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DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF CHLORHEXIDINE AND P-CHLOROANILINE IN VARIOUS PHARMACEUTICAL FORMULATIONS a
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Marco A. Cardoso , Maria L. D. Fávero , João C. Gasparetto , a
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Bianca S. Hess , Dile P. Stremel & Roberto Pontarolo
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Departamento de Farmácia, Universidade Federal do Paraná, Curitiba, Paraná, Brazil Available online: 09 Sep 2011
To cite this article: Marco A. Cardoso, Maria L. D. Fávero, João C. Gasparetto, Bianca S. Hess, Dile P. Stremel & Roberto Pontarolo (2011): DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF CHLORHEXIDINE AND P-CHLOROANILINE IN VARIOUS PHARMACEUTICAL FORMULATIONS, Journal of Liquid Chromatography & Related Technologies, 34:15, 1556-1567 To link to this article: http://dx.doi.org/10.1080/10826076.2011.575979
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Journal of Liquid Chromatography & Related Technologies, 34:1556–1567, 2011 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6076 print/1520-572X online DOI: 10.1080/10826076.2011.575979
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DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF CHLORHEXIDINE AND P-CHLOROANILINE IN VARIOUS PHARMACEUTICAL FORMULATIONS
Marco A. Cardoso, Maria L. D. Fa´vero, Joa˜o C. Gasparetto, Bianca S. Hess, Dile P. Stremel, and Roberto Pontarolo Departamento de Farma´cia, Universidade Federal do Parana´, Curitiba, Parana´, Brazil
& & A simple, rapid and sensitive isocratic reversed-phase (RP) high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of chlorhexidine (CHX) and p-chloroaniline (CAL) in various pharmaceutical formulations. Compound separation was achieved in less than 10 min with an XBridge C18 column that was maintained at 40 C and a mobile phase consisting of 32:68 (v=v) of acetonitrile and a pH 3.0 phosphate buffer solution (a 0.05 M monobasic sodium phosphate solution containing 0.2% of triethylamine). Analyses were performed at a flow rate of 2 mL min1 and at a detection wavelength of 239 nm. The method was shown to be selective, linear, accurate, and precise in intra-day and inter-day analyses. The robustness of the method was shown by slightly changing the flow rate, column oven temperature, and proportion of acetonitrile in the mobile phase. The method was found, however, to be very sensitive to the pH of the mobile phase buffer. The method was successfully validated following the guidelines of the International Conference on Harmonization (ICH). This validation proved that the method was suitable for the determination of CHX and CAL in toothpaste, mouthwash, wound cleanser, and skin and hand disinfectants. Keywords chlorhexidine, HPLC, p-chloroaniline, pharmaceutical forms, validation
INTRODUCTION Chlorhexidine gluconate (2,4,11,13-Tetraazatetradecanediimidamide, N, N 00 -bis(4-chlorophenyl)-3,12-diimino-di-D-gluconate)[1] is an effective antibacterial agent and the most popular biguanide antiseptic.[2] Reviews on plaque control have concluded that the chlorhexidine (CHX), which is used in mouthwashes and toothpastes, is the most efficient chemical antiplaque agent for the treatment of periodontal diseases as a component of Address correspondence to Roberto Pontarolo, Departamento de Farma´cia, Universidade Federal do Parana´, Av. Prefeito Lotha´rio Meissner, 3400, Curitiba, Parana´, Brazil. E-mail:
[email protected]
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topical slow-release vehicles.[3,4] Due to its antiseptic properties and low systemic and dermal toxicity, CHX is also used as a skin disinfectant in various surgical hand scrubs, patient preoperative skin preparation products, personal hand washing products, and wound cleansing products.[5–7] In preparations, a slow decomposition of the CHX molecule into degradation products can be promoted by light, heat, and ionizing radiation.[8] Due to its toxicity and carcinogenicity, p-chloroaniline (CAL) is the degradation product that causes the greatest concern.[9] Liquid chromatography methods have emerged as the preferred methods for CHX analysis. Indeed, the first analytical procedure that used HPLC was described by Bailey et al.[10] HPLC is commonly used to analyze the CHX content in many different pharmaceutical formulations, including ophthalmic solutions,[11,12] topical ointments,[13] and others.[14–16] The basics of the HPLC method, which is used for the determination of the CHX content and that of its degradation product CAL, is only described for oral rinses in the United States Pharmacopoeia (USP). This method uses a gradient elution mode with a high flow rate (1.5 mL min1) and a final run time that is quite long (21 min), thereby requiring a large amount of mobile phase and analysis time.