Stability-indicating HPLC method for the determination of nicardipine in capsules and spiked human plasma. Identification of degradation products using HPLC/MS

2.2.1 Mass spectrometry

Identification of intermediate products was performed by LC/MS. LC/MS experiments were carried out using a Waters Micromass ZQ Detector (Waters, Milford, USA) Quadrupole mass analyzer (mass range 2–2000 amu) equipped with electrospray ionization (ESI) interface in positive mode. The needle voltage was approximately 3500 v and the cone voltage was held at approximately 60 v. MS data acquisition and analysis were performed using MassLynx® 4.0 software Micromass™ version 3.5.

2.2.2 Chromatographic conditions

Separations were carried out using a Waters 2695 Separation Module (Waters, Milford, USA) system consisting of a vacuum degassing module Rheodyne Model 7725 injector equipped with a 20 μL loop, a quaternary pump, column heater and variable wavelength detector. Data acquisition was performed using the MassLynx® 4.0 software Micromass™ version 3.5. The column was Waters Symmetry® C₁₈ 150 mm × 3.9 mm, 5 μm particles size. The column temperature was maintained at 25 °C and the flow rate was 1.0 mL min⁻¹. The detector wavelength was set at 353 nm. The mobile phase consisted of 70% methanol: acetic acid containing 0.01 M triethylamine with pH 4 (Al-Ghannam and Al-Olyan, 2009).

2.2.3 Spectrophotometry

The spectrophotometric analysis was performed on double beam UV/Visible spectrophotometer V-350 (Jasco, Japan) with matched 1-cm quartz cells.

2.3.1 Preparation of standard solutions and calibration graphs

A stock solution containing 1 mg/mL of NIC was prepared in methanol. NIC stock solution was further diluted with the same solvent and then with the mobile phase as appropriate to obtain the working standard solutions ranging between 0.5 and 40 μg/mL. The peak area ratio was plotted versus the concentration of the drug (μg/mL) to get the calibration graph. Alternatively, the corresponding regression equation was derived. The stock solutions were found to be stable for at least two weeks when kept at 4 °C.

2.3.2 Preparation of capsules solutions

Ten capsules from NIC formulation (50 mg/capsule) were weighed and emptied then mixed well. Appropriate amount of the powdered capsules equivalent to 50 mg of the drug was transferred into 50 mL volumetric flask sonicated well with 80 mL methanol for 20 min. Then the solution was completed to the volume with the same solvent to give a stock solution of 1 mg/mL of NIC. This solution was filtered through millipore filter (0.45 μm pore size) and aliquots of this solution were diluted with the mobile phase as appropriate. The nominal contents of the capsules were calculated using either the calibration graph or the corresponding regression equation.

2.3.3 Assay of NIC in human plasma

1.5 mL aliquots of plasma were transferred into a set of centrifugation tubes. Aliquots from the drug stock solution were added so that the final concentration is in the range 5–40 μg/mL. The solution was mixed thoroughly using a vortex mixer. 0.5 mL of 0.5 M borate buffer (pH 9.0) and 5 mL of n-hexane/butanol (12:1 v/v) were added to each tube and gently mixed for 5 min. and centrifuged at 1000 rpm for 10 min. The organic phase was then transferred to a set of small beakers and evaporated under a stream of nitrogen. The residue was dissolved in 10 mL methanol. Aliquots of 30 μL were injected (triplicate) and eluted with mobile phase under the reported chromatographic conditions. A blank sample using plasma without the drug was measured simultaneously. The peak area ratio was plotted versus the concentration of the drug in μg/mL. The corresponding regression equation was derived.

2.4.1 Acid degradation

The effect of strong acid was studied in 10 mL volumetric flask by treating 10 μg/mL solution of NIC with 1 M HCl. This solution was heated at 60 °C for 1 h. Samples were withdraw (1 mL) every 10 min. and then neutralized with the corresponding volume of 1 M NaOH and analyzed by the previously described HPLC/MS method.

2.4.2 Base degradation

The effect of strong alkalis was studied in 10 mL volumetric flask by treating 10 μg/mL solution of nicardipine with different concentrations of NaOH (0.1, 0.2, 0.3, 0.4 and 0.5 M). This solution was heated at (50, 60, 70 and 80 °C) for 1 h. Samples were withdraw (1 mL) every 10 min. and then neutralized with the corresponding volume of 1 M HCl and analyzed by the previously described HPLC/MS method.

