Optimisation and validation of liquid chromatographic and partial least-squares-1 methods for simultaneous determination of enalapril maleate and nitrendipine in pharmaceutical preparations

Simultaneous determination of enalapril maleate (ENA) and nitrendipine (NIT) in pharmaceutical preparations was performed using liquid chromatography (LC) and the partial least-squares-1 (PLS-1) method. In LC, the separation was achieved on a C8 column and the optimum mobile phase for good separation in a gradient elution programme was found to be acetonitrile-water (φ _r = 81: 19) and optimum flow-rate, temperature, injection volume, and detection wavelength were set at 1.0 mL min⁻¹, 25°C, 10 μL, and 210 nm, respectively. Dienogest was selected as an internal standard. In the spectrophotometry, a PLS-1 chemometric method was used. The absorbance data matrix related to the concentration data matrix was established by measurement of absorbances in their zero order spectra with an increment of Δλ = 1 nm in the 220–290 nm range for ENA and with Δλ = 1 nm in the 230–290 nm range for NIT in the PLS-1 method. Following this step, calibration was established by using this data matrix to predict the unknown concentrations of ENA and NIT in their binary mixture. These optimised methods were validated and successfully applied to a pharmaceutical preparation in tablet form and the results were subjected to comparison.     

Chromatographic and electrophoretic determination of thioamides based on thiazole, 1,3,4-thiadiazole, 1,2,4-triazole,and tetrazole

A procedure was developed for the determination of the following thioamides based on thiazole, 1,3,4-thiadiazole, 1,2,4-triazole, and tetrazole: 2-mercaptothiazole (I), 2-mercapto-1,3,4-thiadiazole (II), 2-mercapto-5-methyl-1,3,4-thiadiazole (III), 3-mercapto-1,2,4-triazole (IV), 3-mercapto-4-methyl-1,2,4-triazole (V), and 5-mercapto-1-methyltetrazole (VI). The determination was performed by reversed-phase HPLC on a column (150 × 4 mm) packed with Diaspher-110-C18 (5 μm) using elution with an acetonitrile-acetate buffer solution (pH 4.70) mixture (5: 95). Detection was performed at the light absorption maximums of compounds I (320 nm), II (305 nm), III (310 nm), IV (260 nm), V (254 nm), and VI (245 nm). The calibration graphs were linear over the following concentration ranges (μg/mL): 0.47–11.72 (I), 0.47–11.82 (II), 0.53–13.22 (III), 0.40–10.11 (IV), 0.46–11.52 (V), and 0.46–11.62 (VI). The limits of detection were 0.45, 0.43, 0.50, 0.37, 0.41, and 0.42 μg/mL for compounds I–VI, respectively. Conditions for the separation of a mixture of compounds I and III–V and for the quantitative determination of compounds I–VI by capillary zone electrophoresis (CZE) were optimized. CZE was performed on a quartz capillary of size 60 cm (effective length of 50 cm) × 75 μm at a voltage of 20 kV with a borate buffer solution (pH 9.18). The procedure allowed us to evaluate the concentrations of substances in the ranges of 1.17–93.75 (I), 1.18–94.54 (II), 1.32–105.76 (III), 1.01–101.13 (IV) 1.15–115.16 (V), and 1.16–116.15 (VI) μg/mL with the detection limits of 1.10, 1.11, 1.20, 0.96, 1.01, and 1.02 μg/mL for compounds I–VI, respectively.     

LC Assay for Ciprofloxacin Hydrochloride Ophthalmic Solution

The objective of the current study was to develop and subsequently validate a simple, sensitive and precise reversed-phase LC method for the determination of ciprofloxacin hydrochloride in ophthalmic solution form. The chromatographic separation of ciprofloxacin hydrochloride was achieved on a Symmetry Waters C₁₈ column using UV detection at 275 nm. The optimized mobile phase consisted of 2.5% acetic acid solution: methanol:acetonitrile (70:15:15, v/v/v). The proposed method provided linear responses within the concentration range 1.0–6.0 μg mL⁻¹ for ciprofloxacin hydrochloride. Correlation coefficient (r) for the ciprofloxacin hydrochloride was 0.9994. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 5% in all instances. No interference from any components of pharmaceutical dosage forms was observed.     