[1] A series of methods for the determination of CHX, using ion-pair reversed phase HPLC in either a gradient mode[17–19] or an isocratic mode,[20–25] have been described in the literature. Procedures that do not require the use of ion-pair reagents or working in the typical acid range for reversed phase materials were also reported for both isocratic[26–30] and gradient modes.[31–33] Other methods described in the literature used liquid chromatography tandem mass spectrometry[34–36] and capillary electrophoresis[37] for the determination of CHX. However, there are no reports of HPLC methods that were developed and validated for the simultaneous determination of CHX and CAL in tooth-pastes, mouth-rinses, cleansing solutions, or disinfectant pharmaceutical formulations. The present paper presents a novel and rapid RP-HPLC method that operates in an isocratic mode and that was developed for the separation and quantification of CHX and its main degradation product CAL in toothpastes, mouthwashes, and skin and hand disinfectants. EXPERIMENTAL Chemicals and Reagents A 20% CHX gluconate solution (analytical standard, 99.5%) was provided by Pharmanostra (Campinas, Brazil). CAL (analytical standard, 99%) and anhydrous monobasic sodium phosphate (NaH2PO4) were purchased from Sigma–Aldrich (St. Louis, USA). HPLC grade acetonitrile was
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obtained from J.T. Baker (Phillipsburg, USA). HPLC grade water was obtained by Milli-Q reverse osmosis (Millipore, Bedford, USA). All other chemical reagents were of analytical grade or better. The commercially available samples of toothpaste, mouthwash, wound cleanser, and skin and hand disinfectants were purchased from a local drugstore.
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Preparation of Stock and Working Standard Solutions The CHX stock solution (1 mg mL1) was prepared in a 100 mL volumetric flask by diluting 500 mL of the 20% CHX gluconate solution with a 70:30 (v=v) mixture of water and acetonitrile. The CAL stock solution (0.5 mg mL1) was prepared in a 100 mL volumetric flask by dissolving exactly 50.0 mg of CAL in a 70:30 (v=v) mixture of water and acetonitrile. Both solutions were stored at 4 C, in light-resistant containers; the solutions were allowed to reach room temperature before use. Working solutions of CHX and CAL were freshly prepared as external standards using a diluent mobile phase to obtain final concentrations of 200 and 100 mg mL1, respectively. Sample Preparations Mouthwash, wound cleanser, and skin and hand disinfectant pharmaceutical formulations were directly diluted with the mobile phase to obtain 50 mg mL1 CHX solutions. For the toothpaste sample, accurately weighed portions were diluted with 50 mL of mobile phase in a 100 mL volumetric flask to obtain 50 mg mL1 CHX solutions. The samples were sonicated at 50 C for 15 min and vigorously stirred for 15 min. The volume was then adjusted with the mobile phase. The homogeneous samples were centrifuged for 10 min at 4000 rpm, and the supernatants were analyzed. All samples were filtered through a 0.45 mm Millipore membrane before analysis. Additionally, all samples were protected from direct light throughout the preparations. Chromatography Chromatographic separation was performed on an Agilent 1100 LC system consisting of a G1311A quaternary pump, a G1379A degasser, a G1329A automatic injector, a G1315B diode array detector, and the ChemiStation Rev. A.10.02 [1757] software. In an attempt to achieve the best separation, several C18 (150 4.6 mm, 5 mm particle size) columns (a Zorbax Eclipse XDB, Agilent Technologies, USA, an XBridge and a Spherisorb ODS2, Waters Corporation, Ireland) were separately adapted to the
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equipment during method development. Different flow rates (1.0–2.0 mL min1), pH (2.5–5.0), and oven temperatures (20–50 C) were also tested. Chromatograms were analyzed and compared for a better optimization of the efficiency and run time of the chromatographic method. The conditions that were selected for our method were: an XBridge C18 (150 4.6 mm I.D., 5 mm) column using an isocratic mobile phase composed of 32:68 (v=v) acetonitrile and phosphate buffer solution (a 0.05 M monobasic sodium phosphate solution containing 0.2% of triethylamine, in which the pH was adjusted to 3.0 with 85% phosphoric acid). A constant flow rate of 2.0 mL min1 was applied throughout the analysis. UV detection was set at 239 nm. The column oven temperature was maintained at 40 C and the volume of injection was 20 mL. The mobile phase was filtered through a Millipore 0.45 mm PTFE membrane (Molsheim, France) before use.
