Simultaneous determination of aliskiren and hydrochlorothiazide from their pharmaceutical preparations using a validated stability‐indicating MEKC method

A stability‐indicating MEKC method was developed and validated for the simultaneous determination of aliskiren (ALI) and hydrochlorothiazide (HCTZ) in pharmaceutical formulations using ranitidine as an internal standard (IS). Optimal conditions for the separation of ALI, HCTZ and its major impurity chlorothiazide (CTZ), IS and degradation products were investigated. The method employed 47 mM Tris buffer and 47 mM anionic detergent SDS solution at pH 10.2 as the background electrolyte. MEKC method was performed on a fused‐silica capillary (40 cm) at 28°C. Applied voltage was 26 kV (positive polarity) and photodiode array (PDA) detector was set at 217 nm. The method was validated in accordance with the ICH requirements. The method was linear over the concentration range of 5–100 and 60–1200 μg/mL for HCTZ and ALI, respectively (r²>0.9997). The stability‐indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using the PDA detection. Precision and accuracy evaluated by RSD were lower than 2%. The method proved to be robust by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of ALI and HCTZ both individually and in a combined dosage tablet formulation to support the quality control.     

Simple micellar electrokinetic chromatography method for the determination of hydrogen sulfide in hen tissues

A new method for the determination of hydrogen sulfide in hen tissues has been developed and validated. For estimation of hydrogen sulfide content, a sample (0.1 g) of hen tissue was treated according to the procedure consisted of some essential steps: simultaneous homogenization of a tissue and derivatization of hydrogen sulfide to its S‐quinolinium derivative with 2‐chloro‐1‐methylquinolinium tetrafluoroborate, separation of so‐formed derivative by micellar electrokinetic chromatography with sweeping, and detection and quantitation with the use of UV detector set to measure analytical signals at 375 nm. Effective electrophoretic separation was achieved using fused silica capillary (effective length 41.5 cm, 75 μm id) and 0.05 mol/L, pH 8 phosphate buffer with the addition of 0.04 mol/L SDS and 26% ACN. The lower limit of quantification was 0.12 μmol hydrogen sulfide in 1 g of tissue. The calibration curve prepared in tissue homogenate for hydrogen sulfide showed linearity in the range from 0.15 to 2.0 μmol/g, with the coefficient of correlation 0.9978. The relative standard deviation of the points of the calibration curve varied from 8.3 to 3.2% RSD.     

Simultaneous determination of digoxin and digitoxin by micellar electrokinetic chromatography and application to drug formulations

A simple micellar electrokinetic chromatographic method is described for simultaneous determination of digoxin and digitoxin. The simultaneous analysis of digoxin and digitoxin was performed in Tris buffer (10 mM; pH 9) with 90 mM sodium dodecyl sulfate and 10% isopropyl alcohol as an anionic surfactant and organic modifier. Under these conditions, good separation with high efficiency is achieved in short analysis times. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the Tris buffer and sodium dodecyl sulfate. The linear range of the method for the determination of digoxin and digitoxin was over 0.01–0.3 mg/mL; the detection limit (signal to noise ratio = 3; injection 3.5 kPa 3 s) was 4 and 6 μg/mL, respectively. Application of the proposed method to the determination of digoxin in commercial tablets and in injections proved to be feasible.     

Development and validation of a stability-indicating micellar electrokinetic chromatography method for the determination of ezetimibe in pharmaceutical formulations.

A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 microm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35 degrees C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2-150 microg/mL (R2=0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 microg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography method.     

Optimization and validation of a nonaqueous micellar electrokinetic chromatography method for determination of polycyclic musks in perfumes

A nonaqueous micellar electrokinetic chromatography method was developed for determination of Tonalide®, Galaxolide®, and Traseolide® polycyclic musks (PCMs). These compounds are widely used as fragrance ingredients in cosmetics. The method was optimized by using a three variable Box‐Behnken experimental design and response surface methodology. A modified chromatographic response function was defined in order to adequately weigh the terms in the response function. After optimization of experimental conditions, an electrolyte solution of 195 mM SDS and 40 mM NaH₂PO₄ in formamide was selected for the separation of the three PCMs, and the applied voltage was fixed at 30 kV. The nonaqueous MEKC method was then checked in terms of linearity, limits of detection and quantification, repeatability, intermediate precision and accuracy, providing appropriate values (i.e. RSD values for precision never exceeding 7%, and accuracy 96–107%). Nonaqueous MEKC for determination of the selected compounds was successfully applied to the analysis of commercial perfume samples.     

