Issue 8, 2010

Sildenafil and N-desmethyl sildenafil quantification in human plasma by HPLC coupled with ESI-MS/MS detection: Application to bioequivalence study

Abstract

The present study aims at developing a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LCMS/MS) method for simultaneous quantification of sildenafil and its metabolite N-desmethyl sildenafil in human plasma using sildenafil-d8, N-desmethyl sildenafil-d8 as internal standards (IS). Chromatographic separation was performed on Zorbax SB C18, 4.6 × 75 mm, 3.5 μm column with an isocratic mobile phase composed of 10 mM ammonium acetate and acetonitrile (5/95 v/v), at a flow-rate of 0.6 ml min−1. Sildenafil, sildenafil-d8, N-desmethyl sildenafil and N-desmethyl sildenafil-d8 were detected with proton adducts at m/z 475.2 → 283.4, 483.4 → 283.4, 461.3 → 283.4 and 469.4 → 283.4 in multiple reaction monitoring (MRM) positive mode respectively. Both drug, metabolite and internal standards were extracted by liquid–liquid extraction. The method was validated over a linear concentration range of 1.0–1000.0 ng ml−1 for sildenafil and 0.5–500.0 ng ml−1 for N-desmethyl sildenafil with correlation coefficient (r2) ≥ 0.9998 for sildenafil and (r2) ≥ 0.9987 for N-desmethyl sildenafil. This method demonstrated intra and inter-day precision within 1.5 to 5.1 and 2.2 to 3.4% for sildenafil and within 1.3 to 3.1 and 2.8 to 4.3% for N-desmethyl sildenafil. This method demonstrated intra and inter-day accuracy for sildenafil within 97.3 to 98.3 and 96.7 to 97.2% and for N-desmethyl sildenafil within 95.3 to 96.3 and 95.0 to 97.2%. Both analytes were found to be stable throughout three freeze/thaw cycles, bench top and postoperative stability studies. This method was used successfully for the analysis of plasma samples following oral administration of 100 mg in 43 healthy Indian male human volunteers under fasting conditions.

Graphical abstract: Sildenafil and N-desmethyl sildenafil quantification in human plasma by HPLC coupled with ESI-MS/MS detection: Application to bioequivalence study

Article information

Article type
Paper
Submitted
26 Jan 2010
Accepted
10 May 2010
First published
06 Jul 2010

Anal. Methods, 2010,2, 1043-1050

Sildenafil and N-desmethyl sildenafil quantification in human plasma by HPLC coupled with ESI-MS/MS detection: Application to bioequivalence study

B. R. Challa, B. Z. Awen, B. R. Chandu, M. Khagga, C. K. Bannoth, K. Kanala, R. P. Shaik, R. Peraman and R. Gogineni, Anal. Methods, 2010, 2, 1043 DOI: 10.1039/C0AY00062K

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