Full pdf article l bioanalytical method development and validation of letrozole by rp-hplc method

Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/008 ISSN 0974 – 9446 BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF LETROZOLE BY RP-HPLC
V.Sekar *1, S.Jayaseelan1, N.Subash1,
E.Udhaya kumar1,P.Perumal1.R.P.Venkatesh2. J.K.K.Nataraja College of pharmacy, Natarajapuram, Komarapalayam (P.O)-638183, Namakkal (D.T), Tamil Nadu, India. J.S.S College of Pharmacy, Ooty, Tamil Nadu E-mail: subash23pharma@gmail.com
ABSTRACT
A rapid high-performance liquid chromatography method has been developed for bioanalytical method
development and validation of letrozole in human plasma. letrozole (CGS 20 267), a potent aromatase
inhibitor for treatment of oestrogen- depentent diseases. Letrozole was found with symmetrical peak
shapes on a analytical column Phenomenex Luna C18 column using 75% 0.02M Phosphate buffer
at PH 5.5 and 25% acetonitrile as the mobile phase. The retention times of Letrozole and fluconazole
the internal standard were 4.29 and 7.47 min respectively. Linear calibration curves were obtain for
each compound across a range of 50.55-120.00 ng/ml. the limit of detection was 12.5ng/ml and the limit
of Quantification was 37.5ng/ml for Letrozole. Greater than 85% recoveries were obtain for Letrozole.
The intra and interday relative standard deviation (%RSD) were <5%. It uses less biological material
and applicable.

Keywords: Letrozole, Fluconazole.


International Journal of Pharma Research and Development – Online Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/008 ISSN 0974 – 9446
INTRODUCTION AND MATERIAL AND METHOD
1. Introduction
Letrozole (CGS 20 267) is a potent aromatase inhibitor under development as a drug for the treatment
of oestrogen-dependent diseases, such as breast cancer in post-menopausal women. A high-
performance liquid chromatography (HPLC) method for the determination of letrozole in biological fluids
has been described previously [1], but it appeared that it was not sensitive enough to measure the
unchanged compound after administration of low doses (0.5 rag). An enzyme immunoassay (EIA) [1]
more sensitive than this HPLC method (0.7 versus 28 nmol/I, respectively) was developed. Its
application to clinical samples revealed that it was not specific enough. A strong cross-reactivity of the
antibodies with the metabolite CGP 44 645 (I) has been observed with all urine samples and several
plasma samples from patients under repeated administration [1].
This report describes an HPLC method with a fully automated protein precipitation extraction and
fluorescence detection offering improved sensitivity for the determination of letrozole in human plasma.

