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.
