HPLC-DAD stability indicating determination of pentoxyverine citrate. Application to degradation kinetics and assay of syrup dosage form

2.1 Instrumentation

The HPLC-DAD system consisted of Agilent 1200 series (Agilent Technologies, Santa Clara, CA, USA) (quaternary pump, vacuum degasser and diode array and multiple wavelength detector G1315 C/D and G1365 C/D) connected to a computer loaded with Agilent ChemStation Software. A Rheodyne manual injector with 20 μL loop was used. The column used was Waters Symmetry C8 (3.9 × 150 mm, 5 μm particle size).

2.2 Materials

Pentoxyverine citrate was kindly supplied by Borg Pharmaceutical Industries Co., Alexandria, Egypt. HPLC-grade acetonitrile (Scharlau Chemie S.A., Sentmenat, Spain), HPLC-grade methanol (Fisher Scientific UK Limited, Loughborough, Leicestershire, UK), analytical grade of ortho-phosphoric acid, hydrochloric acid, sodium hydroxide, 50% hydrogen peroxide, glacial acetic acid, boric acid and high purity distilled water were used. The pharmaceutical preparation assayed in the study is Cabella® syrup (Borg Pharmaceutical Industries Co., Alexandria, Egypt, BN 034015) labeled to contain 21.3 mg pentoxyverine citrate in each 10 mL syrup.

2.3 General procedure

A gradient mobile phase system consisting of (A) 0.025 M phosphoric acid and (B) acetonitrile was used. The separation was achieved with a linear gradient program as follows: 10% v/v B at zero time; from 0 to 10 min, ramp up to 60% v/v B; from 10 to 15 min, holding 60% v/v B. After 15 min, the gradient program was returned to the initial conditions and the analytical column was reconditioned for 3 min. The flow rate was 1.0 mL/min. The injection volume was 20 μL. The eluant was monitored by the diode array detector from 190 to 400 nm, and chromatograms were extracted at 210 nm. All determinations were performed at 25 °C.

PTV stock solution (1000 μg/mL) was prepared in HPLC-grade methanol. The working solutions were prepared by dilution of the stock solutions with distilled water to reach the concentration range 10–150 μg/mL PTV, respectively. Triplicate injections were made for each concentration and chromatographed under the previously described LC conditions. The peak areas were plotted against the corresponding concentrations to construct the calibration graphs.

2.4 Assay of syrup dosage form

Accurate volumes of Cabella® syrup were diluted with distilled water to obtain final concentrations within the analytical range of PTV (10–150 μg/mL) then treated as under General Procedure. Recovery values were calculated from similarly treated standard solutions. For standard addition assay, sample solutions were spiked with aliquots of PTV standard solution to obtain total concentrations within the previously specified range then treated as under the general procedure. Recovered concentrations were calculated by comparing the analyte response with the increment response attained after addition of the standard.

2.5 Forced degradation and stability-indicating study

Forced degradation studies were carried out on the PTV standard as well as its commercial syrup dosage form according to the following conditions:

1. Acidic and basic conditions: PTV solution was treated with 1 mL of 1 M HCl or 1 M NaOH. The acidic degradation solution was placed in a water bath at 100 °C for 1 h, while the solution in NaOH was kept at room temperature for 1 h. After the specified time, all solutions were neutralized by adjusting the pH to 7.0 and then diluted with distilled water to reach a final concentration of 75 μg/mL of PTV. The same procedure was applied to an aliquot of the syrup dosage form.

2. Oxidation with H₂O₂: PTV solution was treated with 0.5 mL of hydrogen peroxide 5%. The solution was placed in a water bath at 80 °C for 1 h. After the specified time, the solution was diluted with distilled water to reach a final concentration of 75 μg/mL PTV. An aliquot of the syrup dosage form was similarly treated.

3. Dry heat degradation: An amount of PTV powder (50 mg) was kept in an oven at 90 °C for 12 h. After the specified time, the powder was dissolved in methanol, and aliquots of this methanolic stock were diluted with distilled water to reach a final concentration of 75 μg/mL PTV.

After the previous treatments, solutions were filtered with a 0.45 μm filtration disk prior to injection to the column.

