1.034516E+00 105.2 1.95
9.722460E-01 1.055462E+00 108.6
1.982450E+00 2.082455E+00 105.0
100 1.935490E+00 2.038424E+00 105.3 1.51
1.981120E+00 2.138424E+00 107.9
2.376040E+00 2.465841E+00 103.8
120 2.234920E+00 2.442834E+00 109.3 2.93
2.453420E+00 2.554198E+00 104.1

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Res. J. Chem. Environ.

(a)

(b)

Figure 6: Representative Chromatograms of GTI at a) LOD and b) LOQ

Table 6
Precision at LOQ
Injection No. Peak area of GTI
Injection1 894
Injection2 902
Injection3 865
Injection4 900
Injection5 896
Injection6 905
Average 893.667
%RSD 1.63

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Res. J. Chem. Environ.
Table 7
Robustness study concerning flow and column oven temperature
Parameter condition RT(min) ppm %RSD S/N ratio
Actual
(Flow: 0.5 mL/min; 2.46 2.138 2.12 63.21
Temperature 40°C)
Flow rate: 0.45 mL/min
2.68 2.116 4.91 98.34
(Low flow)
Flow rate: 0.55 mL/min
2.12 1.904 2.34 101.52
(High flow)
Column oven temperature 38°C
2.51 1.924 1.73 76.24
(Low)
Column oven temperature 42°C
2.4 1.965 2.50 74.91
(High)

Table 8
Solution stability study at room temperature (240C to 260C) and 2-8 0C
Temperature Initial After
Component Solutions Difference
Conditions (ppm) 24hours (ppm)
Standard 1.912 1.986 -0.074
Room temperature
Sample 2.135 2.124 0.011
(24-26°C)
Spiked ND ND
GTI
Standard 1.785 1.778 0.007
2-8°C Sample 2.208 2.134 0.074
Spiked ND ND
ND: Not detected

Thus the newly developed method was demonstrated for its
feasibility. Validation was performed. Specificity, precision,
accuracy, LOQ, LOD, linearity and stability of the solution
were verified by following guidelines as per FDA and ICH.
The system precision of the method was performed by
injecting six preparations of standard solution. System
precision was calculated based on the average relative
deviation (RSD). The precision of the method is assessed
with preparing six different samples of same concentration.
The RSD of six preparations obtained was calculated to
determine the method precision of GTI.
The intermediate precision was performed with the same lot
as method precision but by a different analyst, column,
mobile phase and other UPLC-MS/MS on a different day
after the method precision. The overall RSD of GTI content
obtained from the six preparations of method precision (MP)
and intermediate precision (IP) was calculated to determine
the ruggedness of the developed method.
Linearity is achieved by linear dilution of the impurity stock
solution into the specified concentration from LOQ to 150%.
The solutions were diluted at minimum seven concentration
(LOQ to 150%) levels. The linear regression analysis was
calculated using the least square methods. The calculation
value obtained from the retrospective analysis was used in
the calculation of the same predicted sample responses. The
accuracy of proposed method was assessed with the standard
addition method, spiking the known amount of impurity at
different levels i.e. from LOQ to 120% at the specified limit.
The percentage recovery and the % RSD were calculated for
the three preparations.
The limit of detection and the limit of quantification were
tested considering the concentration of impurity which
yields S/N ratio values 3:1 and 10:1 respectively. The
precision at limit of quantification value was verified, testing
the six preparations of impurity solution at a fixed
concentration. The robustness of the developed method was
assessed by varying the flow and temperature conditions of
column. In addition, an analysis of solutions at time intervals
was performed to determine the stability of the solution.
Conclusion
The proposed method is simple and precise. This method
was first developed and validated for the quantification of
Raltegravir GTI by direct UPLC-MS/MS. The better
sensitivity of the method was provided by selecting and
utilizing MRM mode for the quantitation. The described
method was fully validated and provided acceptable
precision and good linearity. The separation indicates
specificity; the amount of GTI recovered shows accuracy.
Variation in chromatographic parameter does not show
significant variation in results and proved to be robust and it
is also found to be sensitive and selective. The method show
very low, 0.15 and 0.42 ppm for GTI impurity as LOD and
LOQ respectively. The proposed and developed method can
be used successfully in identification, separation, monitoring
and quantification of GTI in the pharmaceutical batches of
Raltegravir during its manufacturing.

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Acknowledgement
The authors would like to thank Koneru Lakshmaiah
Education Foundation,Green Fields, Vaddeswaram, Andhra
Pradesh for providing necessary facilities to carry out this
research work.
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