Sains Malaysiana 42(2)(2013): 135–142


Optimization of Extraction Methods for Determination of Phosphodiesterase-5 (PDE5)

Inhibitors in Premix Coffee

(Pengoptimuman Kaedah Pengekstrakan bagi Penentuan Perencat Fosfodiesterase-5

(PDE5) dalam Kopi Pracampuran)


Zailina binti Abdul Majid*

National Public Health Laboratory, Ministry of Health, Lot 1853, Kg. Melayu, 47000 Sg. Buloh, Selangor, Malaysia


Aminah Abdullah & Mohd Ambar Yarmo

School of Chemical Science and Food Technology, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia


Diserahkan: 21 Februari 2012 / Diterima: 9 Oktober 2012



An efficient analytical technique capable of analyzing three most common phosphodiesterase-5 (PDE5) inhibitors (vardenafil, sildenafil and tadalafil) simultaneously in premix coffee was developed. Sample extractions using either acetonitrile or methanol with two different extraction techniques (with and without evaporation steps) were evaluated. Identification and quantitation was conducted by high performance liquid chromatography with photo-diode-array (HPLC-DAD) at different wavelengths; 230 nm, 245 nm and 290 nm; and by time of flight mass spectrometry (LC-MS-TOF). Extraction with acetonitrile (without evaporation with nitrogen) showed recovery ranging from 105% to 113% (± <10%) for HPLC-DAD at 245 nm and 93% to 102% (± <2.5%) for LC-MS-TOF. Chromatogram separation was best achieved with mobile phase consisted of water (0.1% formic acid) and acetonitrile (0.1% formic acid) with gradient elution within 20 min. Thus, the results indicated that extraction using acetonitrile without evaporation step was the most efficient technique for determination of PDE5 inhibitors in premix coffee.


Keywords: HPLC-DAD; LC-MS-TOF; optimization; PDE5 inhibitors; premix coffee



Satu kaedah pengekstrakan yang ringkas dan cekap telah berjaya dibangunkan untuk mengesan kehadiran perencat fosfodiesterase-5 (PDE5) iaitu vardenafil, sildenafil dan tadalafil dalam produk kopi pracampuran. Pengekstrakan sampel menggunakan asetonitril atau metanol dengan dua kaedah pengekstrakan (melalui dan tanpa melalui penyejatan bernitrogen) dinilai. Penentuan dan kuantifikasi sebatian adalah menggunakan kromatografi cecair prestasi tinggi (HPLC) dengan pengesan susunan diod (DAD) pada panjang gelombang 230 nm, 245 nm dan 290 nm serta kromatografi cecair spektrometri jisim masa penerbangan (LC-MS-TOF). Pengekstrakan sampel menggunakan asetonitril (tanpa melalui penyejatan bernitrogen) menunjukkan peratusan perolehan semula antara 105% hingga 113% (± <10%) dengan HPLC-DAD pada panjang gelombang 245 nm dan 93% hingga 102% (± <2.5%) dengan LC-MS-TOF. Pemisahan kromatogram paling baik diperoleh dengan kombinasi fasa bergerak air (0.1% asid formik) dan asetonitril (0.1% asid formik) dengan kecerunan elusi dalam tempoh 20 min. Ini membuktikan kaedah pengekstrakan sampel menggunakan asetonitril tanpa langkah penyejatan bernitrogen merupakan teknik yang paling cekap dalam menentukan perencat PDE5 dalam produk kopi pracampuran.


Kata kunci: HPLC-DAD; kopi pracampuran; LC-MS-TOF; pengoptimuman; perencat PDE5



Aboul-Enein, H.Y. & Ali, I. 2005. Determination of tadalafil in pharmaceutical preparation by HPLC using monolithic silica column. Talanta 65: 276-280.

Abdel-Hamid, M.E. 2006. Determination of sildenafil, tadalafil, and vardenafil in tablets and adulterated herbal products by ESI-MS-MS. Journal of Liquid Chromatography & Related Technologies 29: 591-603.

Bogusz, M.J., Hassan, H., Al-Enazi, E., Ibrahim, Z. & Al-Tufail, M. 2006. Application of LC-ESI-MS-MS for detection of synthetic adulterants in herbal remedies. Journal of Pharmaceutical and Biomedical Analysis 41: 554-564.

Cheng, C.L & Chou, C.H. 2005. Determination of tadalafil in s mall volumes of plasma by high-performance liquid chromatography with UV detection. Journal of Chromatography B 822: 278-284.