2.4.3 Oxidative degradation

The effect of hydrogen peroxide was studied in 10 mL volumetric flask by treating 10 μg/mL solution of nicardipine 5% with H₂O₂. This solution heated at (30, 40 and 50 °C) for 5 h. Samples were withdraw (1 mL) every 1 h then neutralized and analyzed by the previously described HPLC/MS method.

2.4.4 UV degradation

For the kinetic study of photo degradation, the experiments were carried out at room temperature (25 ± 2 °C) in a box containing a UV light system. A 4-Watt, short/long wave Ultraviolet lamp, two separate tubes (254 and 365 nm), intensity, (μw/cm²): 760/720 at 3″ (Cole Parmer, USA). 10 μg/mL Nicardipine methanolic solutions was placed in a cylindrical, quartz cell of 2.4 mL capacity and 1 cm path length, and then irradiated from a source (245 nm) at a distance of 13 cm for up to 3.5 h. This solution was measured every half hour spectrophotometrically (to follow the time needed for complete drug degradation) and by HPLC/MS (to identify the intermediate compounds).

3.3.1 Acid hydrolysis

Nicardipine appears to be stable to acid degradation which is in agreement with acid degradation of isradipine (Abdel Ghany et al., 1998). It was observed that on heating at 60 °C in 1 M HCl, there was no degradation peak product eluted in the chromatogram. Fig. 2A shows the chromatogram after 1 h of heating the reaction mixture at 60 °C with HCl.

3.3.2 Alkaline hydrolysis

From the preliminary study conducted on the degradation of nicardipine in 0.4 M NaOH at 60 °C for 1 h, it is evident that the drug underwent decomposition. The rate of alkaline hydrolysis was fast with significant reduction in the peak area, and new peaks of the degradation products were observed. The drug was found to be highly susceptible to alkaline hydrolysis. The reaction in 0.4 M NaOH at 60 °C was so rapid that 50% of the drug was degraded within 1 h, forming degradation products at RT 6.35 and 6.92 in addition to that of remaining nicardipine RT = 8.93 min. Fig. 2B. Study of the kinetic parameters for the degradation of nicardipine involved measuring the peak height of nicardipine only.

Table 1 and Fig. 3 show the increase in the value of the reaction rate constant (K) and the decrease in the half-life time (t_1/2) upon increasing the concentration of sodium hydroxide.

Close

Figure 3

Effect of concentration of sodium hydroxide on the rate of degradation of Nicardipine HCl (10 μg/mL) at room temperature.

Export to PPT

The combination of effect of the concentration of sodium hydroxide with the effect of time and temperature was studied. Fig. 4 shows the effect of the combination of the three factors for the concentration of 0.2 M of NaOH. Table 2 is a collection of the data representing the effect of increasing the concentration of NaOH at different temperature; the values of the slopes; reaction rate constant; half-life time and activation reaction are listed. As showed from the results the values of activation energy were 55.27–65.67 J/mol K in agreement with the corresponding values for the hydrolysis of esters (Conners et al., 1979; Ibrahim et al., 2010).

Close

Figure 4

Three-dimensional plot representing log (a/a–x)-degradation time relationship for nicardipine HCl (10 μg/mL) at different temperatures in 0.2 M NaOH.

Export to PPT

3.3.3 Oxidation

Fig. 2C shows the total ion chromatogram from the hydrogen peroxide 5% degradation after 5 h of heating the reaction mixture at 40 °C. A single major component is observed at a retention time of 7.8 min. and comprises 100% of the original sample. Table 3 listed the data of kinetic parameters for this degradation at different temperature.

3.3.4 Photolytic degradation

The most undesirable property of 1,4-DHPs from pharmaceutical point of view is their high photochemical sensitivity which can involve molecular changes leading to decrease of therapeutic effect and even some toxic effects after administration (Barand et al., 2006). In this part, the methanolic solution of nicardipine (10 μg/mL) was exposed to the UV light at 245 nm to identify the photo degradation products for the drug. As shown in Fig. 5, the exposure to UV light at wavelength = 245 nm resulted in photo induced modifications of the drug chromophore, as indicated by the significant changes of the UV spectral absorption of the solutions (irradiation time 0–210 min).