2.6 Kinetics study

3.1 Optimization of chromatographic conditions

A gradient liquid chromatographic method coupled with diode array detection was developed to provide a suitable procedure for the routine quality control analysis of PTV in its complex syrup dosage form. The most important aspect in LC method development is the achievement of sufficient resolution of the target drug from all other compounds present in the sample with acceptable peak symmetry in a reasonable analysis time. To achieve this goal, several experiments were carried out in order to optimize both the stationary and mobile phases. For optimization of the stationary phase, several reversed phase columns (Zorbax SB-C8 (4.6 × 250 mm), Zorbax SB-C18 (4.6 × 250 mm), Zorbax Eclipse XDB-C18 (4.6 × 150 mm), Waters Symmetry C18 (3.9 × 150 mm) and Waters Symmetry C8 (3.9 × 150 mm)) were tested. The best clear separation between the eluting peaks, sharper symmetric PTV peak and relatively shorter retention time were attained by using the Waters Symmetry C8 column; hence, it became the column of choice for this study. Other columns especially those of 250 mm length resulted in longer retention times for the eluting peaks and broader PTV peak.

The mobile phase composition was evaluated using various proportions of 0.025 M phosphoric acid solution and acetonitrile. Gradient elution was found necessary in order to ensure complete separation between PTV and all other substances in the analyzed samples (forced degradation products or inactive constituents of the syrup dosage form) in a reasonable run time and acceptable peak shape. The best chosen gradient program started with 10% (by volume) acetonitrile ramped up linearly to 60% in 10 min then kept at this percentage afterward. Flow rate was kept constant at 1.0 mL/min all over the run, and temperature was adjusted at 25 °C. It is noteworthy to mention that the applied gradient program produced stable baseline without any drift or deformation.

Quantification was achieved using diode array detection based on peak area measurement. PTV is considered a weak UV absorbing compound. It exhibits considerable absorbance only in the short UV region (below 230 nm); consequently, 210 nm was found suitable to record all chromatograms in this study. The above described chromatographic conditions showed symmetric PTV peaks with good system suitability parameters. Fig. 2A shows a typical chromatogram for standard PTV. The drug elutes at retention time 7.03 ± 0.056 min with retention factor (k) = 3.18. The column performance (apparent efficiency) can be expressed by the number of theoretical plates (N) for the analyte peak which equals about 63780.

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Figure 2 HPLC chromatogram of 20 μL injection of 75 μg/mL standard PTV solution (A). Chromatograms of solutions of 75 μg/mL PTV after exposure to acid degradation (1 M HCl/100 °C for 1 h) (B), base degradation (1 M NaOH/room temp for 1 h) (C), oxidative degradation (5% H₂O₂/80 °C for 1 h) (D) and dry heat degradation (90 °C for 12 h) (E).

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Figure 2 HPLC chromatogram of 20 μL injection of 75 μg/mL standard PTV solution (A). Chromatograms of solutions of 75 μg/mL PTV after exposure to acid degradation (1 M HCl/100 °C for 1 h) (B), base degradation (1 M NaOH/room temp for 1 h) (C), oxidative degradation (5% H₂O₂/80 °C for 1 h) (D) and dry heat degradation (90 °C for 12 h) (E).

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3.2 Validation of the proposed method

3.2.1

3.2.1 Linearity and concentration range

The linearity of the proposed HPLC procedure was evaluated by analyzing a series of different concentrations of PTV (n = 7) (Fig. 3). The linear regression equation was generated by least squares treatment of the calibration data. Under the optimized conditions described above, the measured peak areas were found to be proportional to concentrations of the analyte. Table 1 presents the performance data and statistical parameters including linear regression equation, concentration range, correlation coefficient, standard deviations of the intercept (S_a), slope (S_b) and standard deviations of residuals (S_y/x). Regression analysis shows good linearity as indicated from the correlation coefficient value (0.9998). In addition, deviation around the slope can be further evaluated by calculation of the RSD% of the slope (S_b%) which was found to be less than 1.0%. The analysis of variance test for the regression line reveals that, for equal degrees of freedom, an increase in the variance ratio (F values) means an increase in the mean of squares due to regression and a decrease in the mean of squares due to residuals. The greater the mean of squares due to regression, the steeper is the regression line. The smaller the mean of squares due to residuals, the less is the scatter of experimental points around the regression line. Consequently, regression lines with high F values (low significance F) are much better than those with lower ones (Table 1).

