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EN
An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed for rapid and simultaneous separation and estimation of 3 antidiabetic drugs, namely, metformin, pioglitazone, and glimepiride, in human plasma within 3 min. Separation was carried out on a MAGELLEN 5U C18 (5 μm, 150 mm × 4.60 mm) using a mobile phase of MeOH–0.025 M KH2PO4 adjusted to pH 3.20 using ortho-phosphoric acid (85:15, v/v) at ambient temperature. The flow rate was 1 mL/min, and the maximum absorption was measured at 235 nm. The retention time of metformin, pioglitazone, and glimepiride was noted to be 1.24, 2.32, and 2.77 min, respectively, indicating a very short analysis time compared to that of other reported methods. Also, limits of detection were reported to be 0.05, 0.26, and 0.10 μg/mL for metformin, pioglitazone, and glimepiride, respectively, showing a high degree of method sensitivity. The method was then validated according to the FDA guidelines for the determination of the three drugs clinically in human plasma, in particular, regarding pharmacokinetic and bioequivalence simulation studies.
EN
A reversed-phased high-performance liquid chromatography–diode-array detection (HPLC–DAD) method has been developed for investigating the stress-dependent degradation of pantoprazole (PTZ) by a photolytic and oxidative mechanism. The developed method separated PTZ from its degradation products on a C18 column with a mobile phase consisted of methanol and water (60:40, v/v; pH 3.0) at a flow rate of 1 mL/min. The linear regression coefficient of 0.9995 was obtained for a concentration range from 5 to 25 μg/mL. The % relative standard deviation for repeatability and intermediate precision were below 0.5% and 1.5%, respectively, while the sensitivity of the method was demonstrated by a limit of detection value of 0.25 μg/mL. The stress sample analyses for PTZ results revealed the formation of a total of 18 degradation products, and out of them, 9 degradation products were common for both photolytic and oxidative degradations. Further, the oxidation by azobisisobutyronitrile produced the highest number of degradation products (11 impurities), 3 of which are more hydrophobic than PTZ. In photolytic degradation, 8 and 7 degradation products were observed with UV radiation and sunlight exposure, respectively. Furthermore, the degradation of pantoprazole sodium injection formulation was carried out under the same stress conditions, and it revealed the formation of 3 common impurities under both stress conditions, but other impurities were not detected in the formulations. Finally, 3 common impurities formed in formulations of PTZ injections, viz., sulfone, N-oxide, and N-oxide sulfone impurities, were identified by spike analyses.
EN
High-performance liquid chromatography (HPLC) is a widely used technique for the simultaneous detection and quantification of different drugs. The purpose of the current study was to develop a simple and cost-effective reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of tizanidine (TZN) HCl and meloxicam (MLX) in rabbit's plasma. Assay of TZN and MLX was performed after extraction of drug from plasma by liquid–liquid extraction technique using methanol and diethyl ether as protein precipitants. Isocratic elution was performed in a Kromasil® C18 column (dimension, 250 × 4.60 mm; particle size, 5 μm) with mobile phase consisting of methanol–water (8:2). Orthophosphoric acid was used to adjust the pH of the mobile phase 3.0, and detection was done at 228 nm. Flow rate was 0.8 mL/min with ambient temperature and average operating pressure of 1400 psig. Retention time of TZN was 2.612 min and that of MLX was 6.960 min with a resolution of 3.18. Both drugs showed satisfactory linearity in the range of 10 to 50 ng/mL with correlation coefficients (R2) of 0.9989 and 0.9972 for TZN and MLX, respectively. The developed method was validated successfully for linearity, system suitability, intra-day and inter-day accuracy, and precision, robustness, and specificity following International Conference on Harmonization (ICH) guidelines. Conclusively, a precise, stable, reproducible, economical, and suitable method for estimation of pharmacokinetic evaluation was developed and validated.
EN
A simple and convenient reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous separation, identification, and determination of sodium metabisulfite and sodium benzoate in pharmaceutical formulation has been developed and validated. Chromatographic separation was achieved on RP column Zorbax Extend C-18 (150 × 4.6 mm i.d., 3.5 μm particles), and mixture of 0.1% phosphoric acid and acetonitrile in the ratio 62:38 (v/v) was used as a mobile phase. The flow rate was set at 1.0 mL/min with detection wavelength of 275 nm. The method was successfully validated according to International Conference on Harmonization (ICH) guidelines acceptance criteria. The method is selective, as no interferences were observed at retention times corresponding to these analytes. Results of regression analyses (r) and statistical insignificance of calibration curve intercepts (p) proved linearity of the method in defined concentration ranges for sodium metabisulfite and sodium benzoate (0.05–0.15 mg/mL). Relative standard deviations calculated for both analytes in precision testing were below the limits defined for active pharmaceutical ingredients (analysis repeatability: <2%; intermediate precision: <3%). Recovery values were between 98.16% and 101.94%. According to results of robustness testing, chromatographic parameters are not significantly influenced by small variation of acetonitrile content in mobile phase, column temperature, and flow rate. Finally, the method was applied for quantitative determination of investigated preservatives in real sample analysis.