Validation of the Analytical Method The proposed method was validated by determining the selectivity, limits of detection (LOD) and quantification (LOQ), linearity, precision (intra-day and inter-day), accuracy, and robustness of the method as described in the guidelines of the International Conference on Harmonisation (ICH).[38] Selectivity was evaluated by comparing the chromatograms obtained for samples spiked with CHX and CAL with chromatograms obtained for individual injections of placebo samples of toothpaste, mouthwash, wound cleanser, and pharmaceutical formulations of skin and hand disinfectant. The LOD and LOQ were determined using a triplicate of injections of decreasing concentrations of CHX and CAL. The LOD was determined as the smallest detectable peak at a signal to noise ratio of 3:1. The LOQ was determined as the smallest detectable peak at a signal to noise ratio of 10:1. Linearity was determined using triplicates of injections of ten different concentrations of CHX (0.5 to 200 mg mL1) and CAL (0.25 to 100 mg mL1). The data were subjected to regression analyses, and calibration curves were generated to confirm the linear relationship between the peak areas and the analyte concentrations. The slope, y-intercept, and correlation coefficient were calculated for both the CHX and CAL analytical curves. Accuracy and intra- and inter-day precision were assessed from the results obtained from the triplicates measurements of the three concentration levels of CHX (40, 50, and 60 mg mL1) and CAL (0.5, 2.5, and 5 mg mL1). Precision was expressed as the percentage of the relative standard deviation (RSD %), and accuracy was expressed as the percentage of recovery of known amounts of analytes that were spiked into the samples. The robustness evaluation was based on the percentage of recovery and
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RSD (%) values that were obtained by deliberately changing critical chromatographic parameters of the optimized method. More specifically, the following modifications were performed: change in flow rate by 0.1 mL min1, change in pH of the buffer by 0.5 units, change in column oven temperature by 2 C, and change in the acetonitrile composition of the mobile phase by 0.5%. System suitability was determined by injecting a toothpaste sample six times, which consisted of a 10 mg mL1 CHX solution that was obtained by dissolving toothpaste in the mobile phase. Subsequently, this sample was spiked with a 40 mg mL1 CHX solution and a 25 mg mL1 CAL solution. In this study, the number of theoretical plates, resolution, and asymmetry were assessed according to USP 31 and the Food and Drug Administration (FDA) guidelines.[39] All standard solutions and samples were prepared using the mobile phase as the diluent and filtered through PVDF syringe filters (0.45 mm) prior to the injections. Additionally, all samples were protected from direct light throughout the experiments. RESULTS AND DISCUSSION Optimization of Chromatographic Conditions To obtain an adequate resolution of the target analytes under isocratic conditions without employing ion-pairing reagents, different columns with various properties were evaluated. Numerous experiments were also conducted using various combinations of a wide range of mobile phases that had different acetonitrile proportions and contained buffers of different pHs. The flow rate was initially maintained at 1.0 mL min1, and the col umn oven temperature was kept at 25 C. The initial results of the column and mobile phase screening studies provided a few conditions that could separate CHX and CAL with a resolution of not less than 3.0, as described in USP 31.[1] Spherisorb ODS2 C18 and Zorbax Eclipse C18 XDB columns did not lead to a good separation of CHX, CAL, and the other components present in the pharmaceutical preparations. XBridge C18 exhibited an adequate resolution for CHX and CAL peaks and also resulted in a satisfactory separation of these two compounds from the other components. However, the peak tailing of CHX was significantly high, thereby affecting quantification. To obtain a sharper peak of CHX, 0.2% of triethylamine was added into the mobile phase. This significantly improved the shape of the peaks that corresponded to the compounds of interest. Retention of CHX and CAL significantly increased with decreasing acetonitrile concentrations. Ratios of an organic modifier in the mobile phase ranging below 30% (v=v) resulted in an analysis time that was too long for routine work. The optimal combination of acetonitrile and phosphate buffer for
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FIGURE 1 Representative chromatogram of a standard mixture, containing chlorhexidine gluconate and p-chloroaniline, which was obtained with the developed HPLC method.