Close

Figure 5

Change in the absorption spectrum of nicardipine solution (10 μg/mL) in methanol after UV irradiation (0–210 min).

Export to PPT

In particular a decrease of the absorption band at λ = 353 nm was observed in agreement with a previous study on related 1,4-dihydropyridine derivatives (Albini and Fasani, 1998). This decrease of absorption suggests a significant modification (aromatization) of the 1,4-dihydropyridine chromophore (Alvarez-Lueje et al., 2003). Moreover, increased absorption at λ = 280 and 310 nm can be observed and is attributed to formation of degradation products.

HPLC–UV chromatograms, acquired at 353 nm, of the photo exposed solutions Fig. 2D showed the formation of four photo degradation products at RT 4.40, 4.98, 6.06 and 7.13. The high percentage of unchanged nicardipine showed that this compound was much more stable compared with the other 1,4-dihydropyridine compounds for which no parent compound was detectable after 2 h of light exposure (Barand et al., 2006; Albini and Fasani, 1998; Alvarez-Lueje et al., 2003; Pomponio et al., 2004).

3.4.1 Identification of base degradation

The Chromatogram of NIC degradation showed two peaks in addition to that of NIC. It is postulated that the decomposition pathway as shown below:

Close

Export to PPT

Upon heating NIC in alkaline medium, it undergoes the following changes, the two ester groups attached to the dihydropyridine ring are hydrolyzed, resulting in the formation of CH₃OH (P3) which is not detectable, and N-methyl-N-(2-hydroxylethyl)benzylamine (P2).

At the same time, NIC undergoes photochemical decomposition through auto-oxidation reduction resulting in reduction of the nitro group to nitroso and oxidation of the dihydropyridine ring (P1), as previously reported (Albini and Fasani, 1998).

Compound (P1), the acid fraction (present as sodium salt) is highly polar, which have peak with high intensity more than compound (P2) or nicardipine itself (Walash et al., 2007). Fig. 6 shows the mass spectrum of degradation of nicardipine with sodium hydroxide (0.4 M) at 60 °C for 1 h, it illustrate that compound (P1) has peak intensity of 100% compared to the intensity of residual nicardipine which was 50%.

Close

Figure 6

Positive ion electrospray MS of nicardipine (10 μg/mL) methanolic solution incubation with sodium hydroxide (0.4 M) for 60 min at 60 °C, MS conditions: 30 V for cone voltage, 3300 V for capillary voltage, 1.5 V for extraction voltage.

Export to PPT

The LC/MS under the same conditions Fig. 2B shows two peaks produced from the degradation which have same intensity, therefore, it is proposed that two peaks appear to be isomers since they have the same molecular weight 353 (P2) appears in less intensity and at molecular weight 165.

3.4.2 Identification of oxidative products

As mentioned before this oxidation produced one product. Fig. 7 shows the mass spectra of nicardipine after degradation by H₂O₂ 5% at (a) t = 0 and (b) t = 5 h. It illustrates that the major compound appear at molecular weight 496. It proposed that product result from the oxidation of dihydropyridine to pyridine derivative in addition to the oxidation of the tertiary amine to the N-oxide (Milcent and Chau, 2002). The increase in molecular weight of 16 would tend to support this assignment which is equal the molecular weight of hydroxyl [M + OH]. And the chromatogram is agreed with the obtained mass spectrum where the intensity of the produced compound is equal to 75% and the intensity of residual nicardipine is equal to 50%. The proposed structure of oxidation product of nicardipine with hydrogen peroxide is suggested as follows:

Close

Export to PPT

Close

Figure 7

LC–MS spectrum of nicardipine solution (10 μg/mL) incubation with hydrogen peroxide for 5 h at 40 °C, (a) 0 h and (b) 5 h, MS conditions: 30 V for cone voltage, 3300 V for capillary voltage, 1.5 V for extraction voltage.