Micellar electrokinetic capillary chromatography as an alternative method for determination of hydrocortisone and its most important associated compounds in local pharmaceutical preparations

Summary A new, simple, and accurate micellar electrokinetic chromatographic (MEKC) method is described for quantification of hydrocortisone, hydrocortisone hemisuccinate, hydrocortisone acetate, mystatin, oxytetracycline, Zn-bacitracin, polymyxin B, and lidocaine in ocular and cutaneous pharmaceutical products. The separation was performed at 25°C and 25 kV, with 15mm phosphate +15mm borate buffer, pH 8.2, and 60mm sodium dodecylsulfate (SDS) in 10∶1 (%,v/v) methanol-water as background electrolyte. Under these conditions the analysis time is approximately 23 min. The method has been used for quantification of these compounds in different commercial pharmaceutical products and gave good results when compared with reference spectrophotometric and HPLC methods.     

Stability indicating HPLC method for the simultaneous determination of moxifloxacin and prednisolone in pharmaceutical formulations

ABSTRACT: BACKGROUND: A simple, specific, and fast stability indicating reverse phase liquid chromatographic method was established for instantaneous determination of moxifloxacin and prednisolone in bulk drugs and pharmaceutical formulations. RESULTS: Optimum chromatographic separations among the moxifloxacin, prednisolone and stressinduced degradation products were achieved within 10 minutes by use of BDS Hypersil C8 column (250 X 4.6 mm, 5 mum) as stationary phase with mobile phase consisted of a mixture of phosphate buffer (18 mM) containing 0.1% (v/v) triethylamine, at pH 2.8 (adjusted with dilute phosphoric acid) and methanol (38:62 v/v) at a flow rate of 1.5 mL min-1. Detection was performed at 254 nm using diode array detector. The method was validated in accordance with ICH guidelines. Response was a linear function of concentrations over the range of 20-80 mug mL-1 for moxifloxacin (r2 [greater than or equal to] 0.998) and 40-160 mug mL-1 for prednisolone (r2 [greater than or equal to] 0.998). The method was resulted in good separation of both the analytes and degradation products with acceptable tailing and resolution. The peak purity index for both the analytes after all types of stress conditions was [greater than or equal to] 0.9999 indicated a complete separation of both the analyte peaks from degradation products. The method can therefore, be regarded as stabilityindicating. CONCLUSIONS: The developed method can be applied successfully for simultaneous determination of moxifloxacin and prednisolone in pharmaceutical formulations and their stability studies.     

Stability indicating HPLC method for the simultaneous determination of moxifloxacin and prednisolone in pharmaceutical formulations

Background

Obtaining new pharmaceutical materials with enhanced properties by using natural compounds and environment-friendly methods is a continuous goal for scientists. Ficaria verna Huds. is a widespread perennial plant with applications in the treat of haemorrhoids and to cure piles; it has also anti-inflammatory, astringent, and antibiotic properties. The goal of the present study is the obtaining and characterization of new F. verna extract/??-cyclodextrin complexes by using only natural compounds, solvents, and environment-friendly methods in order to increase the quality and acceptability versus toxicity indicator. Thus, the flavonoid content (as quercetin) of Ficaria verna Huds. flowers and leaves from the West side of Romania was determined and correlated with their antioxidant activity. Further, the possibility of obtaining ??-cyclodextrin supramolecular systems was studied.

Results

F. verna flowers and leaves extracts were obtained by semi-continuous solid-liquid extraction. The raw concentrated extract was spectrophotometrically analyzed in order to quantify the flavonoids from plant parts and to evaluate the antioxidant activity of these extracts. The F. verna extracts were used for obtaining ??-cyclodextrin complexes; these were analyzed by scanning electron microscopy and Karl Fischer water titration; spectrophotometry was used in order to quantifying the flavonoids and evaluates the antioxidant activity. A higher concentration of flavonoids of 0.5% was determined in complexes obtained by crystallisation method, while only a half of this value was calculated for kneading method. The antioxidant activity of these complexes was correlated with the flavonoid content and this parameter reveals possible controlled release properties.

Conclusions

The flavonoid content of F. verna Huds. from the West side of Romania (Banat county) is approximately the same in flowers and leaves, being situated at a medium value among other studies. ??-Cyclodextrin complexes of F. verna extracts are obtained with lower yields by crystallisation than kneading methods, but the flavonoids (as quercetin) are better encapsulated in the first case most probably due to the possibility to attain the host-guest equilibrium in the slower crystallisation process. F. verna extracts and their ??-cyclodextrin complexes have antioxidant activity even at very low concentrations and could be used in proper and valuable pharmaceutical formulations with enhanced bioactivity.     

