Abstract Development, validation and comparison of two stability-indicating LC methods, one with photodiode array detector (DAD) and the other with evaporative light scattering detector (ELSD), were performed for simultaneous determination of candesartan cilexetil (CANC) and hydrochlorothiazide (HCTZ), in pharmaceutical samples. A RP-18 column (125 mm × 4 mm, 5 μm) was used for separation of CANC, HCTZ and its major degradation products, using acetonitrile and phosphate buffer (pH 6.0) for DAD method and acetonitrile and water with acetic acid and triethylamine (pH 4.1) for ELSD method, as mobile phase in a gradient mode. The response with ELSD was fitted to a power function and the DAD response by a linear model over a range of 32–160 μg/mL for CANC and 25–125 μg/mL for HCTZ. The precision and accuracy of the methods were similar, with RSD below 3.0% and recovery between 98.1% and 103.9%. The drugs were subjected to stress conditions of hydrolysis, oxidation, photolysis, humidity and temperature. The degradation products were satisfactory separated from the main peaks and from each other. Both drugs mainly degrade by hydrolysis, showing the formation of one degradation product for HCTZ and two for CANC; its identification was conducted by LC/MS/MS. The methods were successfully applied to the analysis of CANC and HCTZ in combined commercial tablets. The performance of DAD and ELSD methods are comparable, therefore both methods are suitable for stability study and determination of CANC and HCTZ in pharmaceutical samples. Introduction Candesartan cilexetil (CANC) and hydrochlorothiazide (HCTZ) (Figure 1) are drugs used in the management of a highly prevalent disease such as hypertension. They are used alone or as combination therapy in the treatment of patients whose blood pressure is not adequately controlled with any of the substances alone. CANC is a prodrug with little pharmacological activity until hydrolyzed during absorption in the gastrointestinal tract to candesartan (CAN) (1). Both drugs have chemical structures susceptible to degradation, so it is important to have suitable analytical methods to assess its stability. HCTZ is degraded mainly to 4-amino-6-chloro-1,3-benzendisulfonamine (DSA) by hydrolysis (2–6). CANC is degraded principally by hydrolysis, oxidation and photolysis, with formation between three and eight degradation products (7–10), among which the most important are CAN and hydroxy CANC (7–9). The differences between these results may be due to degradation conditions; the drug should ideally not degrade more than 20%, to obtain a relevant degradation product (11). Figure 1. View largeDownload slide Chemical structures of CANC and HCTZ. Figure 1. View largeDownload slide Chemical structures of CANC and HCTZ. The most commonly used detector for pharmaceutical analysis is the UV detector since a majority of pharmaceutical compounds have some type of chromophore. Degradation may produce compounds without chromophores, therefore, these degradation products will not be detected by photodiode array detector (DAD). Evaporative light scattering detector (ELSD) is an alternative to UV detection, its response is independent of the chemical structures of the compounds. ELSD measures the intensity of light scattered by solids that remain after the solvent has been evaporated (12). Some studies were done to evaluate the performance of ELSD versus UV in pharmaceutical analysis (13). In the present study we present a detailed comparison between both detectors in stability study of drugs. The stability of a pharmaceutical is defined by its resistance to different chemical, physical and microbiological reactions that may change their original properties (14, 15), chemical degradation of the active constituent often results in a loss of potency; so the clinical use of a pharmaceutical cannot be advisable if the degradation is relatively high (14). In order to assess the chemical stability of a compound it is necessary to have a stability-indicating method (15). The main target