Introduction
Rilpivirine hydrochloride is a di-amino pyrimidine derivative. Chemically, it is 4-[[4-[[4-[(E)-2-cyanoethenyl]-2,6-dimethylphenyl]amino]-2-pyrimidinyl]amino]benzonitrile mono hydrochloride structure are shown in (Figure 1).
Figure 1: Structure
of Rilpivirine hydrochloride.
Rilpivirine (TMC278) is Non –nucleoside reverse
transcriptase, which was approved by the FDA in May 2011 [1-3]. It is a basic,
white, amorphous powder which is readily soluble in methanol, dichloromethane
and insoluble in water. Rilpivirine hydrochloride is not official in Indian
Pharmacopoeia and British Pharmacopoeia. A thorough literature survey has
revealed that UV spectroscopy [4-6], HPLC [7-9] method for Rilpivirine
hydrochloride with combination of other drugs, UPLC [10], LC-MS [11,12] for its
estimation in bulk, pharmaceutical dosage forms and biological samples.
Rilpivirine is a poorly soluble drug with intermediate
permeability in vitro studies and its biological half-life 34-50 hrs [13,14].
Thus, bioavailability of poorly water-soluble drugs will be affected positively
when their dissolution rate is increased. These drugs show serous adverse
clinical effects like non-steady absorption due to variability among patients
and individual patient dosing.
Till date there is no report to estimation of
Rilpivirine in saliva samples. The aim of present research work for the
development and validation of a HPLC method. The present work describes the
development and validation of HPLC method, an attempt was made to develop a
simple, accurate, precise and rapid method for the Rilpivirine hydrochloride in
human saliva.
Experimental
Materials
and reagents
Rilpivirine was gift sample from Hetero drugs.
Methanol Grade was purchased from Merck Chemical Company. HPLC Grade water was
purchased from India mart. The 0.45 ?m pump Nylon filter was obtained from
Advanced Micro Devices (Ambala Cantt, India) & whatman no 5 filter
paper was obtained from Modern Science lab, (Nashik, India). Glasswares used
were Class A grade.
Sample
preparation
In order to investigate the effects of medium on
calibration curve linearity and equation parameter, working standard solutions
of Rilpivirine are prepared in acetonitrile and saliva matrices. Human saliva
are obtained from healthy volunteers and stored at -20 °C until
analysis. Stock standard solution of Rilpivirine was prepared by dissolving
10mg in 10ml acetonitrile and stored at -20 °C for 1 month and
protected from light. Further dilutions were prepared, by diluting stock
solution with mobile phase to achieve calibration concentration (25-1000 ng/mL-1).
Extraction
process
Trail
1: To 0.2ml of saliva samples, 50µL of Ortho
phosphoric acid and 3ml of n-hexane was added. The sample were mixed in a
mechanical shaker for 20 min and centrifuged at 1000 rpm for 10 min. After
centrifuged, supernant layer was separated and make up to 10ml with mobile
phase [acetonitrile:water (80:20) v/v].
Trail
2: To 0.2ml of saliva samples, 50µL of ortho
phosphoric acid and 3ml of methanol was added. The sample were mixed in a
mechanical shaker for 20 min and centrifuged at 1000 rpm for 10 min. After
centrifuged, supernant layer was separated and make up to 10ml with mobile
phase [acetonitrile:water (80:20) v/v].
Out of 2nd trial performed, the 2nd
trial was selected for further studies because 2nd trial was found
good separation of saliva, with good peak symmetry.
Standard
preparation
Weigh accurately about 10 mg of Rilpivirine
hydrochloride working standard to a 10 ml volumetric flask. Add about 5 ml
diluent to dissolve it completely (sonicate if necessary), make up the volume
with diluents it gives 1000 µg/ml. Further dilute, 1ml of this solution to 10ml
with diluents it gives 100µg/ml.
Standard
stock solution preparation
Further dilutions were prepared, by diluting stock
solution with mobile phase to achieve calibration concentration (0.25-25
µg/ml).
Preparation
of sample solution
To the extract solution, different concentration of
Rilpivirine hydrochloride was spiked to get the concentration of 0.25-25 µg/ml.