Label‐free aptamer‐based partial filling technique for enantioseparation and determination of dl‐tryptophan with micellar electrokinetic chromatography

In this study, a simple and reproducible method for enantioseparation and determination of dl ‐tryptophan (dl ‐T rp) was developed by using a partial filling technique in combination with MEKC . The corresponding l ‐T rp specific DNA aptamer was used as a chiral selector. Sodium cholate was used to form the chiral micelles and to enhance the enantioseparation of the enantiomers. Effects of aptamer concentration, filling time, buffer composition, and separation voltage on the enantioseparation were evaluated. The M g²⁺ and Na⁺ concentration in separation buffer was found to effectively affect the separation efficiency and reproducibility. Under the optimal conditions, d ‐ and l ‐T rp were completely enantioseparated in less than 9 min. This aptamer‐based partial‐filling approach has the potential to be extended to the separation of other enantiomers after the replacement of corresponding specific aptamers.     

Separation and determination of acrylamide in potato chips by micellar electrokinetic capillary chromatography

A simple and rapid method using micellar electrokinetic capillary chromatography (MEKC) was developed for the separation and determination of acrylamide in potato chips at low levels for the first time. The experimental conditions for the separation and quantification of acrylamide were optimized at first. The optimized conditions were: 50 mmol/L Na₂B₄O₇ and 40 mmol/L SDS at pH 10.0, 12 kV applied voltage, 76 cm total length (67 cm effective length) and 75 μm i.d. capillary, 198 nm wavelength, 15 cm high 25 s hydrodynamics sample injection, 20 °C air-cooling. The linear response of acrylamide concentration ranges from 0.5 to 100 μg/mL with high correlation coefficient (r = 0.9986, n = 9). The LOD and LOQ were estimated to be 0.1 and 0.33 μg/mL based on S/N = 3 and 10. The precision values (expressed as R.S.D.) of intra- and inter-day were 0.86-4.35% and 2.61-9.65%, respectively. Recoveries spiked at levels 2, 20, 60 μg/mL ranged between 90.86% and 99.6% with R.S.D. less than 6.5%. Finally, the developed method has been applied to the analysis of real samples and has achieved satisfactory results. All of these indicated that it was a reliable method for the quantification of acrylamide in potato chips.     

A novel stability‐indicating analytical method development for simultaneous determination of carboplatin and decitabine from nanoparticles

Drug resistance is one of the main problems of cancer treatment. For this reason, combination therapy is commonly used for years. The combination of a chemotherapeutic, carboplatin, and the epigenetic drug decitabine is a new approach to modulate drug resistance. Nanoparticulate systems can overcome the drawbacks associated with the drug combinations. An analytical method that can detect and quantify carboplatin and decitabine which is encapsulated into the nanoparticles is necessary for nanoparticle development. In the literature, there is no analytical method in which carboplatin and decitabine are determined simultaneously. The primary purpose of this study is to develop and validate a novel, and stability‐indicating high‐performance liquid chromatography method for simultaneous determination of carboplatin and decitabine in pharmaceutical preparations in addition to developing the first nanoformulation for this drug combination. Therefore, various experimental parameters were optimized. The chromatographic separation was achieved using an XSelect^® CSH C18 (250 × 4.6 mm I.D., 5 µm) column and a mobile phase consisting of methanol:water (containing 0.1% phosphoric acid) (3:97, v/v). The mobile phase pH was adjusted to 7.0 with 5 M NaOH. The developed method was successfully applied for the simultaneous determination and quantification of carboplatin and decitabine co‐encapsulated in nanoparticles and released into in vitro dissolution medium.     

Sweeping technique combined with micellar electrokinetic chromatography for the simultaneous determination of flunitrazepam and its major metabolites

A sweeping technique, in conjunction with micellar electrokinetic chromatography, for the simultaneous determination of flunitrazepam and its major metabolites, 7-aminoflunitrazepam and N-desmethylflunitrazepam, is described. The optimized conditions for the sweeping and separation were a pH 9.5 buffer, 25mM borate, 50mM cetyltrimethylammonium bromide, 30% MeOH (v/v), and a 151-mm injection length. The calibration functions were all linear with the coefficient of determination (r(2)) exceeding 0.996 for the three target compounds. Using the sweeping procedure, the limits of detection were determined to be 13.4, 5.6, and 12.0ng/mL for flunitrazepam, 7-aminoflunitrazepam, and N-desmethylflunitrazepam, respectively, and the sensitivity enhancement for each compound was within the range of 110-200 fold. The RSDs for the retention time and the peak area were less than 4.10%. The optimized sweeping method was also used to examine a spiked urine sample. We conclude that sweeping with micellar electrokinetic chromatography has considerable potential use in clinical and forensic analyses of flunitrazepam and its metabolites.     

