Persistent phototransformation products of vardenafil (Levitra®) and sildenafil (Viagra®)

At the same time, the results obtained after the analysis of model urine samples containing vardenafil (Fig.

What should I know about storage and disposal of this medication?

The mobile phase was given in linear gradient mode, at a rate of 0.4 ml/minute. Duration of the analysis was 25 minutes. Detection of vardenafil and sildenafil in samples was performed using Agilent 6120 single quadrupole mass spectrometer with electrospray ionization. Mass spectral analysis was performed in the m/z range: 50–500 with the fragmentation of 50 m/z under positive ionization. Ion source for mass spectrometric detection: API-ES.

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The temperature of nitrogen (gas drier) was 300°C; pressure on a nebulizer was 40 psi, speed of nitrogen in an ion source—10 l/minute. Two groups of animals were selected for the study: control and study [10 rats each (males) weighing 190–200 g]. The study and control group of animals had free access to food and water. The rats’ urine from both groups was collected for 24 hours. The study group of animals was administered an aqueous suspension of “Levitra” for 0.28 mg/kg once.

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The aqueous suspension was obtained by dissolving the Levitra tablets (5 mg) in 10 ml of water. Nearly, 5 ml of urine were taken for one study. The rats were kept at the Vivarium Center of Danylo Halytsky Lviv National Medical University. Experiments adhered to ethical standards and approved by the ethical committee of mentioned university (Approval File No. Animal urine was analyzed immediately after collection or stored in a frozen state until used. 2) biological fluid samples of rats taking Levitra (Fig.

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3) were compared with samples of pure rat urine (Fig. Major metabolites were noted on the chromatograms of urine samples of rats taking Levitra, the peaks of which deviated and were absent on the chromatograms of control urine samples.

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After the test was added, the columns were washed with 2 ml of a universal buffer solution (pH 7.4) and 1 ml of distilled water. Within 5 minutes, the sorbent was dried in a stream of nitrogen; elution was performed with 2 ml of methanol. The speed of all liquids passing through the sorbent was 1 ml/minute. The resulting eluate was evaporated to dryness in a stream of nitrogen and dissolved in 500 μl of methanol. Nearly, 10 μl methanol solution was injected into the chromatograph.

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Control urine samples were examined in parallel according to the same mode. Validation of the elaborated technique for the quantitative determination of vardenafil in urine was carried out in accordance with the FDA and EMA guidelines on validation of bioanalytical methods (European Medicines Agency, 2011; Food and Drug Administration, 2013). To prove the specificity of the technique, the chromatograms of control samples of rat urine and those added to the internal standard and working solutions of vardenafil were compared. The concentration of the working solution of vardenafil corresponded to the lower range of the quantitative determination—7 ng/ml, and sildenafil—200 ng/ml. The linearity of the method was studied on model urine mixtures in the range of concentrations of vardenafil 7–500 ng/ml.

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Applying the least squares method, the equation of the line, which describes the relationship between the ratio of peak areas of vardenafil to the internal standard (sildenafil) and the concentration of vardenafil in urine, ng/ml, was calculated. The limit of detection (LOD) was determined on five model urine samples with a signal-to-noise ratio of at least 3:1. The limit of quantification (LOQ) was defined as the lowest concentration of vardenafil, which can be accurately measured [coefficient of variation (CV) of less than 20%]. The study was conducted on five model urine samples containing 7 ng/ml vardenafil and 200 ng/ml sildenafil (internal standard), independent of the calibration curve. The matrix effect is a direct or non-direct change in the instrument signal due to the presence of other analytes in the sample. Their identification was carried out with the obtained mass spectra.

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Mass spectra of extracted metabolites are shown in Figure 6. From 11.27 ± 0.28 to 13.60 ± 0.30 ng of vardenafil in 1 ml of daily urine of animals were determined by the proposed method.

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Standard solutions of vardenafil (1 mg/ml) and sildenafil (internal standard, 1 mg/ml) were prepared by dissolving the accurately weighed quantity of standard substances in methanol (solutions A). Obtained standard solutions can be used within 3 months, keeping them at 4°C in a dark place. Methanol solutions of vardenafil and sildenafil containing 10 μg/ml (solution B) were prepared from standard solutions A. A solution of vardenafil containing 1 μg/ml (solution C) was prepared by diluting the solution B in methanol. For analysis, three parallel series of urine samples with a concentration of vardenafil from 7 to 500 ng/ml (7.0, 50.0, 100.0, 200.0, 300, 400, and 500.0 ng/ml) was prepared.

