Analytical QbD for the optimization of a multimode HPLC method for the investigation of hydrochlorothiazide, diltiazem and propranolol release from 3D printed formulation.

Since 3D printing technology is an emerging field in pharmaceutical technology, the present study aimed at the development of a mixed-mode liquid chromatographic method for the separation and determination of hydrochlorothiazide, diltiazem, and propranolol to investigate their in-vitro release performance from 3D printed tablets. Due to the unique properties of the mixed-mode stationary phase, the three drugs were separated in less than 8 min under isocratic elution. Method development was accomplished following the Analytical Quality by Design principles and was evaluated using risk assessment and multivariate analysis. The influences of critical method parameters on critical method attributes (were screened using a 2-level fractional factorial design and subsequently optimized through a central composite design. The method operable design region was approved by the establishment of a robust zone using Monte Carlo simulation and capability analysis. The validation of the HPLC method was performed based on the total error concept. The relative bias was varied between ─ 11.6 % and 10.5 % and the RSD values for repeatability and intermediate precision were below 4.4 % in all cases. The limits of detection (LOD) ranged between 0.17 - 0.90 µg/mL and were adequate for the specific application. The developed method was successfully applied to the analysis of the studied drugs in in-vitro drug release samples obtained from 3D-printed tablets combining the above-mentioned active pharmaceutical ingredients (APIs).

Resolution of overlapping spectra using chemometric manipulations of UV-spectrophotometric data for the determination of Atenolol, Losartan, and Hydrochlorothiazide in pharmaceutical dosage form.

Simultaneous determination of atenolol (ATN), losartan potassium (LOS), and hydrochlorothiazide (HCZ) in presence of HCZ impurity B was conducted by chemometric approaches and radial basis function network (RBFN) using UV-spectrophotometry without preliminary separation. Three chemometric models namely, classical least-squares (CLS), principal component regression (PCR), and partial least-squares (PLS) along with RBFN were utilized using the ternary mixtures of the three drugs. The multivariate calibrations were obtained by measuring the zero-order absorbance of the mixtures from 250 to 270 nm at the interval of 0.2 nm. The models were built covering the concentration range of (4.0 to 20.0), (3.8 to 20.2), and (0.9 to 50.1) µg mL-1 for ATN, LOS, and HCZ, respectively. The regression coefficient was calculated between the actual and predicted concentrations of the 3 drugs using CLS, PCR, PLS and RBFN. The accuracy of the developed models was evaluated using the root mean square error of prediction (RMSEP) giving satisfactory results. The proposed methods were simple, accurate, precise and were applied efficiently for the quantitation of the three components in laboratory-prepared mixtures, and in dosage form showing good recovery values. In addition, the obtained results were compared statistically with each other using ANOVA test showing non-significant difference between them.

Facile immunochromatographic assay based on metal-organic framework-decorated polydopamine for the determination of hydrochlorothiazide adulteration in functional foods.

Herein, a novel immunochromatographic assay (ICA) based on metal-organic framework-decorated polydopamine (MOF@PDA) was firstly developed for the determination of hydrochlorothiazide (HCTZ) adulteration in functional foods. The coupling rate of MOF@PDA carrier to HCTZ antibody was as high as 91.7 %. The detection limits of the developed MOF@PDA-ICA in functional tablets and capsules were 5.93 and 4.72 µg/kg, the linear ranges were 11.2-91.91 µg/kg and 9.11-86.78 µg/kg, respectively. The sensitivity was 27-fold higher than that of the reported ICA. The recovery was 82.5-116.6 %, and coefficient of variation was 6.9-14.2 %. The results can be achieved and analyzed in 8 min with the smartphone-based detection device. The parallel tests of 23 commercial functional tablets and capsules showed that the results of the MOF@PDA-ICA were consistent with that of the LC-MS/MS (R2 > 0.99). Therefore, our method is facile, sensitive, portable, and can provide a reliable technical mean for the detection of HCTZ adulteration in functional foods.

Diuretic and Antiurolithic Effect of Garcinia humilis (Vahl) C.D. Adams Leaves, a Medicinal Plant Native to South American Countries.