while developing these methods is to have a single method for separation between CANC, HCTZ and their degradation products. A stability-indicating method for the simultaneous determination of CANC and HCTZ has not been reported; therefore, comes the need to develop this method. Veeranjaneyulu et al. (16) report a stability-indicating method, but the separation between CANC, HCTZ and their degradation products was not demonstrated. Besides, there are few methods (no stability-indicating) for simultaneous determination of CANC and HCTZ; for analysis in pharmaceutical formulations (17–19) and plasma (19). Stability-indicating LC methods for the simultaneous determination of HCTZ with enalapril (2), losartan (3, 4), quinapril (20), ramipril (21), spironolactone (22), irbesartan (23), aliskiren (24), losartan and atenolol (25) and valsartan with amlodipine (26) have been reported. Stability-indicating LC methods for the individual determination of CANC as a drug substance and pharmaceutical dosage form (7–10) were reported. None of these methods is for the simultaneous determination of CANC, HCTZ and their degradation products. In the present study, we validated and compared two stability-indicating methods, one by LC/DAD and the other by LC/ELSD for the simultaneous determination of CANC and HCTZ in pharmaceutical samples. Both methods were applied to the analysis of real commercial tablets and the stability study of CANC and HCTZ. Moreover, a characterization of degradation products formed under stress testing was performed by LC/MS/MS in order to confirm the identity of the compounds. Experimental Instrumentation and reagents Chemicals and reagents Acetonitrile and methanol LC grade, KH2PO4, sodium hydroxide, triethylamine, acetic acid, hydrochloric acid and hydrogen peroxide p.a. grade were purchased from Merck (Darmstadt, Germany). Standards of CANC, valsartan and DSA (> 99.0% purity) were obtained from USP (Rockville, MD, USA). Standard of HCTZ (>99.0% purity) was obtained from Sigma (St. Louis, MO, USA). HCTZ drug substance was obtained from Diprolab (Santiago, Chile) and CANC drug substance was obtained from Indukern (Spain). Milli-Q grade water was used for the preparation of mobile phase. The commercial drug tablets containing 8, 16 and 32 mg of CANC and 12.5 mg of HCTZ were purchased from a Chilean pharmacy. Instrumentation The LC/DAD method was performed on a Series Flexar HPLC system (Perkin Elmer, Norwalk, CT, USA). The system consists of a binary LC pump, a DAD and a column oven, equipped with a manual injector and a 20-μL loop. Chromera software was used for the data collection. The LC/ELSD method was performed on a YL9100 HPLC system (Young Lin Instrument, Anyang, Korea). The system consists of a YL9110 quaternary pump, a YL9101 vacuum degasser, a YL9130 column compartment and a Sedex model 85 LT-ELSD (low temperature evaporative light scattering detector) (Sedere S.A., Alfortiville Cedex, France), equipped with a manual injector and a 20-μL loop. YL-Clarity Software version 126.96.36.1994 was used for the data collection. LC–MS/MS analyses were carried out with a Shimadzu® HPLC system (Tokyo, Japan) equipped with a quaternary LC-10ADVP pump with a FCV-10ALVP elution unit, a DGU-14 A degasser unit, a CTO-10AVP oven. A UV-VIS diode array spectrophotometer Shimadzu® model SPD-M10AVP coupled in tandem with a 3200 QTrap LC/MS/MS Applied Biosystems® (MDS Sciex,California, USA). Instrument control and data collection system were carried out using a CLASS-VP DAD Shimadzu Chromatography Data System and Analyst software (version 1.5.2) for MS2 analysis. Chromatographic conditions HPLC analyses were carried out on a Purospher® RP-18 column (125 mm × 4 mm, 5 μm; Merck, Darmstadt, Germany). Valsartan was used as internal standard (IS) at 70.0 μg mL−1. For LC/DAD method the mobile phase consisted of acetonitrile (A) and phosphate buffer (pH 6.0; 0.05 M) (B) in a gradient mode; Tmin/A%; T0/12; T5/12; T9/65; T13/65; T16/12 with 10 min for column re-equilibration. The flow rate was