the separation of the peaks of interest with the desired resolution was 32:68 (v=v). Silica-based particles are unstable at low pHs (pH < 2). Therefore, a pH range from 2.5 to 5 was initially chosen. As a result, it was observed that the retention time (tr) of CAL increased with an increase in pH, but no change in the tr of CHX was observed. A pH of 3.0 was finally selected, as it was a good compromise between compound separation, analysis time, and peak shape. Varying the column temperature further optimized the separation of specific mixtures. The column temperature was set at 40 C, which was better for the chromatographic column, as it decreases the backpressure during routine work. Due to the relatively low backpressure (1500 psi) of the XBridge C18 column at 1.0 mL min1, the flow rate was increased to 2.0 mL min1. Adequate retention times were thus observed for CHX and CAL (2.1 and 2.6 min, respectively). Finally, the best chromatographic conditions were chosen as follows: an XBridge C18 col umn (150 4.6 mm, 5 mm) kept at 40 C, a mobile phase composed of 32:68 (v=v) acetonitrile=phosphate buffer (0.05 M; pH 3.0), an isocratic elution method at a flow rate of 2.0 mL min1, and UV detection at 239 nm. Under these conditions, all compounds were well separated in less than 10 min. A representative chromatogram of the separation of the target compounds under the optimized working conditions is shown in Figure 1.
Method Validation System Suitability The system suitability was assessed by injecting six toothpaste samples that were spiked with CHX and CAL. The values for the number of theoretical plates, resolution (Rs), and asymmetry (Tf) are presented in Table 1. The results obtained are all within the acceptable limits disclosed in the
1562 TABLE 1
M. A. Cardoso et al. System Suitability Results
Parameter Repeatibility—retention timea Repeatibility—areaa Theoretical platesa Resolution (Rs)a Asymmetry (Tf)a
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a
RSD % RSD %
Chlorhexidine
p-Chloroaniline
Criteria
0.32 0.13 6669 2.55 1.24
0.04 0.44 11624 6.05 0.94
X < 5% X < 5% N > 2000 Rij > 1.5 Tf < 2
One sample injected six times.
USP 31[1] and the FDA guidelines.[39] In other words, the chromatographic method that was developed leads to good resolution and reproducibility. Selectivity Selectivity was evaluated using the matrix comparison method. Chromatograms (Figure 2) showed that no placebo peaks co-eluted with either CHX or CAL and that both analytes were well separated in the toothpaste samples and all of the other formulations tested. Therefore, we conclude that this method is selective and suitable for the identification and quantification of CHX and CAL in the different pharmaceutical formulations that were tested. Limits of Detection (LOD) and Quantification (LOQ) LOD and LOQ were assessed using the signal-to-noise ratio. According to the results presented in Table 2, the developed method demonstrated
FIGURE 2 Representative overlaid chromatograms of (A) placebo toothpaste spiked with chlorhexidine gluconate and p-chloroaniline, (B) placebo toothpaste, (C) placebo wound cleanser, (D) placebo skin and hand disinfectant, and (E) placebo mouthwash, which were obtained with the developed HPLC method.
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RP-HPLC Determination of Chlorhexidine and P-Chloroaniline TABLE 2
Linearity, LOD, LOQ, and Sample Stability of Chlorhexidine and p-Chloroaniline
Parameter Linearitya
LOD (mg mL1) LOQ (mg mL1) Sample stability 22 Cb
Correlation coefficient Intercept Slope
% of recovery
Chlorhexidine
p-Chloroaniline
0.9996 22.3503 17.3765 0.2 1.0 99.30
0.9999 1.5405 39.2564 0.05 0.25 98.82
Linearity range of 0.5 to 200 mg mL1 chlorhexidine and 0.25 to 100 mg mL1 p-chloroaniline, both from three replicates. b One-day stability.