This study evaluated the diuretic and antiurolithic effect of methanolic extract (MEGHL), dichloromethane (DCM), and ethyl acetate (EtA) fractions obtained from the leaves of Garcinia humilis, a medicinal plant known as achachairu and native to South American countries such as Bolivia, Peru, and Brazil. For the analysis of diuretic effect, the female rats received the treatment with MEGHL (3, 10, and 30 mg/kg), DCM (1, 3 and 10 mg/kg), EtA (1, 3, and 10 mg/kg), hydrochlorothiazide (HCTZ; 10 mg/kg), or vehicle (VEH) after an overload of saline solution. At the end 8 h of the experiment, the urinary parameters were measured. Additionally, the antiurolithic effect was analyzed, in which sodium oxalate was added in synthetic urine in the presence or absence of MEGHL, DCM, and EtA in different concentrations (0.1, 0.3, and 1 mg/mL). MEGHL, DCM, and EtA were able to promote 8-h diuresis in rats. MEGHL treatment at dose 30 mg/kg was accompanied by increased urinary Na+ , K+ and Cl- excretion. Moreover, the DCM and EtA fractions treatment increased K+ and Cl- excretion in the urine, although it does not cause any change in Na+ elimination. All the preparations were able to exert an antiurolithic effect in vitro, decreasing the number of calcium oxalate crystals of the monohydrate and dihydrate types. Taking together, the results presented herein showed that the preparations of G. humilis leaves are promising strategies to induce diuresis and antiurolithic effects.

Simultaneous determination of hydrochlorothiazide, amlodipine, and telmisartan with spectrophotometric and HPLC green chemistry applications.

For the quantifiable amounts of Telmisartan (TLM) and Hydrochlorothiazide (HYD) in the presence of Amlodipine (AML) in a ternary mixture of synthetic laboratory mixture, a novel, sensitive, quick, and practical reversed-phase high-performance liquid chromatography (RP-HPLC) method was given. In order to separate, a Waters Spherisorb ODS-2 C18 column was used. For HYD, TLM, and AML, these techniques were viable over linearity ranges of 4-12 µg/mL, 4-25 µg/mL, and 5-40 µg/mL, respectively. The mobile phase system was acetonitrile:methanol: phosphate buffer at pH 2.5 (65:5:30 v/v/v), and the flow rate was 1.5 mL/min. Novel spectrophotometric methods were applied for active substances to determine simultaneously. The first method is absorptivity centering using factorized spectrum, and the second method is dual amplitude difference coupled with absorbance subtraction. These approaches have been effectively applied to bulk, laboratory synthetic mixtures to employ active components quantitatively. Correlation coefficients were found to be higher than 0.99 and the limit of detection values lower than 0.49 µg/mL in both spectrophotometric methods. The methodologies were validated following ICH recommendations. In the developed HPLC method, the limit of detection values was found to be 0.01 µg/mL for HYD and 0.02 µg/mL for AML and TLM. The correlation coefficients for the HPLC method were found to be 0.9971 for HYD, 0.9990 for AML, and 0.9983 for TLM. The suggested HPLC technique is a simple, effective, sensitive, environmentally friendly, and time-saving approach for determining TLM and HYD in the presence of AML.

Eco-friendly spectrophotometric evaluation of triple-combination therapies in the treatment strategy of patients suffering from hypertension during coronavirus pandemic - Spectralprint recognition study.

