Sensitive and rapid HPLC-UV method with back-extraction step for the determination of sildenafil in human plasma

https://doi.org/10.1016/j.jchromb.2015.11.059Get rights and content

Highlights

  • HPLC-UV method for quantification of sildenafil in human plasma has been developed.

  • Liquid–liquid extraction procedure followed by a back-extraction was used.

  • Full method validated according to EMEA validation guideline was carried out.

  • The total run time does not exceed 10 min per sample.

  • The lower limit of quantitation was 2 ng/ml.

Abstract

In this work we provided a selective, sensitive and rapid HPLC-UV method for quantification of sildenafil in human plasma. We have adopted a simple liquid–liquid extraction procedure followed a back-extraction in 5% perchloric acid solution. Chromatographic separation was achieved on a BDS C-18Column (150 mm × 4.6 mm, 5 μm) using a mobile phase consisted 63% water, 37% acetonitrile and 0.1% triethylamine (pH 7.7). The analysis was detected at 230 nm. The achieved lower limit of quantification was 2.00 ng/ml. The method showed linear calibration curve over the range of 2.00–200 ng/ml. Intra- and inter day precision (CV%) were less than 6.80 and 5.19%, respectively. Whilst intra- and inter day accuracy% were ranged between (98.3 and 105%) and (99.4 and 103%), respectively. Tests confirmed the stability of sildenafil in plasma at room temperature for 24 h, during three freeze–thaw cycles, after 24 h in autosampler at 10 °C and after 60 days in plasma at −30 °C. The recovery of sildenafil was greater than 78.4%. The described simple UV method achieved very low limit of quantification and by using simple and inexpensive extraction procedure, complete separation was obtained within short run time. Having demonstrated the validity and novelty of our method, thus it is applicable for the clinical and pharmacokinetic studies of sildenafil in human volunteers especially in laboratories in countries where cost of modern techniques and instrumentation is prohibitive.

Introduction

Sildenafil (1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethox-yphenyl]sulfonyl]-4-methylpiperazine) is phosphodiesterase inhibitor used predominantly for erectile dysfunction treatment (Fig. 1). Sildenafil is selective competitive inhibitors of phosphodiesterase type 5 (PDE-5), an enzyme that breaks down cyclic guanosine monophosphate (cGMP) [1], [2]. PDE-5 is widely presented in the smooth muscle cells of the blood vessels of the corpus cavernosum and in the pulmonary blood vessels. cGMP is a second messenger of nitric oxide (NO), which is released with sexual stimulation. Therefore, the major actions of sildenafil is prolongation of penile erection [3].

Far beyond erectile dysfunction, many recent clinical studies suggest some very promising new applications of sildenafil. Sildenafil is currently approved for the treatment of pulmonary hypertension [4]. Moreover, in the central nervous system, it exerts its neuroprotective effects in multiple sclerosis and has a significant memory enhancing action. Sildenafil also significantly enhances neurogenesis. Thus, it may be potentially useful for the treatment of neurodegenerative illnesses, such as Alzheimer's disease [4], [5]. This poses critical need of chromatographic method for pharmacokinetic investigation of drug with such vast popularity in human plasma and biological samples.

In this regard, various chromatographic methods have been used for sildenafil determination in biological samples with different detector types. Electrochemical [6], ultraviolet [7], [8], [9], [10] and mass-spectrometry [11], [12], [13], [14], [15].

Compared to HPLC methods with UV detection, liquid chromatography–mass spectrometry (LC/MS) and liquid chromatography–tandem mass spectrometry (LC/MS/MS) are more expensive and unavailable in many laboratories. Nevertheless, most of the available HPLC methods with UV detection do not have good limit of quantification compared to (LC/MS) and (LC/MS/MS). Moreover, the vast majority of sildenafil methods use solid-phase extraction for sample preparation, this can be attributed to insufficient selectivity of sample preparation procedure which is one of the difficulties in determination of sildenafil in biological samples [8], [12], [13].

Therefore, it is essential to have a sensitive HPLC method with simple liquid–liquid extraction technique. In this work we afford a sensitive, rapid, simple HPLC method with Limit of quantification comparable to HPLC with mass spectrometry methods. This method should add an important contribution to the field of clinical assays and bio-analytical researches with labor, time and money saving advantages. The method validated according to EMEA validation guideline.

Section snippets

Chemicals and reagents

Sildenafil citrate (purity 99%) and etoricoxib (purity 99%) were purchased from (Sigma). Chemical structures are presented in Fig. 1.

HPLC-grade acetonitrile, Triethylamine (TEA) and water were obtained Merck. We used analytical-grade sodium carbonate, perchloric acid (PCA), phosphoric acid (H3PO4) and diethyl ether (Sigma–Aldrich).

HPLC

The work was carried out on a Finnigan Surveyor high performance liquid chromatography with ChromQuest software 4.2.34, equipped with Solvent delivery systems pump

Separation

Chromatograms of the extracted blank plasma, spiked plasma with IS, spiked plasma with sildenafil are shown in Fig. 2. The retention times of sildenafil and etoricoxib were approximately 8.3 and 5.8 min, respectively with 10 min run time. The chromatograms reveal symmetrical and completely separated peaks without any interfering peaks. Thereby, the two-step extraction procedure is sufficient to isolate sildenafil and etoricoxib from the plasma without any interfering endogenous peaks.

Specificity and selectivity

No

Discussion

Selective inhibitor of PDE5, sildenafil has been successfully used by millions of men worldwide for erectile dysfunction treatment. Sildenafil is also currently used for pulmonary hypertension and evaluated in treatment of many medical conditions, such as neurodegenerative illnesses, depression, cardiovascular disease and Raynaud's phenomenon [17], [18].

Therefore, having a sensitive, accurate and simple HPLC method for determination of sildenafil in plasma is essential in clinical,

Conclusion

A sensitive, reliable and rapid HPLC method with UV detection and simple liquid–liquid extraction procedure was developed and validated for sildenafil determination in human plasma. The method uses an economic and relatively fast sample preparation that results with good recovery of sildenafil from plasma. The total run time does not exceed 10 min per sample. The validation of the assay in human plasma was performed according to the EMEA guideline and the obtained results were within the

Acknowledgment

The authors would like to thank University of Petra for the endless help and support.

References (19)

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