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The volumes of biological fluid were adjusted to 5 ml with urine. Samples were stirred for 3 minutes in an orbital shaker and then incubated at 37°C for 60 minutes. The obtained solutions were used to construct calibration graphs using the internal standard method in the concentration range of 7–500 ng/ml. Similarly, urine specimens were prepared for validation of the method, in which the vardenafil concentration was 7, 200, and 500 ng/ml, and each was subjected to 100 μl of the internal standard. Model samples and validation solutions were subjected to further sampling according to the scheme described below.

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The urine samples were adjusted to pH 7.5 with 30% sodium hydroxide solution, followed by double extraction with 1,2-dichloroethane (5 ml), the duration of single extraction was 10 minutes. The combined dichloroethane fractions were evaporated to dryness. The dry residue was dissolved in 0.4 ml of methanol and diluted to 2 ml with water. The entire volume of the methanol-water solution was purified through SPE cartridges. The cartridges were conditioned with 1 ml of methanol and 1 ml of distilled water. The total metabolites were about 50% of the concentration level of the medication. In the analysis of urine specimens, chromatograms do not show peaks with the retention time corresponding to vardenafil, its metabolites, and sildenafil.

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The short-term and long-term stability of vardenafil, as well as the stability of vardenafil during three freeze-defrost cycles, were studied on model urine samples containing 7 and 500 ng/ml of the medication. The retention time of the internal standard (sildenafil) was 6.103 ± 0.052 minutes. Urine sample chromatogram without medications, as well as urine sample chromatogram with vardenafil concentration of 300 ng/ml and the internal standard (sildenafil) of 200 ng/ml, are shown in Figures 1 and 2. The mass spectra of vardenafil and sildenafil are shown in Figures 4 and 5. The proposed method for the extraction and determination of vardenafil was used to analyze urine specimens of laboratory rats taking the study medication. The mass spectrum of vardenafil extracted from the urine corresponds to the mass spectrum of the standard sample of vardenafil and it is characterized by such signals of ions—489, 491 m/z. Quantitative analysis was carried out by the internal standard method.

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In the study of this criterion, six series of biological matrices (urine) were used. The influence of the generic levitra pills canada biological matrix on the ionization efficiency of the analyte and the internal standard was calculated by analyzing the ratio of the peak area of the analyte in the biological sample to the peak area of the analyte in the methanol solution. Normalized by the internal standard, the matrix effect was determined as the ratio of the matrix effect of the analyte to the matrix effect of the internal standard. The matrix effect was studied on the lower (7 ng/ml), middle (200 ng/ml), and upper (500 ng/ml) levels of vardenafil concentrations. To determine the accuracy and precision of urine samples, the amounts of vardenafil were given, which corresponded to the lower range of quantitative analysis by this method (7 ng/ml), middle (200 ng/ml), and upper level of concentration (500 ng/ml), as well as 200 ng/ml of sildenafil (internal standard).

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Six samples were prepared with each concentration level. Concentrations of medications in samples were calculated by the equation of calibration graphs; afterward, the obtained values of concentrations were compared with their nominal value. Samples were analyzed on the day of preparation and 24 hours after (samples were stored at 4°C). The degree of extraction of vardenafil from the urine was determined by calculating the ratio of the amount of vardenafil extracted from the biological sample to the amount introduced into the dry residue of the control sample. The same concentration levels were used to determine the accuracy and precision; six samples for each level were prepared. Applying the least squares method, the equation of the line, which describes the relationship between the ratio of peak areas of vardenafil to the internal standard and the concentration of vardenafil in urine, ng/ml, was calculated. The linear dependence for vardenafil was in the range of 7–500 ng/ml. The equation of the calibration graph in the concentration ranges from 7 to 500 ng/ml is described by the dependence Y = 0.0038 X − 0.0116, where: Y—the ratio between peak areas of vardenafil and internal standard, X—concentration of vardenafil, ng/ml.

Formulation	Average Cost per Dose	Convenience	Onset Speed	Storage	Notes
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The LOD of vardenafil by HPLC/MS on chromatograms was determined by the signal-to-noise ratio of 3:1 and the LOQ by the ratio of 5:1.

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The results of the precision and accuracy of the evaluation of vardenafil in urine samples by the HPLC-MS are presented in Table 1, and in the urine of the rats taking Levitra tablets in Table 2.

Ingredient	Concentration	Purpose	Notes
Vardenafil Hydrochloride	20 mg/mL	Active component for erectile dysfunction relief	Prescribed dose varies
Glycerin	10%	Solvent and base	Enhances absorption
Purified Water	Q.S. to 100%	Diluent	Ensures stability
Flavoring Agents	-	Improves taste	Commonly mint or berry

The developed method is accurate and reproducible since the data are repeatable within one day and on different days.

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The matrix effect, normalized by the internal standard for six samples of biological fluid, is on average 1.14; the average absolute deviation of the matrix effect is 2.54.

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