Recent studies have reported that using certain antihypertensive therapies such as angiotensin II receptor blockers (ARBs) and calcium channel blocker (CCBs) is associated with reduction of fatal outcomes and improving clinical characteristics of patients suffering from hypertension during coronavirus pandemic. Thus, in the current work an effective, innovative and eco-friendly spectrophotometric manner namely, parent spectrum extraction (PSE)was established for evaluation of recommended triple antihypertensive combination therapies incorporate valsartan (VAL) as ARBs, amlodipine besylate as CCBs (AML) and hydrochlorothiazide (HCT)as diuretic into single-pill in challengeable ratio. PSE manner composed of two complementary steps, auxiliary resolution coupled with data analysis resolution(DAR)and it is characterized by resolving the spectral bands of the drugs and extraction of their discrete parent spectra (D0); accordingly, enabling determination of each analyte at its λmax. Auxiliary resolution of AML in triple mixture was applied to decrease complexity of overlapped spectra via constant multiplication (CM) followed by spectrum subtraction (SS) to obtain resolved mixture of VAL and HCT while data analysis resolution (DAR) of this binary mixture was applied via one of three novel methods namely, absorbance extraction (AE), peak-amplitude extraction (PE) and ratio extraction (RE) along with SS method. The proposed methods had analyzed VAL, AML and HCT in the range of 4.0-44.0 µg/mL, 4.0-40.0 µg/mL and 2.0-24.0 µg/mL, respectively with an excellent correlation coefficient (r ≥ 0.9999). Further, the proposed methods in PSR manner were validated as stated by ICH guidelines and it was found that accuracy and precision results are within the acceptable limit. The suggested procedures were effectively utilized for the concurrent quantification of VAL, AML and HCT in synthetic mixtures and tablets. The greenness of the proposed spectrophotometric methods was evaluated by National Environmental Methods Index (NEMI), the Analytical Eco-Scale, the Green Analytical Procedure Index (GAPI) and Analytical greenness metric (AGREE) where the four tools affirmed the eco-friendly nature of the proposed methods. A comparison between the outcomes of the studied methods with the official and reported ones was performed and no statistical difference was arisen between the methods regarding to accuracy and precision.The achieved results along with the simplicity, affordability and low-cost of the proposed methods recommended their appropriateness for the regular quality control examination and analysis of pure materials and pharmaceutical formulations as well as their applicability for the spectralprint recognition of the studied drugs.

Development of a multivariate model with desirability-based optimization for determination of atenolol and hydrochlorothiazide by eco-friendly HPLC method with fluorescence detection.

Determination of a widely used antihypertensive combination of atenolol and hydrochlorothiazide was achieved by rapid and eco-friendly high-performance liquid chromatography method combined with fluorescence detection. The response surface methodology is conducive to the complete separation of the two drugs in a shorter analysis time. The separation of the mixture was achieved using an Inertsil C18 analytical column (150 × 4.6 mm, 5 µ). The mobile phase used was ethanol: potassium dihydrogen phosphate at pH 3 (65:35 v/v) and the flow rate was 0.7 mL/min. The fluorescence detector operated at excitation and emission wavelengths of 230 and 310 nm (atenolol) and 270 and 375 nm (hydrochlorothiazide). The linearity of the developed method covered a concentration of atenolol of 0.05-5 µg/mL and a concentration of hydrochlorothiazide of 0.02-1 µg/mL. The greenness of the developed method was evaluated by analytical eco-scale and the recently reported evaluation method, that is, green analytical procedure index, and it was found to be an excellent, sensitive, and green alternative to the reported methods. The developed method was validated according to the ICH guidelines and compared with the reference method. No significant difference was found in terms of accuracy.

Mathematical Modeling for HPLC Separation and Quantification of an Anti hypertensive Combination by Response Surface Methodology.

BACKGROUND: Determination of a multi-component mixture by HPLC requires many preliminary runs for method development which is both time-consuming and expensive due to the usage of large solvent volumes. OBJECTIVE: In the present study, the main objective was to reduce the preliminary runs that are required for optimizing the method conditions and also shorten the run time of analysis to be suitable for quality control laboratories where there are a large number of samples to be analyzed. METHODS: That was achieved using a two-factor, three-level response surface experiment which is a multivariate design that predicts the significant factors for optimizing the studied responses. RESULTS: The response surface design suggests that both acetonitrile ratio and flow rate are significant factors for full resolution of the studied mixture: atenolol, amiloride, and hydrochlorothiazide. The studied mixture was fully separated and determined in less than 5 min with perfect resolution. CONCLUSION: Experimental design is a very beneficial tool for optimization of the method conditions in HPLC, especially if the studied mixture ingredients have overlapping peaks. For atenolol, amiloride, and hydrochlorothiazide, acetonitrile and flow rate were found to be the significant factors that affected the resolution of the studied mixture. HIGHLIGHTS: Response surface design is a powerful tool that could be used for predicting the significant factors for separation in HPLC. Optimization of the method conditions was done using a limited number of preliminary runs. The studied ternary mixture was fully separated in less than 5 min with the aid of experimental design.