set to 1 mL min−1, with UV detector wavelength fixed at 225 nm and the column temperature was set a 30°C. For LC/ELSD method the mobile phase consisted of acetonitrile (A) and water with acetic acid (0.175 M) and triethylamine (0.06 M) (pH 4.1) (B) in a gradient mode; Tmin/A%; T0/8; T7/65; T11/65; T14/8; with 10 min for column re-equilibration. The flow rate was set to 0.8 mL min−1 and the column temperature was set a 35°C. ELSD evaporation temperature was set at 40°C, the gain was 7 and the nebulizer gas pressure was kept at 3 bar. The optimum column temperature, evaporation temperature and gas pressure were obtained using an experimental design procedure by means of the software Statgraphics Centurion XV, version 15.2.05. The mass spectrometer was equipped with an electrospray ionization (ESI) source. The HPLC effluent entered the MS through a steel ionization needle set at 4,000 V (in positive—or negative—ion mode) and a heated capillary set at 350°C. The nebulizer gas was set at 30 psi and auxiliary gas at 20 psi. The ion source and ion optic parameters were optimized with respect to the positive or negative molecular related ions of CANC or HCTZ standards. The molecular mass peaks from the HPLC system were detected by use of positive ion full-scan ESI-MS analysis in the mass range of 100 to 1,200 m/z, in order to obtain the better sensitivity. Mass resolution was 0.2 uma. Standard solutions A stock solution of CANC and HCTZ were independently prepared at 1.0 mg mL−1 in acetonitrile and methanol, respectively. A stock solution of IS was prepared at 2.0 mg mL−1 in acetonitrile and a stock solution of DSA was prepared at 1 mg ml−1 in methanol. Standard solutions were prepared from the stock solutions after adequate dilution with water. Sample solution To prepare the sample solution, 20 tablets were weighed and powered, a portion equivalent to 2.4 mg of CANC and 1.9 mg of HCTZ was accurately weighed and transferred to a 25 mL volumetric flask, then 10 mL of acetonitrile was added. The solution was vortexed for 15 s and sonicated for 15 min. A portion of 0.875 mL IS (2.0 mg mL−1) was added, then the volume was made up to the mark with water and finally the solution was filtered by sample filter (0.45 μm nylon membrane filter) (final concentration of 96.0 μg mL−1, 76.0 μg mL−1 and 70.0 μg mL−1 for CANC, HCTZ and IS, respectively). Methods Method validation The method was validated according to the ICH Q2(R1) guideline (27). Linearity, precision, accuracy, selectivity, limit of detection and limit of quantification, and solution stability were used as the validation parameters. The results of validation parameters obtained from LC/DAD and LC/ELSD methods were statistically compared at the 95% confidence level; F- and t-test were used to compare precision and accuracy, respectively. Linearity Linearity was investigated for the calibration curves in which the concentrations of the drugs were plotted against the peak areas relation of CANC and HCTZ to the IS. Five calibration solutions (32-64-96-128-160 μg mL−1 for CANC, and 25-50-75-100-125 μg mL−1 for HCTZ) were prepared by serial dilution of stock solution (each solution was injected 3 times). Precision The precision was calculated by relative standard deviation (RSD %). The intra-day precision was determined by carrying out three independent assays in three concentrations on the same day, and inter-day precision was studied by comparing the assays on three different days. Accuracy To evaluate the accuracy, recovery test were performed by adding know amounts of standard of CANC and HCTZ equivalent to 80%, 100% and 120% of the nominal levels in the tablets (three replicates of each level) to a mixture of common tablet excipients (lactose, starch and magnesium stearate). The accuracy was determined by comparing the found amount with the added amount (results were expressed as the percentage recovered from the matrix). Selectivity The selectivity was evaluated through the stress studies in order to demonstrate the separation between