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Figure 3 3-Dimentional plot of HPLC chromatograms of serial concentrations of standard PTV: 10, 20, 40, 60, 80, 100 and 150 μg/mL.

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Table 1 Analytical parameters for the determination of PTV using the proposed HPLC-DAD method.

Parameter	PTV
Wavelength (nm)	210
Linearity range (μg/mL)	10–150
Intercept (a)	−2.30
Saa	12.42
Slope (b)	20.72
Sbb	0.16
RSD% of the slope	0.77
Correlation coefficient (r)	0.99986
Sy/xc	18.66
Fd	17721
Significance F	4.54 × 10−10
LODe (μg/mL)	2.28
LOQf (μg/mL)	7.60

a Standard deviation of the intercept.

b Standard deviation of the slope.

c Standard deviation of residuals.

d F Equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals).

e Limit of detection.

f Limit of quantification.

3.3 Analysis of syrup dosage form

The optimized HPLC-DAD procedure was applied for the assay of PTV in the pharmaceutical formulation available in the local market (Cabella® syrup). Accurate volumes of the syrup were diluted with distilled water to reach the concentration range of PTV (10–150 μg/mL) then solutions were injected to the column. A representative chromatogram obtained from the syrup sample solution is shown in Fig. 4A. PTV eluted at its specific retention time, and the inactive ingredients of the formulation appeared as well-resolved peaks at retention times 5.37 min (saccharin sodium), 7.43 min (methyl paraben) and 10.10 min (propyl paraben). Resolution (R_s) is a measure of the degree of separation between adjacent peaks. A resolution value of 1.5 is usually regarded as sufficient for the baseline separation of closely eluted peaks (Kazakevich and Lobrutto, 2007). Resolution (R_s) was calculated between PTV and its nearest peak at 7.43 min, and it was found to be 4.41. Additionally, resolution was found to be 9.84 between PTV and the preceding peak at 5.37 min. The diode-array detector enables peak purity verification of the analyte where no signs of co-elution from any of the inactive components were detected. Recoveries were calculated using both external standard and standard addition methods. The assay results revealed satisfactory accuracy and precision as indicated from % recovery, SD and RSD% values (Table 3). The resolution and quantification of PTV in its complex syrup dosage form without any interference from the inactive ingredients can be considered as clear evidence for the specificity of the proposed method.

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Figure 4 HPLC chromatogram of 20 μL injection of a solution containing 85.2 μg/mL PTV obtained from Cabella® syrup (A). Chromatograms of solutions of 85.2 μg/mL PTV obtained from Cabella® syrup after exposure to acid degradation (1 M HCl/100 °C for 1 h) (B), base degradation (1 M NaOH/room temp for 1 h) (C) and oxidative degradation (5% H₂O₂/80 °C for 1 h) (D).

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Figure 4 HPLC chromatogram of 20 μL injection of a solution containing 85.2 μg/mL PTV obtained from Cabella® syrup (A). Chromatograms of solutions of 85.2 μg/mL PTV obtained from Cabella® syrup after exposure to acid degradation (1 M HCl/100 °C for 1 h) (B), base degradation (1 M NaOH/room temp for 1 h) (C) and oxidative degradation (5% H₂O₂/80 °C for 1 h) (D).

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A reference HPLC method was adopted for the determination of PTV in its commercial product (Dönmez et al., 2011). Recovery data obtained from the developed HPLC method were statistically compared with those of the reference method using the Student’s t- and the variance ratio F-tests. In both tests, the calculated values did not exceed the theoretical ones at the 95% confidence level which indicated that there were no significant differences between the recoveries obtained from both methods (Table 3). It is evident from these results that the proposed method is applicable to the analysis of PTV in its syrup commercial formulation with minimum sample preparation and satisfactory level of accuracy and precision.