Development and validation of a stability-indicating UPLC method for the determination of olmesartan medoxomil, amlodipine and hydrochlorothiazide degradation impurities in their triple-combination dosage form using factorial design of experiments.

The current work describes the development and validation of a stability-indicating UPLC method for the determination of olmesaratan medoxomil (OLM), amlodipine besylate (AMB), hydrochlorothiazide (HCT) and their degradation products in the triple-combination tablet dosage form. The separation was achieved using a Zorbax Eclipse plus C8 RRHD (100 mm × 3.0 mm), 1.8 µm column with gradient elution of mobile phase A containing 0.02 m of sodium phosphate buffer (pH 3.35) and mobile phase B as acetonitrile and water (90:10, v/v). The detector signal was monitored at UV 250 nm. Analytical performance of the optimized UPLC method was validated as per International Conference on Harmonization guidelines. The linearity ranges for OLM, AMB and HCT were 0.59-240, 0.30-60 and 0.37-150 µg/ml, respectively, with correlation coefficients >0.999. The dosage form was subjected to forced-degradation conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal and photodegradation. The method was proved to be stability indicating by demonstrating the specificity of the drugs from degradation products. The robustness of the method was evaluated through a two-level, three-factorial design with a multivariate approach. Statistical data analysis with best model fit P-value < 0.05 from an ANOVA test indicated that the influence of individual factors is relatively higher than the interaction effects. The method is useful for the analysis of drug products.

High performance liquid chromatographic assay of amlodipine, valsartan and hydrochlorothiazide simultaneously and its application to pharmaceuticals, urine and plasma analysis.

A Simple, Specific, Precise, Accurate, Linear, Rugged, Robust High Performance Liquid Chromatographic method of analysis for simultaneous determination of assay of Amlodipine, Valsartan and Hydrochlorothiazide drugs in the pharmaceuticals tablet formulations using Pioglitazone as a common internal standard was developed and validated. The assay was accomplished using a mixture of acetonitrile & methanol in the volume ratio of 20:80 v/v (mobile phase B) and Ammonium acetate buffer (Mobile phase A) in gradient flow as mobile phase on an Hibar RP-18e, 250 × 4.6 mm, 5µ as chromatographic column at a flow rate of 1.300 mLmin-1, injection volume 10 µL and at a wavelength 235 nm with UV detector. Linearity of the analytical method was evaluated at a concentration range of 2.5-45.3 µg/ml for Amlodipine, 32.0-720.1 µg/ml for valsartan and 5.0-112.6 µg/ml for Hydrochlorothiazide respectively with Correlation coefficient (r) value more than 0.9997. The limit of detection (LOD) for Amlodipine, Valsartan and Hydrochlorothiazide was found to be 1.1 µg/ml, 8.0 µg/ml & 1.0 µg/ml respectively. Specificity, Method Precision, System Precision, Ruggedness, Robustness, Recovery, Stability of analytical solution, Filter paper selection study, Stress testing (Force Degradation) at various conditions were performed as per the ICH (Q2) recommendations. The chromatographic method may also be applied for simultaneous estimation of analytes in plasma and urine.

Analysis of quinary therapy targeting multiple cardiovascular diseases using UV spectrophotometry and chemometric tools.