CANC, HCTZ and their degradation products. It was also evaluated by observing any interference from excipients used in the tablets; therefore samples of the commercial products were analyzed. The peak purity was evaluated using DAD detector, to confirm that there was no co-eluting analytes. LOD and LOQ LOD and LOQ were calculated using signal/noise ratios of 3:1 and 10:1, respectively. The LOQ were validated by triplicate analysis of samples prepared at a concentration close to that obtained experimentally. Robustness To evaluate the robustness, the flow rate and pH of aqueous phase of mobile phase were intentionally altered in 0.2 units below and above nominal value, and the resolution, theoretical plate number and peak tailing factor were evaluated as comparison parameters. Solution stability The standard solution stability of CANC was evaluated at room temperature (23 ± 2°C), + 8 ± 1°C and −20 ± 0.5°C, for 4, 14 and 25 days, respectively. The standard solution stability of HCTZ was previously evaluated by us, finding that it was stable in all three conditions (2). Stress testing The stress study was carried out in accordance with the ICH guideline Q1A(R2) (28). Forced degradation studies of HCTZ were previously evaluated by the authors, using LC with UV detection (2); in the present study the stress testing of HCTZ was conducted with the use of DAD and ELSD detection, which allow peak purity determination and detection of possible degradation products undetected by UV detector, respectively. CANC and HCTZ were stressed under various conditions until to facilitate approximate 10–20% degradation, in order to obtain a relevant degradation product (primary degradation product) (11). CANC and HCTZ are practically insoluble in water, therefore, it was necessary the addition of acetonitrile for CANC and methanol for HCTZ as a co-solvent. Peak purity analysis of stressed samples was checked using DAD detector, to ensure the absence of co-eluting analytes. Hydrolysis Acid, alkaline and neutral degradation of CANC and HCTZ were carried out at 400 μg mL−1 in HCl (0.1 N), water and NaOH (0.1 N). About 3 mL of these mixtures were kept on a hot plate at 70°C for different periods of time, then they were cooled to room temperature, neutralized and transferred to a 10 mL volumetric flask, then an aliquot of 0.35 mL of IS was added, and finally they were diluted to volume (final concentration: 120 μg mL−1). Oxidation The oxidative degradation was carried out at 400 μg mL−1 in 3% H2O2, this mixture was kept at room temperature (25 ± 2°C) for 7 days in the dark, then 3 mL was transferred to a 10 mL volumetric flask and then proceeded in the same way as indicated in the hydrolysis. Thermal degradation and humidity For thermal degradation and humidity, solid drugs were spread in a thin layer on a petri-plate and subjected to dry heat at 70°C in an oven and at 70°C/75% RH over a saturated NaCl solution for 30 days. After this time, a solution of 400 μg mL−1 was prepared, then 3 mL was transferred to a 10 mL volumetric flask, and then proceeded in the same way as indicated in the hydrolysis. Photodegradation Photodegradation studies were carried out according to option 2 of the ICH Q1B guidelines (29). Samples of 400 μg mL−1 and solid drug in 1 mm layer on a petri-plate, were exposed to light for an overall illumination of 1.2 million lux hours and a integrated near ultraviolet energy of 200 watt hour m2−1. Then, solutions of 120 μg mL−1 were prepared as indicated previously. Control samples were protected from light with aluminum foil, and exposed concurrently. Results Method development and optimization For method development and optimization, different conditions were evaluated, such as elution mode (isocratic vs. gradient), pH of aqueous phase and organic modifier of mobile phase. The best chromatographic conditions, with sharp and symmetric peaks and appropriate resolution, was obtained using mobile phase of acetonitrile as soltion A and phosphate buffer (pH 6.0; 0.05 M) as solution B in a gradient