Development and validation of RP-HPLC method for simultaneous estimation of prednicarbate,mupirocin and ketoconazole in topical dosage forms

A simple and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for simultaneous estimation of prednicarbate (PC), mupirocin (MP) and ketoconazole (KT) in topical dosage forms. This combination is preferred for topical delivery of anti-inflammatory, antibacterial and antifungal agents for treatment of various skin disorders. The proposed RP-HPLC method utilizes a Hypersil GOLD C18, 5 μm, 250 mm × 4.6 mm i.d. column, mobile phase consisting of methanol-water (80: 20, v/v) adjusted to pH 5.0 with orthophosphoric acid in isocratic mode at the flow rate of 0.5 mL/min and UV detection at 243 nm. The method does not require any specific sample preparation except extraction of active pharmaceutical ingredients from the developed topical emulgel formulations using dichloromethane. Linearity was found in the range of 0.05–0.3 mg/L for PC and 0.4–2.4 mg/L for each of MP and KT with R ² > 0.999. The method is precise with low RSD%, accurate (overall average recovery yields: 99.92% for PC, 99.44% for MP and 99.74% for KT) and selective. Due to its simplicity and accuracy, the method is suitable for simultaneous analysis of PC, MP and KT in topical dosage forms.     

RP-HPLC and NMR study of antioxidant flavonoids in extract from <Emphasis Type="Italic">Gentiana piasezkii</Emphasis>

A reversed-phase high performance liquid chromatography (RP-HPLC) separation with photodiode array detection (PDA) was developed for the simultaneous determination of five flavonoids, lutonarin (LO), saponarin (SA), isoorient (IO), 7-O-feruloylorientin (FO) and luteolin (LE) in the extract of Gentiana piasezkii, a popular forage grass, pharmaceutical and ornamental plant in China. The optimized method was achieved for the separation and detection of selected constituents, using acetonitrile-1% acetic acid as the mobile phase with gradient elution at a flow rate of 0.8 mL/min and 350 nm as the detection wavelength. The contents of the five flavonoids, i.e., LO, SA, IO, FO and LE in the plant of G. piasezkii were 0.1524, 0.0673, 0.300, 0.0552 and 0.0052%, respectively. The compounds possess similar structures and have been studied by nuclear magnetic resonance (NMR) spectroscopy.     

Optimization of the Extraction Conditions and Simultaneous Quantification by RP-LC of Six Alkaloids in Evodiae Fructus

A reversed phase liquid chromatographic (LC) method coupled with DAD (250 nm) has been developed and validated for simultaneous quantification of six alkaloids, dehydroevodiamine (1), wuzhuyuamide-I (2), 5-hydroxyrutaecarpine (3), 14-formyldihydrorutaecarpine (4), evodiamine (5) and rutaecarpine (6), in 12 batches evodiae fructus [the dried, unripe fruits of Evodia rutaecarpa (Juss.) Benth. or E. rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang, E. rutaecarpa (Juss.) Benth. var. bodinieri (Dode) Huang] as a traditional Chinese medicine. The method was carried out by a C₁₈ column (250 × 4.6 mm) with a gradient mobile phase of methanol, acetonitrile, and phosphoric acid–triethylamine–buffer solution. The contents of 1–6 in the evodiae fructus could easily be determined within 70 min. The experimental results were satisfactory for the intra-day and inter-day precision and accuracy of the method for simultaneous determination. The linear calibration ranges of 1–6 were 40–1,000, 20–500, 1–100, 10–500, 40–1,000 and 80–1,000 μg mL⁻¹. The recoveries of 1–6 were 97.43–103.73% with RSDs from 0.21 to 1.99%. The limits of detection for 1–6 were 2.0, 2.0, 0.1, 1.0, 5.0 and 5.0 μg mL⁻¹, and the limits of quantification were 6.6, 6.6, 0.3, 3.3, 16.5 and 16.5 μg mL⁻¹. The method was successfully applied to the quantification of six alkaloids in the evodiae fructus.     