3.4 Forced-degradation and stability-indicating aspects

Forced degradation studies help in identification of the likely degradation products of a drug substance, which can in turn help in establishing the degradation pathways and the intrinsic stability of the molecule and validate the stability-indicating power of the proposed analytical procedure (Klick et al., 2005). Also, forced degradation studies are useful when the degraded products or impurities are unknown or not available (Klick et al., 2005). Forced degradation experiments were carried out on PTV standard solutions in order to produce the possible relevant degradation products and test their chromatographic behavior using the developed method. Hydrolytic, using strong acidic (1 M HCl) and strong basic (1 M NaOH) media, oxidative (5% H₂O₂) and dry heat degradation experiments were conducted, and the resulting chromatograms were compared with that obtained from standard untreated solution of the drug (Fig. 2A).

No signs of degradation of PTV could be observed in 1 M HCl medium at room temperature for 24 h; however, about 37% decrease in peak area as well as the appearance of a degradation product peak at 11.03 min were clearly noticed after heating with 1 M HCl at 100 °C for 1 h (Fig. 2B). On the other hand, PTV was susceptible to degradation by strong alkali at room temperature. Treatment of PTV with 1 M NaOH and keeping the solution at room temperature for 1 h resulted in about 26% degradation and the appearance of the same previously mentioned degradation peak at 11.03 min (Fig. 2C). Being an ester, PTV is expected to hydrolyze in both acidic and basic media with the formation of the hydrolysis product (1-phenylcyclopentanecarboxylic acid) which appears as a common degradation peak at 11.03 min. Another extra peak at 14.54 min appears only in the chromatograms of basic degradation. This peak shows an absorption spectrum which is very similar to that of the intact drug. This suggests that the drug chromophore was not altered, and this impurity is probably produced from a reaction in the side chain. Further investigation revealed that this extra peak appears only when the solution contains methanol (the solvent used for preparation of PTV stock), while aqueous PTV solutions does not produce this extra peak. This can be explained by the in situ formation of the methyl ester of the hydrolysis product (1-phenylcyclopentanecarboxylic acid). This esterification reaction can occur as an artifact reaction when acid/base hydrolysis reactions are performed using an alcohol co-solvent system such as methanol (Baertschi, 2005). This side reaction does not occur in acid degradation solutions, simply because acid hydrolysis is initiated only at high temperature (100 °C) where methanol evaporates and does not remain in the solution.

PTV was found stable to oxidative degradation at room temperature after 24 h, however, about 7% decrease in peak area as well as the appearance of a degradation product peak at 7.35 min were obviously detected after heating with 5% H₂O₂ at 80 °C for 1 h (Fig. 2D). Resolution between the peaks of PTV and the oxidative degradation product at 7.35 min was found to be 2.95. Hydrogen peroxide reacts with tertiary amines to form tertiary amine oxides (Baertschi, 2005). The degradation peak at 7.35 min shows an absorption spectrum which is identical to that of the intact drug. This implies that the drug chromophore is not involved in the reaction with H₂O₂, and the new peak is most likely for a side chain oxidation product (e.g. N-oxide of the tertiary nitrogen of PTV). Dry heat forced degradation of PTV revealed slight degradation of the drug after heating at 90 °C for 12 h. After the specified time, solutions were prepared from the powder, and main peak appeared at its specific retention time with area about 90% of that of the standard of the same concentration (i.e. about 10% degradation), additionally, two small extra peaks were observed at 2.83 and 14.07 min (Fig. 2E).

Similar forced degradation experiments were carried out on aliquots of the syrup dosage form. As mentioned earlier, a hydrolysis product peak at 11.03 min could be seen in the chromatograms of the acid and base degradations (Fig. 4B and C). Also, obvious changes were noticed for the inactive ingredients peaks especially in acidic medium. These changes included decrease in the peak areas and the emergence of several new degradation peaks at retention times: 2.98, 3.05, 3.65, 4.07 and 4.57 min (Fig. 4B and C). Oxidative degradation of the syrup produced similar observations to those obtained with the standard PTV (lower peak area for the drug and the evolution of a well resolved degradation product peak at retention time of 7.30 min), besides, no signs of change could be seen for the inactive ingredients peaks at 5.44, 7.43 and 10.11 min. Fig. 4D shows the chromatogram of the dosage form solution after treatment with 5% H₂O₂/80 °C for 1 h.

It is noteworthy to mention that peak purity test results obtained from the diode-array detector (DAD) confirm that PTV peak was homogenous and pure in all the analyzed samples subjected to forced degradation conditions.