mode for DAD method, and acetonitrile as solution A and water with acetic acid (0.175 M) and triethylamine (0.06 M) (pH 4.1) as solution B in a gradient mode for ELSD method. The DAD and ELSD methods enabled separation between CANC, HCTZ and their degradation products, therefore they proved to be stability-indicating. Chromatograms are shown in Figure 2. System suitability parameters showed that are within the suitable range for both methods: Rs > 3.2 between all peaks (range Rs ≥ 2) and T (peak tailing factor) between 1.2 and 1.4 for CANC and HCTZ (range 1 ≤ T < 1.5). Figure 2. View largeDownload slide Chromatograms with DAD and ELSD detection: (1) degradation product of HCTZ (DSA); (2) HCTZ; (3) alkaline degradation product of CANC (candesartan); (4) IS (valsartan); (5) acidic and neutral degradation product of CANC (desethylcandesartan cilexetil); (6) CANC. Figure 2. View largeDownload slide Chromatograms with DAD and ELSD detection: (1) degradation product of HCTZ (DSA); (2) HCTZ; (3) alkaline degradation product of CANC (candesartan); (4) IS (valsartan); (5) acidic and neutral degradation product of CANC (desethylcandesartan cilexetil); (6) CANC. Sample preparation studies A study was done to determine the optimum sample treatment for complete leaching of CANC and HCTZ from tablets. Different order of solvent addition (ACN and water in one or two steps) and sonication times (5, 10, 15 and 20 min) were evaluated. Also, the use of sample filter, in order to demonstrate that does not affect the recovery, was evaluated. When mixtures of acetonitrile-water (40:60, v/v) were added in one step (with 20 min. sonication time), recovery was less than 70% for CANC and HCTZ, therefore it was necessary solvent addition in two steps in order to have a complete release of CANC and HCTZ from excipients. Whit less than 15 min sonication time, CANC recovery was low (less than 95%), therefore it was necessary 15 min sonication time. The results using or not sample filter were compared using the t-test (n = 6, α = 0.05); the calculated t values were less than the tabulated revealing that no significant difference between results, showing that the filter does not affect the recovery (over 98% for CANC and HCTZ). Optimal results were obtained with addition of 10 mL of ACN with 15 min sonication time, then addition of water and filtration with sample filter, with recovery of 98.1% and 101.6% for CANC and HCTZ, respectively. Method validation Linearity The DAD response is linear; the equations of the calibration curves were y = 0.0178x + 0.0004; r2 = 0.9985 for CANC, and y = 0.0277x − 0.0951; r2 = 0.9976 for HCTZ. According to statistical analyses by ANOVA, both calibration curves were linear (P < 0.005). The ELSD response was fitted to a power function; the equations of the calibration curves were y = 0.057×1.628; r2 = 0.998 for CANC, and y = 0.078×1.588; r2 = 0.996 for HCTZ. As the response is nonlinear, a log–log transformation was applied producing linear curves with r2 > 0.997. The ELSD response is in agreement with previous research reporting a nonlinear relationship for analysis of gabapentin drug (12). Precision The results of precision study are shown in Table I. The obtained values with DAD and ELSD show a suitable precision for both methods and were compared using F-test; the calculated F values are less than the tabulated, revealing that no significant difference between precision of both methods for CANC and HCTZ. These results differ from other research which shows that ELSD is less precise than UV detector for determination of anti-diabetic drugs, specially at lower concentrations (13). Table I. Precision and Accuracy Study with DAD and ELSD Sample level (%) DAD ELSD RSD (%) Recovery RSD (%) Recovery Intra-daya Inter-dayb (%) intra-daya inter-dayb (%) CANC 80 0.43 1.25 98.6 1.00 1.41 98.9 100 1.56 2.99 98.1 0.42 1.47 103.0 120 2.90 2.09 101.9 1.80 1.47 98.3 HCTZ 80 1.06 2.52 100.0 1.09 1.92 101.1 100 2.78 2.60 101.1 2.61 1.67 100.1 120 2.08 0.61 100.1 2.17 1.43 103.9 Sample level (%) DAD