Export to PPT

3.4.3 Identification of photo degradation

In contrast to chemical degradation photo degradation shows the formation of four different products Fig. 2B. The major photo degradation product form through the conversion of the dihydropyridine ring to a pyridine ring via loss of 2H⁺. This product has been reported as the major photo degradation product from all of the dihydropyridine based calcium channel blockers studied to date (Keaneth et al., 1984; Ibrahim et al., 2010).

Close

Export to PPT

The nicardipine mass spectra are shown in Fig. 8a along with a scheme of fragmentation, represented of the fragmentation pattern of all the main photoproducts. Fig. 8b shows reduction of the peak at m/z = 466 resulting from the reduction of nitro to nitrous group. Fig. 8c shows the proposed degradation products of nicardipine obtained after the exposure of a methanolic solution (10 μg/mL) to UV (245 nm) light for 210 min.

Close

Figure 8

LC–MS spectrum of nicardipine solution (10 μg/mL) in methanolic solution after different time of exposure to UV light: (a) 0 min, (b) 90 min and (c) 210 min, MS conditions: 30 V for cone voltage, 3300 V for capillary voltage, 1.5 V for extraction voltage.

Export to PPT

3.5.1 Linearity

To establish linearity and range, a stock solution containing 1.0 mg/mL of NIC was diluted at different concentration and response for the NIC was found to be linear in the concentration range between 0.5 and 40 μg/mL. Linear regression analysis of the data gave the following equation: $$P=0.4046+0.6062C(R^2=0.999)$$ where C is the concentration in μg/mL, P is the peak area ratio and R is the correlation coefficient. The result shows an excellent correlation existed between the peak area ratio and concentration of the analyte.

3.5.2 Specificity and selectivity

The specificity of the method was established through study of resolution factors of the drug peak from the nearest resolving peak and also among all the other peaks (ICH-Harmonised Tripartite Guideline, 2003). The presence/absence of peaks due to excipients, impurities and degraded products was examined to study the interference from these substances in the assay of the drug. A solution containing all the capsule excipients omitting NIC was prepared and a blank plasma sample was performed under the optimum chromatographic conditions. No interfering peaks appeared in the chromatogram at the retention time of the drug or its degradation product indicating the specificity of the proposed method Fig. 9a.

Close

Figure 9

HPLC chromatograms of (a): plasma blank; (b) NIC (10 μg/mL) in spiked human plasma.

Export to PPT

3.5.3 Precision

For repeatability, a solution of 10 μg/mL was injected three times and the RSD of these three determinations was not more than 1.9% and no change in retention time of the drug. Intraday and inter day dilutions of 5, 15, 30 μg/mL were injected three times in the same day and in three consecutive days, respectively. The overall RSD of both intraday and inter day was not more than 1.2%.

3.5.4 Sensitivity

The limit of detection (LOD) and the limit of quantification (LOQ) of NIC were determined based on signal-to-noise ratio of 3 and 10, respectively (United States Pharmacopoeia, 2007). The baseline noise was measured in a blank experiment in the region of retention time of NIC using chromatographic software. It was found that, the LOD and LOQ values were 0.11 μg/mL and 0.036 μg/mL, respectively.

3.6.1 Dosage form analysis

The proposed method was successfully applied to the assay of NIC in commercial capsules (Pelcard capsules, 50 mg). The average percentage found of different concentrations of the drug was based on the average of three replicate determinations. The mean% recovery of NIC in the examined dosage form was 100.11%. High percentage recovery values revealed that the proposed method is accurate and could be adopted for routine quality control.

3.6.2 Spiked human plasma analysis

Nicardipine is rapidly absorbed from the gastro intestinal tract but is subjected to saturable first-pass hepatic metabolism. Bioavailability of about 35% has been reported following a 30 mg dose at steady state. The terminal plasma half-life is about 8.6 h (Parfitt, 1999). The method could be successfully applied to the determination of NIC in spiked human plasma Fig. 9b. The plasma calibration curve was linear with the following equation: $$P=13.699+0.0713C(R^2=0.9991)\text{;}$$ where P is the peak area ratio, C the concentration of the drug in μg/mL and R is the correlation coefficient. The accuracy of the proposed method was assessed by investigating the recovery of NIC at three concentration levels covering the specified range using spectrophotometric detection. The mean% recovery of NIC was 99.04%.