3.5 Degradation kinetics

The kinetics of acidic hydrolysis of PTV was investigated in 1 M HCl at different temperatures (70, 80 and 100 °C) while basic hydrolysis was studied in NaOH solutions of different strengths (0.1, 0.5 and 1.0 M) at room temperature. Since the decomposition rate of PTV at lower strengths of HCl and/or at room temperature was too slow to obtain reliable kinetic data, solutions of 1 M HCl at elevated temperatures were used to investigate the acidic hydrolysis kinetics. However, significant basic hydrolysis of PTV could be observed at a faster rate compared with that of acidic hydrolysis. Thus milder conditions of NaOH strength at room temperature were applied for basic hydrolysis kinetic study.

3.5.1

3.5.1 Acidic hydrolysis

A regular decrease in the concentration of PTV with increasing time intervals was observed especially at 100 °C. At the selected temperatures (70, 80 and 100 °C), the degradation process followed pseudo-first order kinetics (Fig. 5). From the slopes of the straight lines, it was possible to calculate the corresponding degradation rate constants and half lives (t_1/2) at each temperature, based on the equation (Maher and Belal, 2012):

(1)

$$\mathit{Log}\text{C}=\log {\text{C}}_0-\text{Kt}/2.303$$ where the slope of the line = −K/2.303, C and C₀ are the drug concentrations measured at a given time t and at zero time, respectively and K is the rate constant. These values are gathered in Table 4. Additionally, log K values were then plotted against the reciprocals of the absolute temperature (1/T) to construct the Arrhenius plot (Maher and Belal, 2012; Florence and Attwood, 2006). Arrhenius equation was found to be:

(2)

$$\log \text{K}=13.80-(5286.84/\text{T})$$ Arrhenius plot is presented in Fig. 6. The K₂₅ is used as a measure of the stability of the drug at room temperature. The log K₂₅ value can be obtained by extrapolation on the Arrhenius plot as illustrated in Fig. 6, however, for a more accurate result, it was calculated from Eq. (2) where the log K₂₅ = –3.9411, and therefore K₂₅ had the value of 1.145 × 10⁻⁴ h⁻¹. Accordingly, it was found that half life-time (t_1/2) of PTV in 1 M HCl at room temperature is about 252 days (Table 4). It is clear from these results that PTV can be considered stable at room temperature even in strong acidic medium.

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Figure 5 Kinetic study of the acid-induced degradation of PTV at different temperatures.

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Table 4 Pseudo first-order rate constants (K) and half lives (t_½) for the acidic and basic hydrolysis of PTV.

Temperature (°C)	K (h−1)	t ½ (h)
Acidic hydrolysis using 1 M HCl
100	0.4314	1.61
80	0.0624	11.11
70	0.0253	27.39
25	1.145 × 10−4	6052.40 (252 days)
NaOH strength (M)	K (h−1)	t½ (h)
Basic hydrolysis at room temp
1.0	0.2724	2.54
0.5	0.1272	5.45
0.1	0.0330	21.00

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Figure 6 Arrhenius plot for the acid-induced degradation of PTV.

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3.5.2

3.5.2 Basic hydrolysis

For each NaOH strength (0.1, 0.5 and 1.0 M), log concentration of intact PTV remaining was plotted against time (Fig. 7) and it was found that the degradation followed pseudo first order kinetics. In each case, pseudo first order rate constants were calculated from the slopes of the corresponding plot, Eq. (1). The corresponding (t_1/2) values were also calculated and presented in Table 4.

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Figure 7 Kinetic study of the base-catalyzed hydrolysis of PTV at different sodium hydroxide strengths.

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3.5.3

3.5.3 pH-rate profile study

The effect of different pH values on the degradation of PTV was studied using Britton–Robinson buffer of pH values ranging from 2 to 12 at 100 °C for different time intervals. The apparent first order degradation rate constants were calculated at each pH and plotted versus the pH values resulting in pH-rate profile curve (Fig. 8). Obviously, PTV hydrolyzes in alkaline pH values faster than in acidic pH solutions. The lowest degradation rate constant is within the pH range 4–6 at which the drug solution is most stable.

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Figure 8 pH-rate profile curve of PTV using Britton Robinson buffer at 100 °C.

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