Validated spectrophotometric method for quantitative determination of simvastatin in pharmaceutical formulations and human serum

A simple ultraviolet spectrophotometric method for the determination of simvastatin in methanol has been devised and compared with the existing pharmacopoeial RP-HPLC method for the estimation of the drug. Analytical parameters such as stability, selectivity, accuracy, and precision have been established for the method, using SIMS® tablets and human serum samples, and evaluated statistically to assess the application of the method. The method was validated under ICH and USP guidelines and was found to comprise the advantages for simplicity, stability, sensitivity, reproducibility, and accuracy for use as an alternative to existing nonspectrophotometric methods for the routine determination of the drug in pharmaceutical formulations and for pharmaceutical investigations involving simvastatin.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

HPLC‐DAD method for the simultaneous determination of zofenopril and hydrochlorothiazide in oral pharmaceutical formulations

An HPLC method with DAD detection was developed and validated for the simultaneous determination of zofenopril and hydrochlorothiazide in tablets. The separation was carried out through a gradient elution using an Agilent LiChrospher C18 column (250×4.0 mm id, 5 μm) and a mobile phase consisting of (A) water–TFA (99.9:0.1 v/v) and (B) acetonitrile–TFA (99.1:0.1 v/v) delivered at a flow‐rate of 1.0 mL/min. 8‐Chlorotheophylline was used as internal standard. Calibration curves were found to be linear for the two drugs over the concentration ranges of 5.0–40 and 1.0–20 μg/mL for zofenopril and hydrochlorothiazide, respectively. Linearity, precision, accuracy, specificity and robustness were determined in order to validate the proposed method, which was further applied to the analysis of commercial tablets. The proposed method is simple and rapid, and gives accurate and precise results.     

Different sample stacking strategies for the determination of ertapenem and its impurities by micellar electrokinetic chromatography in pharmaceutical formulation

Ertapenem, a Group 1 carbapenem, is most recently introduced into the market. It is a beta-lactam antibiotic that possesses a broad antibacterial spectrum including common community-acquired Gram-positive and Gram-negative aerobic and anaerobic pathogens, but low activity against some nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter spp., enterococci and methicillin-resistant staphylococci. The elaborated method of micellar electrokinetic chromatography (MEKC) of ertapenem separation from its impurities was successfully performed using normal stacking mode (NSM) and stacking with reverse migrating micelles (SRMM), followed by UV absorption detection at 214 nm. The best results were obtained with 60mM sodium dihydrogen phosphate and 20mM boric acid buffer pH 6.0, as background electrolyte. Uncoated fused-silica capillary and neutral-coated capillary with normal and reverse polarity, and voltage values of +18 and -12 kV, respectively, were used throughout the investigation. Sodium dodecyl sulfate was employed as the pseudostationary phase. A comparison of applied techniques, including sensitivity enhancement factors and limits of detection (LOD), is presented. The optimized method was validated in terms of linearity, accuracy and precision. Comparable LOD was obtained using both stacking methods (0.3 microg/mL) but better efficiency of ertapenem peak was obtained using NSM. Under the optimum stacking conditions, about 183-4.75-fold and 1289-4.07-fold improvements in peak areas were obtained for NSM and SRMM, respectively, compared to the usual hydrodynamic sample injection (10s). The reproducibility, expressed by relative standard deviations (RSD) of the migration times, for NSM was about 0.96-1.25 and for SRMM was 0.32-0.45. The RSD of corrected peak areas, for NSM was about 1.07-8.14 and for SRMM was 0.74-8.12. The difference in separation time between the two techniques was not obvious. Satisfactory separation was possible after less than 11min of electrophoresis. The evaluated MEKC method was applied in the analysis of medicinal product containing ertapenem: Invanz-ertapenem for injection.     