Herein, UV spectrophotometry assisted by multivariate chemometric analysis have been presented for quantitative determination of complex quinary therapy containing atenolol, ramipril, hydrochlorothiazide, simvastatin and aspirin without any prior separation. Such combination is very useful for treating various cardiovascular diseases (CVD) including high blood pressure, hypercholesterolemia in addition to its antiplatelet aggregating activity. Calibration (15 samples) and validation (10 samples) sets were prepared of different concentrations for these drugs via implementing partial factorial experimental design. The zero order UV spectra of these sets were recorded and then subjected for further chemometric analysis. Partial least square (PLS) with/without variable selection procedure i.e. genetic algorithm (GA) were employed to untangle the UV spectral overlapping of these mixtures. The performance of these chemometric techniques were compared in terms of accuracy and predictive abilities using cross-validation and external validation methods. It was found that PLS provides good recoveries with prompt predictive ability albeit GA-PLS exhibited better analytical performance owing to its capability to remove redundant variables i.e. the number of absorbance variables had been reduced to about 19-28%. The developed methods allowed reliable determination of such complex therapy in its laboratory prepared mixtures and pharmaceutical preparation within comparable results to those reported by HPLC method, posing these chemometric methods as valuable and indispensable analytical tools in in-process testing and quality control analysis of many pharmaceutical compounds targeting CVD.

Photostability study of multicomponent drug formulations via MCR-ALS: The case of the hydrochlorothiazide-amiloride mixture.

The kinetics and photodegradation mechanism of the pharmaceutical mixture of hydrochlorothiazide (HCT) and amiloride (AML) has been studied in depth using a chemometric approach. Water solutions of HCT and AML, separately or in binary mixtures, were irradiated with forced light at different pH values (3, 7, 9 and 12). Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) modelling has been applied to the experimental data recorded by UV spectrophotometry and HPLC-UV/MS. 78 data sets were collected and their chemometric processing has allowed the simultaneous determination of the behaviour of the two drugs in the mixture when exposed to light and the dependence of their photodegradation kinetics on pH. MCR-ALS has been applied using three different implementations. Soft-MCR-ALS and hybrid Hard/Soft-MCR-ALS have been used to resolve the experimental data and to get the equilibrium and kinetic parameters of the investigated chemical processes. A third implementation of the MCR-ALS method has been used in the analysis of the incomplete data sets obtained when UV spectrophotometric and HPLC-UV/MS data were simultaneously analysed, using a row- and column-wise incomplete augmented data matrix arrangement. In these matrices, information from HPLC-UV detector was used as a bridge between the data recorded by UV spectrophotometry (acid-base and kinetic reactions monitoring) and the data obtained by HPLC-MS.

Simultaneous spectrofluorometric analysis of tablets containing hydrochlorothiazide combined with timolol maleate or amiloride hydrochloride.

Green and sensitive spectrofluorometric methods have been developed and validated for the determination of timolol maleate (TML)/hydrochlorothiazide (HCT) and amiloride hydrochloride (AMH)/hydrochlorothiazide in tablets. The proposed spectrofluorometric procedures were found to be linear in the range of 4-12, 5-35 and 0.025-0.2 mg L-1 for HCT, TML and AMH, resp. The excitation and emission wavelengths for HCT, TML and AMH at room temperature were 270 and 375, 295 and 435, 330 and 415 nm, resp. The methods were validated with respect to ICH guidelines. The AMH showed higher sensitivity with lower values of LOD and LOQ values compared to HCT and TML. The proposed methods were applied to two pharmaceutical formulations; the method for HCT and AMH has proven as reliable assaying method, whereas the method for TML, when combined with HCT, is applicable to screening semi-quantitative analyses.

HPLC method transfer study for simultaneous determination of seven angiotensin II receptor blockers.

In this study, a sensitive high-performance liquid chromatography method was developed and validated for the simultaneous determination of seven angiotensin II receptor blockers, namely, hydrochlorothiazide, chlorthalidone, eprosartan mesylate, valsartan, losartan potassium, irbesartan, and candesartan cilexetil. Different chromatographic parameters were tested and fully optimized. Best chromatographic separation was accomplished on a reversed-phase octadecylsilyl column (250 × 4.6 mm id; 5 µm) under gradient elution using methanol/sodium phosphate monobasic buffer (0.01 M, pH 6.5) as mobile phase. The detection of target analytes was obtained at 254 nm. The pH of the buffer has been selected according to Marvin® sketch software. The proposed method was validated according to ICH guidelines and showed good precision (relative standard deviation < 1), good linearity (square of correlation coefficient ≥ 0.999), and high accuracy (between 98 and 102%) with detection limit and quantitation limit (40 and 160 ng/mL, respectively) for all the detected analytes.