Cheng, C.L., Kang, G.J. & Chou, C.H. 2007. Development and validation of a high-performance liquid chromatographic method using fluorescence detection for the determination of vardenafil in, small volumes of rat plasma and bile. Journal of Chromatography A1154: 222-229.

Choi, D.M., Park, S. & Yoon, T.H. 2008. Determination of analogues of sildenafil and vardenafil in foods by column liquid chromatography with a photodiode array detector, mass spectrometry, and nuclear magnetic resonance spectrometry. Journal of AOAC International 91(3): 580-588.

Eardley, I. & Sethia, K. 2003. Erectile Dysfunction: Current Investigation and Management. 2nd ed. London: Elsevier Ltd.

Fleshner, N., Harvey, M., Adomat, H., Wood, C., Eberding, A., Hersey, K. & Guns, E. 2005. Evidence for contamination of herbal erectile dysfunction products with phosphodiesterase type 5 inhibitors. The Journal of Urology 174: 636-641.

Gratz, S.R., Flurer, C.L. & Wolnik, K.A. 2004. Analysis of undeclared synthetic phosphodiesterase-5 inhibitors in dietary supplements and herbal matrices by LC-ESI-MS and LC-UV. Journal of Pharmaceutical and Biomedical Analysis 36: 252-533.

Liang, Q., Qu, J., Luo, G. & Wang, Y. 2006. Rapid and reliable determination of illegal adulterants in herbal medicines and dietary supplements by LC/MS/MS. Journal of Pharmaceutical and Biomedical Analysis 40: 305-311.

Mikami, E., Ohno, T. & Matsumoto, H. 2002. Simultaneous identification/determination system for phentolamine and sildenafil as adulterants in soft drinks advertising roborant nutrition. Forensic Science International 130: 140-146.

Reepmeyer, J.C. & d’Avignon, D.A. 2009. Structure elucidation of thioketone analogues of sildenafil detected as adulterants in herbal aphrodisiacs. Journal of Pharmaceutical and Biomedical Analysis 49: 145-150.

Reepmeyer, J.C. & Woodruff, J.T. 2007. Use of liquid chromatography-mass chromatography and a chemical cleavage reaction for the structure elucidation of a new sildenafil analogue detected as an adulterant in an herbal dietary supplement. Journal of Pharmaceutical and Biomedical Analysis 44: 887-893.

Savaliya, A.A., Shah, R.P., Prasad, B. & Singh S. 2010. Screening of Indian aphrodisiac ayurvedic/herbal healthcare products for adulteration with sildenafil, tadalafil and/or vardenafil using LC/DAD and extracted ion LC-MS-TOF. Journal of Pharmaceutical and Biomedical Analysis 52: 406-409.

Stehlik, J. & Movsesian, M. 2009. Combined use of PDE5 inhibitors and nitrates in the treatment of pulmonary arterial hypertension in patients with heart failure. Journal of Cardiac Failure 15(1): 31-34.

Venhuis, B.J., de Kaste, D. & Blok-Tip, L. 2008. Designer drugs in herbal aphrodisiacs. Forensic Science International 177: e25-e27.

Wang, J., Yang, D., Wang, Z., Chen, B. & Yao, S. 2009. Simultaneous of illegal additives in dietary supplements medicines by high performance liquid chromatography–ionization mass spectrometry. Food Chemistry 113: 227-232.

Zhu, X., Xiao, S., Chen, B., Zhang, F., Yao, S., Wan, Z., Yang, D. & Han, H. 2005. Simultaneous determination of sildenafil, vardenafil and tadalafil as forbidden components in natural dietary supplements for male sexual potence by high-performance liquid chromatography-electrospray ionization mass spectrometry. Journal of Chromatography A 1066: 89-95.

Zou, P., Oh, S.S.Y., Hou, P., Low, M.Y. & Koh, H.L. 2006. Simultaneous determination of synthetic phosphodiesterase-5 inhibitors found in a dietary supplement and pre-mixed bulk powders for dietary supplements using high-performance liquid chromatography with diode array detection and liquid chromatography–electrospray ionization tandem mass spectrometry. Journal of Chromatography, 1104: 113-122.

Zou, P., Oh, S.S.Y., Hou, P., Chong, Y.M., Bloodworth, B.C., Low, M.Y. & Koh, H.L. 2008. Isolation and identification of thiohomosildenafil and thiosildenafil in health supplements. Journal of Pharmaceutical and Biomedical Analysis 47: 279-284.



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