Determination of methylparaben,propylparaben, clotrimazole and its degradation products in topical cream by RP-HPLC

Summary Reversed-phase, high-performance liquid chromatographic (RP-HPLC) methods with UV detection were developed and validated for determination of compounds in a topical cream. The first method describes determination of the active component clotrimazole and two preservatives present in the cream; methylparaben and propylparaben. The second method describes determination of two degradation products of clotrimazole, imidazole and (2-chlorophenyl) diphenylmethanol, in a topical cream after long-term stability tests. Chromatographic separation was on a Purospher RP-18e column; the mobile phase in Method1 for separation of clotrimazole, methylparaben and propylparaben comprises acetonitrile and water (70:30 v/v). For determination of degradations products-imidazole and (2-chlorophenyl) diphenylmethanol—the optimum composition of mobile phase in Method2 was acetonitrile and water (75:25 v/v) apparent pH^* 2.7. Analysis time was <10 min for both methods. The methods were found to be applicable for routine analysis of the active compound clotrimazole, preservatives and degradation products in the pharmaceutical product: topical cream 1% Clotrimazol Cream. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Speciation analysis of manganese in tea samples using flame atomic absorption spectrometry after cloud point extraction

The speciation of Mn(II) in tea infusion was studied using cloud point extraction (CPE). In tea infusion, the flavonoid-bound Mn(II) was extracted at pH 5.0 using Triton X-100 (TX-100), the remaining free aquated Mn(II) and weakly-complexed Mn(II) in solution were both chelated with 8-hydroxyquinoline (HOx) and CPE-preconcentrated with TX-100. The enriched analyte was determined by flame AAS. The optimal concentrations for CPE of 0.02 ppm Mn were as follows: TX-100, 0.2% (v/v); HOx, 1.0 × 10⁻⁴ M; NaCl, 1.0% (w/v). LOD was 1.9 μg/L with a preconcentration factor of 10–20. The method was validated using a standard XAD-resin separation procedure and applied to synthetic seawater and CRM samples.     

Spectrophotometric multicomponent analysis of a mixture of chlorhexidine hydrochloride and lidocaine hydrochloride in pharmaceutical formulation using derivative spectrophotometry and partial least-squares multivariate calibration

Two spectrophotometric methods were applied to the simultaneous assay of chlorhexidine hydrochloride (CHL) and lidocaine hydrochloride (LIH) in pharmaceutical formulations. Using derivative spectrophotometry, CHL was determined by measurement of its first derivative signal at 290 nm (peak to zero amplitude) in the concentration range 5–9 μg/mL, and LIH was analysed by measurement of its second derivative signals at 272 and 276 nm (peak to peak amplitude) in the concentration range 160–480 μg/mL. With the partial least-squares (PLS-2), the experimental calibration matrix was constructed using 9 samples. The concentration ranges considered were 5–7 μg/mL for CHL and 220, 240, 260 μg/mL for LIH. The absorbances were recorded between 240 and 310 nm at every 5 nm.     

Rapid and Simple Method for Simultaneous Determination of Escin and Diethylamine Salicylate in Pharmaceutical Preparations by Partial Least-Squares Multivariate Calibration

Summary. A partial least-squares calibration (PLS) method has been developed for simultaneous quantitative determination of escin (ES) and diethylamine salicylate (DAS) in pharmaceutical preparations. The resolution of these mixtures has been accomplished without prior separation or derivatisation, by using partial least-squares (PLS-2) regression analysis of electronic absorption spectral data. The experimental calibration matrix was constructed with 9 samples. The concentration ranges considered were 10, 20, 30 (ES) and 40, 50, 60 (DAS) μg cm⁻³. The absorbances were recorded between 200 and 325 nm every 5 nm. Proposed method was compared with conventional spectrophotometric method. The results show that PLS-2 is a simple, rapid, and accurate method applied to the determination of these compounds in pharmaceuticals.     