Analysis of the Overlapped Electrochemical Signals of Hydrochlorothiazide and Pyridoxine on the Ethylenediamine-Modified Glassy Carbon Electrode by Use of Chemometrics Methods.

In this work, the electrochemical behavior of hydrochlorothiazide and pyridoxine on the ethylenediamine-modified glassy carbon electrode were investigated by differential pulse voltammetry. In pH 3.4 Britton-Robinson (B-R) buffer solution, both hydrochlorothiazide and pyridoxine had a pair of sensitive irreversible oxidation peaks, that overlapped in the 1.10 V to 1.20 V potential range. Under the optimum experimental conditions, the peak current was linearly related to hydrochlorothiazide and pyridoxine in the concentration range of 0.10-2.0 µg/mL and 0.02-0.40 µg/mL, respectively. Chemometrics methods, including classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS), were introduced to resolve the overlapped signals and determine the two components in mixtures, which avoided the troublesome steps of separation and purification. Finally, the simultaneous determination of the two components in commercial pharmaceuticals was performed with satisfactory results.

First identification of a diuretic, hydrochlorothiazide, in hair: Application to a doping case and interpretation of the results.

An athlete contested an adverse analytical finding involving hydrochlorothiazide, and requested our laboratory for testing his hair. As there is no reference in the literature about identification of hydrochlorothiazide in hair, several volunteers were first enrolled (4 after a single 25 mg administration and 10 with daily therapeutic treatment). A specific method was developed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS. Hair samples were decontaminated with dichloromethane and 30 mg were incubated in buffer at pH 7.0 for 15 hours at 50°C. Then, 5 mL ethyl acetate was added for extraction. Linearity was observed for hydrochlorothiazide concentrations ranging from 5 to 2000 pg/mg. The limit of quantification was 5 pg/mg. The coefficients of variation (CVs) of repeatability and matrix effect were lower than 20%. Analysis of the 0-2-cm segment of the 4 volunteers having received a single dose, collected 1 month after administration, was negative at the limit of quantification. The hair of the 10 patients (proximal 2 cm) on daily treatment was positive with concentrations ranging from 12 to 1845 pg/mg, with no correlation between daily dose and concentration. The athlete's hair tested positive for hydrochlorothiazide at 36 pg/mg in the segment corresponding to the period of the urinary control. Since a single exposure to hydrochlorothiazide is not detectable in hair and based on the results of the patients on daily treatment, the concentration found in the athlete has been interpreted as corresponding to repeated exposures, where it was not possible to establish the dosage and the frequency.

Multiple incidence of the prescription diuretic hydrochlorothiazide in compounded nutritional supplements.

Diuretic agents are prohibited in sports in- and out-of-competition according to the regulations of the World Anti-Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (µg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA-accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (µg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ-contaminated powder (6.4 µg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid-liquid extraction and liquid chromatography-tandem mass spectrometry determination. Post-administration specimens were found to contain HCTZ at concentrations of 5-230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.

A Capillary Zone Electrophoresis Method with Multiresponse Chemometric Optimization for the Simultaneous Determination of Zofenopril Calcium and Hydrochlorothiazide in Presence of Hydrochlorothiazide Major Impurities.

In the present decade, great importance has been focused on the development of green analytical methods (GAM) as eco-friendly techniques. Minimizing the wastes, analysis time, hazardous reagents, sample size and energy are the main important principles for development of GAM. This manuscript describes a green, novel, rapid, accurate and reliable capillary zone electrophoresis method (CZE) for the simultaneous separation and determination of zofenopril calcium (ZOF) and hydrochlorothiazide (HCT) in presence of two major impurities of HCT, namely; chlorothiazide (CT) and salamide (DSA). Uncoated fused-silica capillary (50 µm i.d. × 48.5 cm and 40 cm effective length) was used. The main factors affecting the separation were the buffer concentration, pH of the buffer and applied voltage. Optimization of the experimental conditions was performed by applying response surface methodology (RSM). The experiments were designed using central composite face-centered design (CCD). The model obtained from the design described the linear, non-linear and interaction effects of factors on the responses. The optimum conditions given by the design were running buffer of sodium borate (pH 9.15; 10 mM) and 17 kV as positive mode applied voltage. Upon applying these conditions, baseline separation for the four compounds with short analysis time of 5.0 min was achieved. UV detection was performed at 225.0 nm and the capillary temperature was maintained at 25°C. The method was validated and applied for quantitative determination of the studied drugs according to the International Conference on Harmonization (ICH) guidelines. Good linearity was obtained in the range of 10.0-100.0 µg/mL for both ZOF and HCT. As for CT and DSA (HCT impurities), linearity range was 5.0-100.0 µg/mL. The proposed method was successfully applied for the analysis of these drugs in their synthetic mixtures and in their co-formulated pharmaceutical formulations.