Validation
of RP-HPLC method
As per the International Conference on Harmonization
(ICH) guidelines, the method validation parameters like linearity, precision,
accuracy, limit of detection, limit of quantitation, specificity and robustness
were experimentally determined and the method was validated.
System
suitability parameters
System suitability tests are an integral part of
chromatographic method. To ascertain its effectiveness, system suitability
tests were carried out by injecting freshly prepared standard stock solution of
10µg/ml Rilpivirne hydrochloride in six replications and the parameters like
retention time, peak area, plate number (N), and peak asymmetry of samples were
calculated.
Specificity
Specificity for an assay ensures that the signal
measured comes from the substance of interest and there is no interference from
excipient and/or degradation products and/or impurities. Specificity of the
method was done by comparing the chromatogram of drug with the chromatogram of
blank (mobile phase).
Linearity
Calibration and quality control samples were prepared
by adding Rilpivirine hydrochloride solution in blank saliva. The amount
corresponded to the saliva concentration of Rilpivirine hydrochloride ranged
from 0.25 to 25 µg/ml. The calibration curves for the saliva spiked by
Rilpivirine hydrochloride were obtained by platting Rilpivirine peak areas for
the concentration range 0.25,0.5,1,5,10,15,20 and 25 µg/ml.
Precision
Precision studies were carried out to ascertain the
reproducibility of the proposed method.
Repeatability
(Method precision)
Repeatability was determined by preparing six
replicates of 10 µg/ml Rilpivirine hydrochloride spiked with saliva separately
inject equal volumes (20 µl) of each solution. Record the chromatograms and
measure the peak response of drug. The results were reported as %RSD.
Intermediate Precision
Interday
precision
Intraday precision study was carried out by preparing
drug solution spiked with saliva of concentration (10 µg/ml) and analysing it
at three different times in a day. Record the chromatograms and measure the
peak response of Rilpivirne hydrochloride. The results were reported as %RSD.
The precision result showed a good reproducibility with percentage relative
standard deviation less than 2.
Accuracy
The accuracy of methods was evaluated by performing
triplicate analyses of concentrations of 125ng/mL, 250ng/mL and 500ng/mL in
saliva spiked drug samples.
Limit
of detection and limit of quantitation
The limit of detection (LOD) and the limit of
quantitation (LOQ) of the drug were derived by calculating the signal-to-noise
ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations
designated by International Conference on Harmonization (ICH) guidelines.
LOD = 3.3 × ?/S
LOQ = 10 × ?/S
Where,
? = the standard deviation of the response and
S = slope of the calibration curve
Was performed and chromatographed. The sample solution
20µl was injected.
Robustness
Robustness studies were carried by changing the flow
rate of mobile phase from 0.9 to 1.1 mL/min, and wavelength from 298 to 302.
Rilpivirne hydrochlorides made in triplicates and were analysed.
Ruggedness
Ruggedness studies were performed by preparing three
replicates of 10 µg/ml of Rilpivirine hydrochloride , analysing by two
different analyst and the results are reported as %RSD. Structure of
Rilpivirine hydrochloride is shown in (Figure 1).
Results
The Eclipse C18 (100 mm x4.6mm, 3.5µm) column was used and the mode of elution was isocratic. The flow rate 1.0m/min, injection volume was 20µL and run time of sample was 5min. Initially various mobile phase compositions were tired, to separate the ingredients. Out of 5 trials performed, the 5th trail was selected because when compared to other trails, the 5th trial had the least in retention time, with good peak symmetry as mention in and (Figures 2 and 3).
Figure 2:
Trails of mobile phases a. Methanol:water (50:50) b. Acetonitrile:water (50:50)
c. Acetonitrile:methanol (50:50) d. Acetate buffer (pH 4):methanol (50:50) e.
Acetate buffer (pH 4): methanol (40:60).
Figure 3: Typical Chromatogram of i. blank saliva ii. Rilpivirine
spiked with saliva.