ELSD RSD (%) Recovery RSD (%) Recovery Intra-daya Inter-dayb (%) intra-daya inter-dayb (%) CANC 80 0.43 1.25 98.6 1.00 1.41 98.9 100 1.56 2.99 98.1 0.42 1.47 103.0 120 2.90 2.09 101.9 1.80 1.47 98.3 HCTZ 80 1.06 2.52 100.0 1.09 1.92 101.1 100 2.78 2.60 101.1 2.61 1.67 100.1 120 2.08 0.61 100.1 2.17 1.43 103.9 aAnalyzed on the same day (n = 3). bAnalyzed on three different days (n = 9). Accuracy The results of accuracy study are shown in Table I. According to t-test (n = 9, α = 0.05) the recovery obtained with DAD and ELSD did not differ from the real value, confirming the accuracy of both methods, and there were no significant differences between both methods. These results differ from other research in which UV detector shows higher recoveries than ELSD for determination of anti-diabetic drugs (13). Selectivity Results from degradation studies indicated that DAD and ELSD methods are selective towards CANC, HCTZ, degradation products and IS as shown in Figure 2; also there is no interference or overlap of the excipients of commercial tablets. The peak purity analysis obtained from the DAD confirmed that all peaks were pure. LOD and LOQ The LOD values were 0.55 and 0.67 μg mL−1 for CANC and 0.61 and 1.00 μg mL−1 for HCTZ with DAD and ELSD, respectively, and the LOQ values were 1.66 and 2.00 μg mL−1 for CANC and 1.85 and 3.03 μg mL−1 for HCTZ with DAD and ELSD, respectively. These values are adequate for determination in pharmaceutical samples. According to these results, both detectors display similar LOD and LOQ for both compounds, but they were slightly higher when ELSD was used, due to the higher noise level observed in the response of this detector. The obtained LOQ was validated with recoveries between 99.5 and 112.6% and RSD lower than 5%. Robustness After modifications of flow rate and pH, the Rs, T and N were practically not affected, demonstrating that the methods are robust, except at flow rate 0.6 mL min−1 and pH 3.9 for CAN with ELSD in which the theoretical plate number decreases significantly, probably due to inefficient evaporation of the solvent when mobile phase flow is modified and moreover, changes in pH directly affects the peak shape obtained. Solution stability The results confirm that all CANC solutions were stable, as there the concentration remained almost unchanged and there was no formation of degradation products. Stress testing HCTZ and CANC were found to degrade via hydrolysis and were stable under humidity, thermal, photolytic and oxidative conditions. The results of hydrolysis are shown in Table II. According to these results, both detectors display similar degradation for both compounds. It was observed, the formation of one degradation product for HCTZ by DAD and ELSD after acid, alkaline and neutral hydrolysis, this is in agreement with others researches (3–6) and our previous research (2) in which the percentage of HCTZ degradation was lower except in alkaline conditions, probably due to the lower temperature of the study. Preliminary confirmation of identity of this degradation product as DSA was conducted by HPLC-DAD-ESI-MSn analysis, and UV-VIS spectra. In chromatograms of Figure 2, the peak 1 corresponding to DSA was found in mass spectra as the molecular ion [M–H]+ at m/z 283.9. Further DSA fragmentation resulted in the formation of ion at m/z 247.1 corresponding to the loss of chlorine. This results matched with DSA standard and the UV spectrum obtained with DAD, which showed similar UV absorbance spectra. Table II. Hydrolysis with DAD and ELSD DAD ELSD Degradation (%) Degradation (%) NaOH HCl H2O NaOH HCl H2O Time 2 min 30 min 3 h 2 min 30 min 3 h CANC 77.1 33.1 12.1 78.9 37.3 14.5 Time 5 h 3 h 3 h 5 h 3 h 3 h HCTZ 6.3 36.3 28.9 7.4 39.0 24.7 DAD ELSD Degradation (%) Degradation (%) NaOH HCl H2O NaOH HCl H2O Time 2 min 30 min 3 h 2 min 30 min 3 h CANC 77.1 33.1 12.1 78.9 37.3 14.5 Time 5 h 3 h 3 h 5 h 3 h 3 h HCTZ 6.3 36.3 28.9 7.4 39.0 24.7 CANC is highly sensitive to alkaline hydrolysis with fast formation of one degradation product, it