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a
good sensitivity as evidenced by a LOD value of 0.2 mg mL1 and an LOQ value of 1.0 mg mL1 for CHX and a LOD value of 0.05 mg mL1 and a LOQ value of 0.2 mg mL1 for CAL. These compounds can be quantified even though they are present in low concentrations in the pharmaceutical formulations. Linearity The standard curves for CHX (0.5 to 200 mg mL1) and CAL (0.25 to 100 mg mL1) were linear, considering that the correlation value was greater than 0.999 in both cases. The values of the correlation coefficient, y-intercept, and slope, obtained by a least square regression for CHX and CAL, are shown in Table 2. Precision and Accuracy The precision (intra-day and inter-day) and accuracy values obtained for the three different concentrations of CHX and CAL are summarized in Table 3. The values for RSD (%), which were used to assess the intraand inter-day precision, ranged from 1.60 to 3.27% for CHX and 1.34 to 3.79% for CAL. The percentage of recovery, which was used to assess the accuracy of the study, ranged from 98.14 to 102.87% for CHX and 98.26 to 101.17% for CAL. Thus, the method that was developed can be considered precise and accurate. Robustness The robustness of the method was demonstrated by slightly varying the flow rate (0.1 mL min1), column oven temperature (2 C), and proportion of acetonitrile in the mobile phase (0.5%). When the pH of the mobile phase buffer was modified (0.5 units), significant variations in the resolution between CAL and the other detectable compounds were observed. The molecular structure of both components implies that CHX
1564 TABLE 3
M. A. Cardoso et al. Accuracy and Precision of Chlorhexidine and p-Chloroaniline Accuracy
Compound Chlorhexidinea
p-chloroanilinea
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a
Precision
Standard Concentration (mg mL1)
Main Recovery (%)
Intra-Day RSD (%)
Inter-Day RSD (%)
40.0 50.0 60.0 0.5 2.5 5.0
102.87 99.28 98.14 98.26 99.94 101.17
1.70 2.84 3.14 3.79 2.97 2.43
1.60 3.27 2.59 2.25 1.34 2.18
Nine samples injected three times each.
and CAL have different functional groups and, as a result, different pKa values (10.8 and 4.1, respectively). Since CAL is a weak acidic compound (pKa ¼ 4.1), and it is partially ionized at the pH of the mobile phase buffer (pH ¼ 3.0), its retention on the column is more pH dependent than CHX under acidic conditions which makes its retention time pH sensitive. This could explain why changes in the pH of the mobile phase affect only CAL retention time. The retention factor of CAL lowered drastically when the pH of the mobile phase decreased from 3.0 to 2.5, compromising the selectivity of the method. Therefore, pH 3.0 was chosen as an optimum pH because of the reasonable retention times, resolution and separation of all the compounds of interest. Hence, it was concluded that the method is sensitive to the pH of the mobile phase buffer. In other words, the pH of the mobile phase buffer is a critical parameter that must be monitored carefully. Short-Term Stability The stability of CHX and CAL during storage at room temperature was investigated and expressed as the percentage of recovery (Table 2). Spiked samples were stored at room temperature (22 1 C) for approximately 24 hr and protected from light. The results obtained for these samples were then compared to the results obtained for the corresponding freshly prepared samples. No significant degradation of any of the compounds of interest was observed. This result indicates that the analytes were stable under these storage conditions and confirms the applicability of the method to routine analyses. Commercial Sample Analysis Using the proposed method described previously, the contents of CHX and CAL in commercially available samples of toothpaste (Cariax1), mouthwash (Periogard1), wound cleanser (Merthiolate1), and skin and
RP-HPLC Determination of Chlorhexidine and P-Chloroaniline TABLE 4
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Assay of Chlorhexidine and p-Chloroaniline in Different Pharmaceutical Formulations
Compound
Toothpaste
Mouthwash
Wound Cleansing
Skin and Hand Disinfectants
96.40 NQ
104.38 NQ
95.14 NQ
92.66 NQ
Chlorhexidine (%)a p-chloroaniline (mg mL1)
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a Based on the labeled amount. NQ=Under LOQ.
hand disinfectants (Handex Degermante1) were determined. The results, expressed as the percentage of drug compared to the label claim, are shown in Table 4. The results for the drugs assayed were in good agreement with the label claims, demonstrating that the method was a reliable tool and could be applied to the testing of various pharmaceutical formulations.
CONCLUSION The described method was shown to be sensitive and reliable for the quantification of CHX and CAL in toothpaste, mouthwash, wound cleanser, and skin and hand disinfectants. To the best of our knowledge, this is the first study that describes an isocratic, fast, simple, and validated RP-HPLC method for the quantification of compounds in various pharmaceutical formulations. Validation experiments demonstrated that the method is selective, linear, precise, and accurate. The robustness study indicated that the method is very sensitive to pH variations in the mobile phase buffer. Additionally, the solutions of analytes were shown to be stable for over 24 hr at room temperature. Because a large number of samples can be evaluated in a short period of time, the analytical method described herein is an analysis method adequate to be used for routine quality control and for the quantitative determinations of CHX and CAL.
ACKNOWLEDGMENTS The authors gratefully acknowledge CAPES, CNPq, and SETI for their financial support.
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