Rapid and Simple Method for Simultaneous Determination of Escin and Diethylamine Salicylate in Pharmaceutical Preparations by Partial Least-Squares Multivariate Calibration

A partial least-squares calibration (PLS) method has been developed for simultaneous quantitative determination of escin (ES) and diethylamine salicylate (DAS) in pharmaceutical preparations. The resolution of these mixtures has been accomplished without prior separation or derivatisation, by using partial least-squares (PLS-2) regression analysis of electronic absorption spectral data. The experimental calibration matrix was constructed with 9 samples. The concentration ranges considered were 10, 20, 30 (ES) and 40, 50, 60 (DAS) μg cm⁻³. The absorbances were recorded between 200 and 325 nm every 5 nm. Proposed method was compared with conventional spectrophotometric method. The results show that PLS-2 is a simple, rapid, and accurate method applied to the determination of these compounds in pharmaceuticals.     

Liquid chromatographic and spectrophotometric determination of diflucortolone valerate and isoconazole nitrate in creams

A new RP-LC method and two new spectrophotometric methods, principal component regression (PCR) and first derivative spectrophotometry, are proposed for simultaneous determination of diflucortolone valerate (DIF) and isoconazole nitrate (ISO) in cream formulations. An isocratic system consisting of an ACE C18 column and a mobile phase composed of methanol-water (95 + 5, v/v) was used for the optimal chromatographic separation. In PCR, the concentration data matrix was prepared by using synthetic mixtures containing these drugs in methanol-water (3 + 1, v/v). The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbances at 29 wavelengths in the range of 242-298 nm for DIF and ISO in the zero-order spectra of their combinations. In first derivative spectrophotometry, dA/dlambda values were measured at 247.8 nm for DIF and at 240.2 nm for ISO in first derivative spectra of the solution of DIF and ISO in methanol-water (3 + 1, v/v). The linear ranges were 4.00-48.0 microg/mL for DIF and 50.0-400 microg/mL for ISO in the LC method, and 2.40-40.0 microg/mL for DIF and 60.0-260 microg/mL for ISO in the PCR and first derivative spectrophotometric methods. These methods were validated by analyzing synthetic mixtures. These three methods were successfully applied to two pharmaceutical cream preparations.     

Development and validation of the HPLC method for the analysis of trimetazidine hydrochloride in bulk drug and pharmaceutical dosage forms

A simple, economic, selective, precise, and accurate high-performance liquid chromatographic (HPLC) method for the analysis of trimetazidine hydrochloride in both bulk drug and pharmaceutical formulations was developed and validated in the present study. The mobile phase consisted of water: methanol: triethylamine (75: 25: 0.1 v/v/v), and pH 3.3 was adjusted with orthophosphoric acid. This system was found to give a sharp peak of trimetazidine hydrochloride at a retention time of 3.375 ± 0.04 min. HPLC analysis of trimetazidine hydrochloride was carried out at a wavelength of 232 nm with a flow rate of 1.0 mL/min. The linear regression analysis data for the calibration curve showed a good linear relationship with a regression coefficient of 0.997 in the concentration range of 5–90 μg/mL. The linear regression equation was y = 35362x − 8964.2. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.6 and 10.9 μg/mL, respectively. The developed method was employed with a high degree of precision and accuracy for the analysis of trimetazidine hydrochloride. The developed method was validated for accuracy, precision, robustness, detection, and quantification limits as per the ICH guidelines. The wide linearity range, accuracy, sensitivity, short retention time, and composition of the mobile phase indicated that this method is better for the quantification of trimetazidine hydrochloride. The text was submitted by the authors in English.     