also degrades after acid and neutral conditions with formation of other degradation product (the same for both conditions). Some examples of chromatograms are shown in Figure 3. The preliminar identification of degradation products was performed by analysis of mass spectra of full scan and fragmentation pattern. In Figure 4, the corresponding spectra for each compound are shown. A full-scan mass showed the parent ion peak at m/z 611.8 corresponding to CANC and its sodium adduct [M+Na]+ at m/z 633.7, an intense signal at m/z 423.9 was due to the loss of a water molecule ([M–H2O]+) (Figure 4A); while the ion at m/z 441.9 of alkaline degradation was identified as CAN [M+H]+, this is in agreement with previous research (7, 9). Isolation and further fragmentation of m/z 441.9 ion resulted in the loss of tetrazol-5-yl-bencil group to yield a characteristic ion at 295.6 (Figure 4B). Figure 3. View largeDownload slide Representative chromatograms of CANC after acidic, neutral and alkaline hydrolysis with DAD or ELSD: (1) IS (valsartan); (2) acidic and neutral degradation product (desethylcandesartan cilexetil); (3) CANC; (4) alkaline degradation product (candesartan). Figure 3. View largeDownload slide Representative chromatograms of CANC after acidic, neutral and alkaline hydrolysis with DAD or ELSD: (1) IS (valsartan); (2) acidic and neutral degradation product (desethylcandesartan cilexetil); (3) CANC; (4) alkaline degradation product (candesartan). Figure 4. View largeDownload slide Mass spectrometry of (A) standard of CANC; (B) alkaline hydrolysis (degradation product 4 of Figure 3); (C) acid and neutral hydrolysis (degradation product 2 of Figure 3). Figure 4. View largeDownload slide Mass spectrometry of (A) standard of CANC; (B) alkaline hydrolysis (degradation product 4 of Figure 3); (C) acid and neutral hydrolysis (degradation product 2 of Figure 3). In acid and neutral conditions, the obtained mass spectra showed the parent ion peak [M+H]+ at m/z 413.5 which was identified as desethylcandesartan. In these conditions, we also find the ion at m/z 583.1 whose difference from the original compound was 27.9 that is, the lilkely loss of an ethyl group, this degradation product is the desethylcandesartan cilexetil which was also reported (7, 9). Finally, the ion m/z 395.6 was identified as the [M–H2O]+ fragment (Figure 4C). Peak purity test results from the DAD confirmed that the CANC, HCTZ and degradation products peaks obtained from all the stress samples were pure, with values of peak purity index higher than 0.999 for all compounds. Determination of CAN and HCTZ in combined commercial tablets The LC/DAD and LC/ELSD methods were used for the quantification of commercial tablets containing 8, 16 and 32 mg of CANC and 12.5 mg of HCTZ. Five different products were analyzed, for tablets of the same ratios (16 mg CANC/12.5 mg HCTZ) the products are from a different manufacturing laboratory. As shown in Table III both detectors display similar results for all products. According to USP, the amounts found are within the specified limits of 90–110%. Table III. Analysis of Commercial Drugs by DAD and ELSD Declared DAD ELSD Product Content (mg) Found content (%) Found content (%) CANC/HCTZ CANC HCTZ CANC HCTZ 1 8/12.5 96.3 ± 1.8 97.3 ± 0.4 100.6 ± 1.1 98.2 ± 0.3 2 16/12.5 97.0 ± 1.2 97.5 ± 0.9 96.1 ± 2.6 98.2 ± 1.6 3 16/12.5 107.3 ± 1.7 102.3 ± 0.1 105.2 ± 1.2 99.1 ± 0.8 4 16/12.5 97.0 ± 1.0 102.7 ± 2.4 100.9 ± 1.0 105.0 ± 0.3 5 32/12.5 92.2 ± 1.2 94.4 ± 0.9 92. 9 ± 1.9 96.3 ± 1.9 Declared DAD ELSD Product Content (mg) Found content (%) Found content (%) CANC/HCTZ CANC HCTZ CANC HCTZ 1 8/12.5 96.3 ± 1.8 97.3 ± 0.4 100.6 ± 1.1 98.2 ± 0.3 2 16/12.5 97.0 ± 1.2 97.5 ± 0.9 96.1 ± 2.6 98.2 ± 1.6 3 16/12.5 107.3 ± 1.7 102.3 ± 0.1 105.2 ± 1.2 99.1 ± 0.8 4 16/12.5 97.0 ± 1.0 102.7 ± 2.4 100.9 ± 1.0 105.0 ± 0.3 5 32/12.5 92.2 ± 1.2 94.4 ± 0.9 92. 