A Validated High-Performance Thin-Layer Chromatographic Method for the Simultaneous Determination of Zofenopril Calcium and Hydrochlorothiazide in the Presence of the Hydrochlorothiazide Impurities: Chlorothiazide and Salamide.

The chromatographic analysis of either process-related impurities or degradation products is very important in the pharmaceutical industry. In this work, a simple, selective, and sensitive HPTLC method was developed and validated for the simultaneous determination of zofenopril calcium (ZOF) and hydrochlorothiazide (HCT) in the presence of the HCT impurities: A) chlorothiazide (CT) and B) salamide, in raw materials and in pharmaceutical formulation. The separation was carried out on HPTLC silica gel 60 F254 using ethyl acetate-glacial acetic acid-triethylamine (10 + 0.1 + 0.1, v/v/v) as a developing system. The separated bands were scanned densitometrically at 270 nm. Polynomial equations were used for the regression. Calibration curves were constructed for ZOF, HCT, CT, and salamide in the ranges of 0.5-10, 0.2-4, 0.05-1.4, and 0.05-1.0 µg/band, respectively. Different parameters affecting the suggested method, including developing systems of varying composition/ratios and different detection wavelengths, were studied to achieve the best resolution and precision with good sensitivity. System suitability parameters were also tested. The proposed method was validated as per the International Conference on Harmonization guidelines and was successfully applied for the quantification of the studied drugs in their pharmaceutical formulation, with no interference from excipients observed. The results obtained by the developed HPTLC method were compared statistically with those obtained by the reported HPLC method using Student's t and F ratio tests, and no significant difference was obtained, indicating the ability of the proposed method to be used for routine analysis of drug product.

Simultaneous Determination of Acetylsalicylic Acid, Hydrochlorothiazide, Enalapril, and Atorvastatin in a Polypill-Based Quaternary Mixture by TLC.

A new chromatographic-densitometric method has been developed for the qualitative and quantitative determination of the active ingredients in a simulated mixture corresponding to the PolyIran polypill, composed of acetylsalicylic acid, hydrochlorothiazide (HCT), enalapril (ENA), and atorvastatin (ATR), whose efficacy in the treatment and prevention of cardiovascular disease has been documented in clinical trials. Chromatographic separation was performed using TLC silica gel 60 plates with fluorescent indicator F254 as the stationary phase and a mixture of n-hexane-ethyl acetate-methanol-water-acetic acid (8.4 + 8 + 3 + 0.4 + 0.2, v/v/v/v/v) as the mobile phase. Densitometric measurements were carried out at λ = 210 nm when determining ENA and at λ = 265 nm in the case of the other drugs. Peaks of examined substances were well separated in the recorded chromatograms, enabling the evaluation of the results in terms of both qualitative and quantitative analysis. The method was specific for the analyzed components and was characterized by high sensitivity. The LOD was between 0.043 and 0.331 µg/spot, and LOQ was between 0.100 and 0.942 µg/spot. Recovery was in the range of 97.02-101.34%. The linearity range was broad and ranged from 0.600 to 6.000 µg/spot for acetylsalicylic acid, from 0.058 to 1.102 µg/spot for HCT, from 0.505 to 6.560 µg/spot for ENA, and from 0.100 to 1.000 µg/spot for ATR. The method was characterized by good precision, with RSD values that ranged from 0.10 to 2.26%.