A Sensitive and Simple High Performance Liquid Chromatographic Method for Quantification of Tadalafil in Human Serum Bioequivalence and Quality Control Laboratory, Faculty of Pharmacy, Saint-Joseph University,Mathaf, LebanonKEY WORDS: HPLC, tadalafil, assay,
centration range tested (10-800 ng/mL).
Accuracy, precision, and stability studieswere satisfactory. This analytical proce-
ABSTRACT
dure is relatively inexpensive and simple
paraben was used as the internal stan-dard. Optimum conditions for tadalafil
INTRODUCTION
Tadalafil is a potent and selective phos-
single-step liquid–liquid extraction with
vasodilation of erectile tissues.1-3 Oral
less than 10 ng/mL for tadalafil. The cali-
The Journal of Applied Research • Vol. 6, No. 1, 2006
Table 1. Intraday Variability of the Assay for Tadalafil in Serum (4 series)
Concentration Concentration Standard Coefficient Added (ng/mL) Found (ng/mL) Deviation of Variation (%)
Correlation coefficients are 0.9984, 0.9984, 0.9984, and 0.9993 for each of the 4 curves.
Table 2. Interday Variability of the Assay for Tadalafil in Serum (5 series)
Concentration Concentration Standard Coefficient Added (ng/mL) Found (ng/ml) Deviation of Variation (%)
Correlation coefficients are 0.9993, 0.9993, 0.9991, 0.9993, and 0.9993 for each of the 5 curves
oral dosing has not been determined.
life of 17.5 hours in healthy subjects.5-7
Lebanon, and is described in this report.
be determined in biological samples,dietary supplements, or herbal matrices
EXPERIMENT Reagents and Standards
All chemicals used were reagent grade.
tion8-10 or micellar electrokinetic capil-
The Journal of Applied Research • Vol. 6, No. 2, 2006
Figure 1. Chromatograms of (A) standard tadalafil solution, (B) extracted sample at 800 ng/mL,
(C) blank serum, (D) blank serum with internal standard, and (E) extracted sample at 10 ng/mL.
Station, NJ). Tadalafil standard was sup-
water/acetonitrile (50/50, v/v). Internal
Vol. 6, No. 2, 2006 • The Journal of Applied Research
Table 3. Tadalafil Recovery After Extraction:
Tadalafil at Different Concentrations and
USA). The data were collected using thesystem software (Chemstation 1990-
Concentration Average Extraction Coefficient (%) (n=3 for each level) Chromatographic Conditions
5-µm particle size, 250 x 4 mm I.D., with
with glacial acetic acid (0.1 mM, pH 2.5-
UV detection was performed at 280 nm. All analyses were made at room temper-ature. The injection volume was 25 µL,
trile. All stock solutions were protected
through each sample before injection.
from light and kept at -20˚C. They werestable for at least 6 months. Serum Extraction Procedure
stock solution with drug-free serum.
Stability of the drug in serum at –20˚C
a micropipette while gently vortexingthe tubes. To each tube 6 cc of
Instrumentation Table 4. Drug-Free Human Serum Spiked With 3 Different Concentrations of Tadalafil and Stored at –20˚C Over a 6-Month Period Concentration Concentration Obtained (ng/mL) The Journal of Applied Research • Vol. 6, No. 2, 2006
dichloromethane extraction of serumsamples were not less than 75% over the
Assay Validation
For assay validation, tadalafil was mixedwith drug-free human serum over the
Stability
stored at –20˚C did not show significant
ined using 4 series, and interday variabil-
performed on 5 separate days. Tables 1and 2 show the results of within-run and
DISCUSSION AND CONCLUSION
described in this report for the determi-
studied (correlation coefficients 0.998 to
ng/mL were not tested for linearity.
ric detection is not available. The assay
tetraacetic acid in the collection tube.
for determination of tadalafil concentra-
than 10 ng/mL showed CVs more than10% and were not incorporated in the
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