Extraction
process
To 0.2ml of saliva
samples, 50µL of ortho phosphoric acid and 3ml of methanol was added. The
sample were mixed in a mechanical shaker for 20 min and centrifuged at 1000 rpm
for 10 min. After centrifuged, supernant layer was separated and make up to
10ml with mobile phase [methanol:acetate buffer (70:30) v/v]. To the above
solution, different concentration of drug solution was spiked and solution was
injected. (Table 1) shows mobile phase section.
HPLC
Method Validation
As per the
International Conference on Harmonization (ICH) guidelines, the method
validation parameters like linearity, precision, accuracy, limit of detection,
limit of quantitation, specificity and robustness were experimentally
determined and the method validated.
Table
1: Selection
of mobile phase.
|
S.No |
Mobile phase conditions |
Observation |
|
1 |
Water : Methanol (50:50% v/v) |
Broad peaks appear |
|
2 |
Water : Acetonitrile(50:50 v/v) |
Extra peak with tailing |
|
3 |
Acetonitrile : Methanol (50:50 v/v) |
|
|
4 |
0.05M Acetate buffer of pH 4.0 :
Acetonitrile (50 : 50 % v/v) |
Sharp peak with fronting |
|
5 |
0.05M Acetate buffer of pH 4.0 :
Acetonitrile (60 : 40 % v/v) |
Sharp peak with extra peak |
Specificity
Specificity of the
method was done by comparing the chromatogram of drug (drug spiked with saliva)
with the chromatogram of blank (saliva). The chromatogram of the blank was
recorded and it did not show any peaks. The chromatogram of the drug is given
in (Figure 3) respectively.
System
suitability parameters
System suitability
test are an integral part of chromatographic method. They were used to verify
that the reproducibility of the chromatographic system is adequate for the
analysis. To ascertain its effectiveness, system suitability tests were carried
out by injecting freshly prepared standard stock solution of Rilpivirine
hydrochloride. Results are shown in (Table 2).
Table
2: System
Suitability Parameters.
|
Parameters |
Results |
|
Retention time (min) |
2.640 |
|
Theoretical plates |
5962 |
|
HETP |
0.016 |
|
Asymmetry |
1.2 |
Linearity
Standard solutions of Rilpivirine hydrochloride in the concentration range of 0.25-25µg/ml were injected into chromatographic system and peak areas were measured. A graph of peak areas (on Y-axis) versus concentration (on X-axis) was plotted and calibration graph was shown in (Figure 4). The corresponding values are given in (Table 3). The regression equation was found to be y = 1000000x+34542. Coefficient of determination was found to be 0.999.
Figure
4: Calibration
curve of Rilpivirine hydrochloride.
Table
3:
Repeatability studies of Rilpivirine hydrochloride.
|
Concentration (µg/ml) |
Peak area |
Mean =13556868 S.D = 73291.8 %RSD =0.540 |
|
10 |
13593009 |
|
|
10 |
13526588 |
|
|
10 |
13485698 |
|
|
10 |
13685472 |
|
|
10 |
13547892 |
|
|
10 |
13502548 |
Precision
The results were
reported as %RSD. The precision result showed a good reproducibility shown with
percent relative standard deviation less than 2. Corresponding results were
mentioned in (Table 4).
Accuracy
The accuracy of HPLC
analysis tested by the recovery of Rilpivirine hydrochloride in saliva is
summarized in (Table 5).
Robustness
Robustness studies
were carried out by changing the flow rate of the mobile phase from 0.8 to 1.2
mL/min and by changing the wavelength from 238 to 242 nm. 125ng/mL Rilpivirine
hydrochloride was analysed.
Ruggedness
Robustness studies
were carried by changing the flow rate of mobile phase from 0.9 to 1.1 mL/min
and by changing in wavelength from 298 to 300 nm. 10 µg/ml Rilpivirine
hydrochloride was analysed and the %RSD is determined. Referred in (Table 6).