Use of high‐conductivity sample solution with sweeping‐micellar electrokinetic capillary chromatography for trace‐level quantification of paliperidone in human plasma

Paliperidone is a new antipsychotic drug with a relatively low therapeutic concentration of 20–60 ng/mL. We established an accurate and sensitive CE method for the determination of paliperidone concentrations in human plasma in this study. To minimize matrix effect caused by quantification errors, paliperidone was extracted from human plasma using Oasis HLB SPE cartridges with three‐step washing procedure. To achieve sensitive quantification of paliperidone in human plasma, a high‐conductivity sample solution with sweeping‐MEKC method was applied for analysis. The separation is performed in a BGE composed of 75 mM phosphoric acid, 100 mM SDS, 12% acetonitrile, and 15% tetrahydrofuran. Sample solution consisted of 10% methanol in 250 mM phosphoric acid and the conductivity ratio between sample matrix and BGE was 2.0 (γ, sample/BGE). The results showed it able to detect paliperidone in plasma samples at concentration as low as 10 ng/mL (S/N = 3) with a linear range between 20 and 200 ng/mL. Compared to the conventional MEKC method, the sensitivity enhancement factor of the developed sweeping‐MEKC method was 100. Intra‐ and interday precision of peak area ratios were less than 6.03%; the method accuracy was between 93.4 and 97.9%. This method was successfully applied to the analysis of plasma samples of patients undergoing paliperidone treatment.     

Validation of a micellar electrokinetic capillary chromatography method for the determination of imidurea, methyl and propylparabens in a pharmaceutical ointment

The quantitative aspects of a micellar electrokinetic chromatographic (MEKC) method for the determination of three preservatives (imidazolidinyl urea, methyl and propylparabens) in a pharmaceutical ointment are presented. Separation was carried out in a 31.2 cm long (21 cm to the detection window) × 50 μm i.d. fused silica capillary at an electric field of 960 V/cm and 25 °C. The electrolyte was 10 mM sodium dihydrogenophosphate containing 40 mM sodium dodecylsulfate (SDS) and adjusted to pH 6.1. Sample preparation consisted of a simple dispersion of the ointment in an internal standard solution (ethylparaben in the electrolyte). A 3.5 nl volume of the sample solution was introduced hydrodynamically into the capillary. The compounds detected at 200 nm were separated within 1.6 min. The method was validated with respect to specificity, linearity, accuracy, repeatability according to the International Conference on Harmonisation guidelines. Calibration curves were found to be linear for each compound (r²>0.999). Recovery studies performed by spiking an ointment placebo with the preservatives between 50 and 150% of their target concentration on 3 different days gave satisfactory results (mean recovery at each of the five concentrations ranging from 99.23 to 101.40%). The repeatability of sample preparation (n=6) was better than 2% for each of the preservatives.     

Comprehensive stability‐indicating high‐performance liquid chromatography coupled with diode array detection method for simultaneous determination of amprolium hydrochloride and ethopabate in powder dosage form for veterinary use

This research deals with the development of a stability‐indicating high‐performance liquid chromatography method for simultaneous determination of amprolium hydrochloride and ethopabate. To the best of our knowledge, no comprehensive stability‐indicating method has been reported for analysis of this mixture. Separation was achieved using Kromasil cyano column with gradient elution of the mobile phase composed of sodium hexane sulfonate solution and methanol. Quantification was based on measuring peak areas at 266 nm. Amprolium and ethopabate peaks eluted at retention times 10.42 and 18.53 min, respectively. The proposed procedure was validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Linearity ranges for amprolium and ethopabate were 1.5–240 and 1–160 μg/mL, respectively. Analytes were subjected to stress conditions of hydrolysis, oxidation and thermal degradation. The proposed method enabled resolution of drugs from their forced‐degradation products and amprolium related substance (2‐picoline). Moreover, specificity was verified by resolution of the analytes from about 22 drugs used in antimicrobial veterinary products. The validated method was successfully applied to assay of the combined veterinary powder dosage form, additionally it was implemented in the accelerated stability study of the dosage form when stored for six months at 40°C and 75% relative humidity.     

Derivatizing assay for the determination of aldehydes using micellar electrokinetic chromatography

In this work, the use of a novel derivatization agent for the determination of aldehydes (in this particular case: formaldehyde, acetaldehyde, propionaldehyde, and valeraldehyde) using micellar electrokinetic chromatography is reported. The derivatization reaction is based on the reaction of aldehydes with benzhydrazide to form the corresponding derivates with maximum absorbance at 250 nm. The experimental conditions of the derivatization reaction as well of the separation were optimized. The adducts were separated with a +22 kV voltage at a temperature of 29°C. The adducts’ separation was performed in less than 14 min using as the running buffer a mixture containing 110 mmol/L of sodium dodecyl sulfate and 27 mmol/L of sodium tetraborate at pH 9.45. Samples were injected using hydrodynamic mode (50 mbar × 5 s). The calibration curves were linear up to 15.0 mg/L with r ² above 0.99. Intra and inter‐day precisions were in average 3 and 4%, respectively, and recoveries were in average of 95%. Limits of detection and quantification were around 0.5 and 1.5 mg/L, respectively. The developed method was successfully applied in the analysis of low molar weight aldehydes in yogurt and vinegar samples.