MATERIAL AND METHOD
2. Experimental
2.1. Chemicals and reagents
Letrozole and fluconazole (IS) were kindly supplied by CIPLA pharmaceuticals Pvt Ltd., Bangalore. HPLC Grade solvents (Acetonitrile, water) were obtained from S.D.Fine chemicals Ltd., India, Ranbaxy India Ltd. HPLC Grade potassium di hydrogen phosphate and phosphoric acid were purchased from Qualigens Fine Chemicals Ltd., Mumbai. Stock standard solutions of fluconazole and the internal standard were prepared by dissolving appropriate amounts of compounds in a known volume of acetonitrile and ammonium acetate and stored at 4oC. Letrozole (CGS 20 267) Mol. wt.: 285.31 ; CI7HllN5 Fig. 1. Chemical structure of letrozole, 2.2. Equipment
The chromatographic equipment used was LC – 20 AT Prominence solvent delivery system (Pump),
Rheodyne 7725i injector with 20 µl loop, and Phenomenex – Luna, C18 (250 x 4.6 mm i.d., 5µ), a
fluorescence detector (Perkin-Elmer LS 30) to determine the letrozole spectrum (Fig. 2), a fluorescence
detector (Hitachi 1080, Merck) with excitation and emission wavelengths set at 230 nm and 295 nm,
respectively; a data station connected to the detector equipped with Winchrome data station. The
International Journal of Pharma Research and Development – Online Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/008 ISSN 0974 – 9446
mobile phase (0.02 M phosphate buffer, pH 5.5 -acetonitrile, 75:25, v/v) was used at a flow-rate of 1.5
ml/min.
2.3. Sample preparation
Blood samples were collected in heparinized tubes and immediately placed on ice and taken to the lab where they were centrifuged at 5000rpm for 5 min at room temperature. The resulting plasma samples were stored at -30oC until analysis. 2.3.1. Protein Precipitation
The blank plasma sample was prepared by adding 1ml of plasma and 1ml of methanol and vortex for 30sec. Then centrifuge the solution at 4°C, 5000 RPM for 5min. The supernatant liquid is taken and transferred to HPLC vials The blank plasma sample was prepared by adding 1ml of spiked plasma 1ml of methanol and 0.2ml of IS (250µg/ml) and vortex for 30sec. Then centrifuge the solution at 4°C, 5000 RPM for 5min.The supernatant liquid is taken and transferred to HPLC vials. 2.4 Quantification of Letrozole in plasma
A Standard curve was prepared by injecting various concentrations of letrozole in plasma. The concentrations of the plasma and quality control samples were calculated by using the regressed equation of the straightline y=ax+b. The limit of detection (LOD) and limit of quantification (LOQ) were determined as follows: 6 blank samples from six separate subjects for each analyte and matrix were extracted and compared to a low standard of each analyte. Where an obvious peak existed at the same retention time as the analyte, a concentration was calculated for this peak. Where no discreet peak, or a series of small noise peaks existed at the same retention time as the analyte, the height of the noise was measured and compared to the height of the low standard. This provided a “concentration” for the noise. An average of the 6 “noise concentrations” was calculated and multiply by either 3 (LOD) or 5 (LOQ). LOQ values were subsequently confirmed using six replicates spiked at the target concentration as being within an acceptable variance of 20%. Determination of recovery, accuracy and precision
The absolute recovery of letrozole, was determined by comparison of the peak areas from non- extracted and extracted samples of QC-3 in triplicate. The intra-day accuracy and precision were determined at three different concentrations from six replicate QC. The inter-day accuracy and precision were determined at three concentrations from six replicate QC on three independent occasions. The precision was calculated as the relative standard deviation of the mean (R.S.D.) with R.S.D. (%) = (standard deviation of the mean/mean)×100. 2.6. Stability
Short term stock stability
International Journal of Pharma Research and Development – Online Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/008 ISSN 0974 – 9446 A Stock solution of letrozole and IS was kept at room temperature for 6 hours. Long term stock stability
A Stock solution of letrozole and IS was kept at room temperature for 15 days. Bench top stability
The replicate concentration of low and high quality control samples were determined by comparing the mean area ratio of freshly thawed samples which kept at room temperature for about 8 hours. Coolant stability
The replicate concentration of low and high quality control samples were determined by comparing the mean area ratio of freshly thawed samples which kept at room temperature for about 24 hours. Freeze thaw stability
The stability of low and high quality control samples were determined after three freeze thaw cycles. The percent degradation was determined by comparing the mean of area ratio of letrozole. Long term plasma stability
At least three aliquots of each of low and high concentrations at same conditions as study samples. Analyses on three separate occasions. Storage time should exceed the time between the date of first sample collection and the date of last sample analysis. Ruggedness
This includes different analysts, laboratories, columns, instruments, sources of reagents, Ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test condition. The ruggedness of the method was studied by changing the experimental condition such as, Changing to another column of similar type (Phenomenex Gemini C18) Different operation in the same laboratory. 3. RESULTS AND DISCUSSION
The method was validated in terms of limit of quantification, Recovery, Selectivity, Precision, 3.1. Linearity
International Journal of Pharma Research and Development – Online Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/008 ISSN 0974 – 9446 The method was validated over the range of 50.55-120.00ng/ml. The slope, intercept, correlation co-efficient were found to be 0.007791, 0.005728, 0.9983 respectively. 3.2. Sensitivity
In the plasma, the calculated limit of quantification was 37.5ng/ml and limit of detection was 3.3. Recovery
The recovery was determined by comparing the aqueous solution and the spiked drug. The percentage recovery of the drug and the internal standard was calculated and it was 98.55% and 99.16% respectively. 3.4. Precision and Accuracy
The accuracy, precision and intraday precision were carried out by preparing 6 individual samples of HQC, MQC and LQC and the percentage C.V. and percentage nominal was calculated. 3.5. Stability
Stability of the method was carried out by performing short term and long-term stock stability. The percentage mean ratio of the drug and internal standard were calculated. Stability of the plasma samples was carried out by performing coolant, bench top and freeze thaw stability. The percentage mean ratio of the HQC and LQC were calculated. The ruggedness of the method was carried out by changing by column and by different analyst in the same lab. The percentage CV of the HQC and LQC were calculated. The long-term plasma stability was carried out by performing from the initial sample to the date of last sample.The percentage CV of the HQC and LQC were calculated.

FIGURES AND TABLES:

International Journal of Pharma Research and Development – Online Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/008 ISSN 0974 – 9446 Table: 1 PARAMETERS
Fig : 1 chromatogram of LETROZOLE and I.S in human plasma
International Journal of Pharma Research and Development – Online Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/008 ISSN 0974 – 9446
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