9 ± 1.9 96.3 ± 1.9 Discussion The compounds were initially analyzed under isocratic mobile phase, but CANC and its degradation products eluted with a long retention time in comparison to HCTZ and it degradation product which eluted with a short retention time, this is explained by their different physicochemical properties, for example, CANC has a Log P (partition coefficient octanol/water) about 5 in contrast to HCTZ that is −0.1, thus, CANC is a highly lipophilic compound that is more retained than HCTZ in a reverse phase column, therefore it was necessary to use a gradient mobile phase. The gradient program was optimized to provide sufficient selectivity in a short separation time. For DAD method, change in pH of aqueous phase, proportions of acetonitrile and methanol as organic modifier of mobile phase were evaluated before the final chromatographic conditions were selected. A wavelength of 225 nm was chosen to achieve satisfied absortion for both CANC and HCTZ (the CANC signal considerably decreases over 250 nm and the HCTZ signal decreases under 210 nm). For ELSD method, acetic acid and trietilamine were added into the mobile phase to enhance sensitivity and peak shapes. Different concentrations of these compounds were tested in order to optimize the mobile phase (data not shown). The effects of column temperature (35–45°C), evaporation temperature (40–60°C) and gas pressure (3–4 bar), were investigated using a two-level full factorial design (23) with a central point and two replicates, to assess their impact on the ELSD response. The measured response was peak areas and was plotted in relation to each modified factor. The ELSD response was decreased by increasing the column temperature, evaporation temperature and gas pressure, the effects of the factors were statistically significant (α = 0.05), therefore, the lowest values of these parameters were selected. Stress study shows that HCTZ and CANC are degraded via hydrolysis, with formation of one degradation product for HCTZ, identified as DSA, and two degradation products for CANC, identified as CAN and desethylcandesartan cilexetil. In other studies, between three and eight degradation products were found (7–10), this difference may be due to degradation conditions; we try to employ a range of stressors that can mimic the formation of relevant degradation products. A relevant degradation product is considered that resulting from direct degradation, usually it is formed before the drug becomes degraded not more than 10–20%, is representative of the degradation under real conditions (30). For example, the study of Mehta et al. (9) shows the formation of eight degradation products, but the stress conditions were greater (eg. 0.1 N NaOH at 80°C by 1 h). Conclusions This work provides two new stability-indicating LC methods, one with DAD and the other with ELSD detection, for simultaneous analysis of CANC and HCTZ in pharmaceutical samples. The methods were successfully validated according to ICH guidelines. A comparison of performance of both methods was performed, obtaining that both detectors display similar parameters of precision, accuracy, selectivity, LOD and LOQ, and the detector response is different, since DAD shows a linear relationship whereas for ELSD a nonlinear response was observed. The results of stress testing and quantification of commercial product were similar, demonstrating that both methods are suitable for stability study and determination of CANC and HCTZ in pharmaceutical samples. The results obtained from the stress testing show that CANC and HCTZ are unstable under hydrolysis, forming CAN and desethylcandesartan cilexetil as degradation products for CANC and DSA as degradation product for HCTZ. Therefore, caution should be used in the manufacturing process and during storage of these compounds in order to prevent degradation. Acknowledgments Authors would like to thank FONDECYT Grant 1130447 (CONICYT, Chile). References 1 Mc Evoy, G.K. (ed).; AHFS Drug Information . 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Journal of Chromatographic Science – Oxford University Press
Published: Feb 1, 2018
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