Solvent extraction-spectrophotometric determination of trace amounts of fluoxetine in pharmaceutical formulations

A simple, reproducible, and sensitive extraction-spectrophotometric method for the determination of fluoxetine (FL) in pharmaceutical formulations is reported. The FLH⁺ cation, which is formed in an acidic solution, can form an ion-pair with Orange II, (OR II), an anionic dye. The FLH⁺-OR II⁻ ion pair was quantitatively extracted into dichloromethane solvent and its absorption was measured at 482 nm. The calibration graph is linear over the FL concentration range of 0.2–9.0 μg/mL and the regression coefficient is 0.9995. The relative standard deviation (RSD) of ten replicate determinations of 5.0 and 1.4 μg/mL of FL are 0.022 and 0.038, respectively, and the limit of detection (LOD) of the method is 0.17 μg/mL. The method was successfully applied to the determination of an FL amount in pharmaceutical formulations (10.0-and 20.0-mg capsules). The text was submitted by the authors in English.     

Development of a Validated Stability-Indicating LC Method for Nitazoxanide in Pharmaceutical Formulations

A reversed-phase liquid chromatographic (LC) method was developed for the assay of nitazoxanide (NTZ) in solid dosage formulations. An isocratic LC separation was performed on a Phenomenex Synergi Fusion C₁₈ column (250 mm × 4.6 mm, i.d., 4 μm particle size) using a mobile phase of 0.1% o-phosphoric acid solution, pH 6.0: acetonitrile (45:55, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was achieved with a photodiode array detector at 240 nm. The detector response for NTZ was linear over the concentration range from 2 to 100 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method were proved using stress conditions. The RSD values for intra-day precision were less than 1.0% for tablets and powder for oral suspension. The RSD values for inter-day precision were 0.6 and 0.7% for tablets and powder for oral suspension. The accuracy was 100.4% (RSD = 1.8%) for tablets and 100.9% (RSD = 0.3%) for powder for oral suspension. The limits of quantitation and detection were 0.4 and 0.1 μg mL⁻¹. There was no interference of the excipients on the determination of the active pharmaceutical ingredient. The proposed method was precise, accurate, specific, and sensitive. It can be applied to the quantitative determination of drug in tablets and powder for oral suspension.     

Determination of biogenic amines in fermented beverages and vinegars by pre-column derivatization withpara-nitrobenzyloxycarbonyl chloride (PNZ-Cl) and reversed-phase LC

Summary The reagentp-nitrobenzyloxycarbonyl chloride (PNZ-CI) was used for pre-column derivatization of biogenic amines (BAs) at ambient temperature followed by reversed-phase, liquid-chromatographic separation of the derivatives. Optimized derivatization of samples was achieved within 10 min in borate buffer by adding PNZ-Cl in acetonitrile (MeCN). Excess reagent was scavenged by subsequent addition of glycine in water. For LC a Superspher^? RP-18e column and gradient elution using a ternary gradient system containing sodium acetabe buffer (pH 6.1), sodium acetate buffer (pH 4.3) and MeCN, were used. The PNZ-derivatives were quantified by their UV-absorption at 265 nm. Detection limits of BAs were approximately 62–1000 μg L⁻¹ (injected amounts: 53–850 pg) at a signal-to-noise ratio of 3:1. The coefficients of determination were 0.9906–0.9992. Diaminohexane was used as internal standard. Recoveries of BAs ranged from 78–93% depending on the food matrix. This method was applied to the quantitative determination of 2-phenylethylamine, tryptamine, serotonin, putrescine, histamine, cadaverine, tyramine, spermidine, and spermine, in beer, wine, vinegars, and lactic fermented cabbage juice. Parts of the results were presented at the 35th Congress of the “Deutsche Gesellschaft für Ern?hrung”, Kiel, 19^th–20^th March 1998, and the “Regionaltagung der Lebensmittelchemiker”, Giessen, 9^th–10^th March 1998     

Simultaneous quantification of cefpirome and cetirizine or levocetirizine in pharmaceutical formulations and human plasma by RP-HPLC