Table
4: Precision
of Rilpivirine hydrochloride.
|
|
Intraday precision |
Interday precision |
||
|
Concentration (µg/ml) |
Peak area |
Mean 13565961 ±
92006.3 %RSD = 0.678 |
Peak area |
Mean = 13572845 ± 78610.4 %RSD=0.579 |
|
10 |
13526584 |
13524587 |
||
|
10 |
13565845 |
13658742 |
||
|
10 |
13542584 |
13458751 |
||
|
10 |
13658742 |
13548752 |
||
|
10 |
13698521 |
13587498 |
||
|
10 |
13569852 |
13658742 |
||
Table 5: Accuracy studies of
Rilpivirine hydrochloride.
|
Saliva Concentration (µg/ml) |
Amount recovered (µg/ml) |
% Recovery |
% Mean recovery± SD |
%RSD |
|
5 |
4.92 |
98.4 |
97.7 ± 0.989949 |
1.01 |
|
5 |
4.85 |
97.0 |
||
|
5 |
4.81 |
96.2 |
||
|
10 |
9.70 |
97.0 |
97.85 ± 1.20 |
1.22 |
|
10 |
9.87 |
98.7 |
||
|
10 |
9.61 |
96.1 |
||
|
15 |
14.72 |
98.1 |
98.73 ± 1.66 |
1.64 |
Table 6: Ruggedness studies of
Rilpivirine hydrochloride.
|
Concentration
(µg/ml) |
Change
in flow rate (ml/min) |
Retention
time (min) |
Change
in wavelength (nm) |
Retention
time (min) |
||||
|
0.9 |
1.1 |
0.9 |
1.1 |
298 |
302 |
298 |
302 |
|
|
10 |
13526589 |
13548695 |
2.907 |
2.273 |
13569852 |
13587456 |
2.640 |
2.640 |
|
13654857 |
13658745 |
13658745 |
13594526 |
|||||
|
13458752 |
13459854 |
13487536 |
13655421 |
|||||
|
13558745 |
13548752 |
13569852 |
13518742 |
|||||
|
13548754 |
13554874 |
13542658 |
13487521 |
|||||
|
13569854 |
13569854 |
13652158 |
13598521 |
|||||
|
Mean area ± SD |
13552925 ± 63702.64 |
13556796 ± 63396.98 |
13580134 ± 65661.9 |
13573698 ± 60604.5 |
||||
|
%RSD |
0.470 |
0.467 |
0.483 |
0.446 |
||||
Limit
of detection and limit of quantitation
The parameters LOD
and LOQ were determined on the basis of response and slope of the regression
equation. The limit of detection (LOD) and the limit of quantitation (LOQ) of
the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3
for LOD and 10 for LOQ) using the following equations designated by
International Conference on Harmonization (ICH) guidelines.
LOD = 3.3 × ?/S LOQ = 10 × ?/S
Where, ? = the
standard deviation of the response and; S= slope of the calibration curve.
·
LOD = 0.076
·
LOQ = 0.232
Conclusion
The proposed RP-HPLC
method is uncomplicated, quick, accurate, precise, robust, and sensitive. This
process has been observed to be improved over previously reported analytical
methods of Rilpivirine hydrochloride in terms of validation parameters, use of
a cost-effective as well as a mobile phase methanol:acetate buffer (70:30)
v/v], low Rt (speedy analysis), no internal standard and UV detection. A
simple, rapid precise, accurate and robust RP-HPLC-UV method was developed,
validated for the determination of Rilpivirine hydrochloride in saliva and
optimization parameters. The simplicity of the method allows for application in
laboratories that lack sophisticated analytical instruments such as LC-MS/MS or
GC-MS/MS that are complicated, costly and time consuming rather than a simple
HPLC method. The contribution of each of the above mentioned factors indicate
the superiority of the developed method above other described analytical
methods for the regular investigation of Rilpivirine hydrochloride as an active
pharmaceutical ingredient and biological samples like saliva.
Acknowledgements
The
authors are grateful to Management of Malla Reddy College of Pharmacy for
providing necessary research facilities to carry out the research work and to
hetero drugs, India for providing the gift sample of the drug
Declaration of Interest
The author has no
relevant affiliations or financial involvement with a financial interest in or financial with the subject matter or materials discussed in
the manuscript.
Conflicts of Interest
There
is no conflict of interest.
References
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Kiran B, Sanjay R. Development and validation of UV spectrophotometric method
for estimation of rilpivirine hydrochloride in bulk and pharmaceutical
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