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the simultaneous determination and quantification of cefpirome and cetirizine or cefpirome and levocetirizine in pharmaceutical formulations and human plasma without changing the chromatographic conditions is described. Chromatographic separations were performed on a prepacked Nucleosil 120, C₁₈ (5 μm, 12.5 ± 0.46 mm) column using CH₃CN: H₂O (75: 25, v/v) as a mobile phase at a flow rate of 1 mL/min while UV detection was performed at 232 nm for monitoring the effluent. A number of other brands of C₁₈ columns were also employed which had a significant effect on the separation. The method has been validated over the concentration range of 0.5–50 μg/mL (r ₂ > 0.999). The limit of detection (LOD) and quantification (LOQ) for cefpirome and levocetirzine in pharmaceutical formulations and serum were in the range 0.24–1.31 μg/mL. Analytical recovery from human plasma was >98%, and the within and between-day relative standard deviation was <3.1%. The small sample volume and simplicity of preparation make this method suitable for use in pharmaceutical industries, drug research centers, clinical laboratories, and forensic medical centers. The text was submitted by the authors in English.     

Development of a New Analytical Method for Determination of Related Components in Nateglinide

An isocratic reverse phase liquid chromatographic (RP-LC) assay method has been developed for the quantitative determination of nateglinide and its related components namely imp-1 and imp-2 in bulk drug and in pharmaceutical dosage form, used for the treatment of type II diabetes mellitus. The developed method is stability indicating and also can be used for stability testing. The chromatographic separation was achieved on C-8, 150 × 4.6 mm, 3.5 μm stationary phase. The LC method employs solution A as mobile phase. Solution A contains a mixture of phosphate buffer pH 3.0: acetonitrile (50:50 v/v). The flow rate was 1.0 mL min⁻¹ and the detection wavelength was 210 nm. In the developed LC method the resolution between nateglinide and its potential impurities namely imp-1 and imp-2 was found to be greater than 5.0. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid medium, alkaline medium and oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.2%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

Stress Degradation Studies on Tadalafil and Development of a Validated Stability-Indicating LC Assay for Bulk Drug and Pharmaceutical Dosage Form

A stability-indicating HPLC assay method was developed for the quantitative determination of tadalafil in bulk samples and in pharmaceutical dosage forms in the presence of the degradation products. It involved a 250 mm × 4.6 mm, 5 μm C-18 column. The gradient LC method employs solution A and B as mobile phase. Solution A contains a mixture of buffer (phosphate buffer and tetra-n-butyl ammonium hydrogen sulfate) pH 2.5: acetonitrile (80:20, v/v) and solution B contains a mixture of water: acetonitrile (20:80, v/v). The flow rate was 1.0 mL min⁻¹ and the detection wavelength was 220 nm. The retention time of tadalafil is about 17 min. Tadalafil was subjected to different ICH prescribed stress conditions. Degradation was found to occur in hydrolytic and to some extent in oxidative stress conditions, while the drug was stable to photolytic and thermal stress. The drug was particularly labile under alkaline hydrolytic conditions. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The assay of stress samples was calculated against a qualified reference standard and the mass balance was close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and ruggedness.     

Simultaneous Determination of Paracetamol, Caffeine, Guaifenesin and Preservatives in Syrups by Micellar LC

A micellar liquid chromatographic technique allowing the separation and simultaneous determination of the active ingredients paracetamol, caffeine, and guaifenesin, and preservatives benzoic acid, methyl and propyl paraben is described. The separation was effective by using the Kromasil C18 column (150 mm × 4.6 mm, 5 μm) and a mobile phase of 1-butanol:water (1:99, v/v), containing 0.04 M sodium dodecyl sulfate and 0.1% (v/v) trichloroacetic acid, for eluting all compounds. The detection wavelength was set as 260 nm. The column heater was also used, set at 40 °C for these determinations. Under these conditions, separation of the six components was achieved in less than 30 min. The specificity of the method was demonstrated. Analytical characteristics such as limit of detection, limit of quantification, linear range, accuracy, precision (repeatability) and the influence of the various method parameters (robustness study) were evaluated. The developed method was applied to the determination of paracetamol, caffeine, guaifenesin, benzoic acid (sodium benzoate), methyl and propyl paraben in cough-drop syrups. Presented at the International Conference “Modern physical chemistry for advanced materials (devoted to the 100th birthday of Professor Nikolai Izmailov)”, Kharkov, Ukraine, June 2007.