EN
An effective, reliable, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) with diode array detector (DAD) method was investigated for simultaneous determination of polydatin, isoquercitrin, resveratrol, and nicotiflorin in Tetrastigma hemsleyanum. The chromatographic separation of the four compounds was carried out on a Welchrom ODS column (4.6 mm × 250 mm, 5 μm) by gradient elution with phosphoric acid (H3PO4) aqueous solution (0.4%)–methanol as the mobile phase, at the temperature of 30 °C and a flow rate of 1.0 mL/min. The detection wavelength was set at 270 nm. Under optimum conditions, the baseline separation of these four compounds can be performed within 30 min. The developed method was validated in terms of detection limit, quantification limit, linearity, precision, and recovery tests. Eventually, the established HPLC–DAD method was successfully applied to the simultaneous determination of polydatin, isoquercitrin, resveratrol, and nicotiflorin in the extract of herb T. hemsleyanum.
EN
This study presents the development and validation of a new reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of captan, folpet, and metalaxyl residues in table grape samples with ultraviolet–diode array detection (UV–DAD). Successful separation and quantitative determination of analytes was carried out on LiChrospher 60 RP-select B (250 × 4 mm, 5 μm) analytical column. Mixture of acetonitrile–0.1% formic acid in water (65:35, v/v) was used as a mobile phase, with flow rate of 1 mL/min, constant column temperature at 25 °C, and UV detection at 220 nm. The target residues were extracted with acetone by ultrasonication, followed by a cleanup using liquid–liquid extraction (LLE) and solid-phase extraction (SPE). The obtained values for multiple correlation coefficients (R2 > 0.90), relative standard deviation (RSD) of retention times, peak areas and heights (RSD ≤ 2.25%), and recoveries ranging from 90.55% to 105.40%, with RSD of 0.02% to 5.37%, revealed that the developed method has a good linearity, precision, and accuracy for all analytes. Hence, it is suitable for routine determination of investigated fungicides in table grape samples.
PL
Wzrost konsumpcji paliw kopalnych, ich pozyskiwanie oraz eksploatacja niesie ze sobą wiele zagrożeń dla środowiska, dlatego alternatywnym źródłem energii stają się biopaliwa, w tym bio-wodór pozyskiwany w konwersji biomasy ligno-celulozowej, która poddawana jest obróbce wstępnej. Najczęściej wykorzystywaną metodą obróbki wstępnej jest hydroliza alkaliczna, podczas której powstaję bardzo dużo produktów ubocznych, nieprzydatnych do wytwarzania paliw, szczególnie powstałych z hydrolizy ligniny. Najczęściej wykorzystywaną techniką identyfikacji i oznaczania składu hydrolizatów biomasy ligno-celulozowej jest chromatografia cieczowa realizowana w różnych układach faz oraz z wykorzystaniem elucji gradientowej. W przypadku badania hydrolizatów zawierających hydrofobowe składniki, najbardziej korzystne wydają się warunki odwróconych układów faz – RP-HPLC. W niniejszej pracy porównano dwie metodyki wysokosprawnej kolumnowej chromatografii cieczowej w odwróconych układach faz (RP-HPLC) do rozdzielania i wstępnej identyfikacji składników hydrofilowych mieszanin po hydrolizie zasadowej biomasy lignocelulozowej, w celu optymalizacji procesu konwersji biomasy ligno-celulozowej (BMLC) do uzyskania najlepszej efektywności procesu hydrolizy. Wyniki tych badań powinny doprowadzić w przyszłości do procedur pozyskania ubocznych produktów, powstających podczas procesu hydrolizy BMLC, przydatnych użytkowo. Konieczne będą badania uzupełniające, wykonywane w warunkach dwu-wymiarowej elucyjnej gradientowej kolumnowej wysokosprawnej chromatografii cieczowej, z uwzględnieniem spektrometrii Mas (MS), oprócz detektora spektrofotometrycznego z detektorem typu DAD (Diode Array) - 2D-Grad-HPLC-UV-VIS-DAD / MS.
EN
The increase in the consumption of fossil fuels, their acquisition and exploitation carries a lot of threats to the environment, therefore an alternative source of energy are biofuels, including biohydrogen obtained in the conversion of ligno-cellulosic biomass, which undergoes pre-treatment. The most frequently used method of pre-treatment is alkaline hydrolysis, during which a lot of by-products are generated, unsuitable for the production of fuels, especially those resulting from hydrolysis of lignin. The most commonly used technique for identifying and determining the composition of lignocellulose biomass hydrolysates is liquid chromatography carried out in various phase systems and using gradient elution. In the case of testing hydrolysates containing hydrophobic components, the conditions of reversed phase systems - RP-HPLC seem to be most favorable. This paper compares two methods of high performance reverse phase column chromatography (RP-HPLC) for the separation and initial identification of components of hydrophilic mixtures after basic hydrolysis of lignocellulose biomass, in order to optimize the process of converting lignocellulose biomass (BMLC) to the best the effectiveness of the hydrolysis process. The results of these tests should lead in the future to procedures for obtaining by-products of BMLC hydrolysis which are useful for use. Supplementary tests will be required, performed in two-dimensional, elution, gradient, columnar high performance liquid chromatography, including Mas (MS) spectrometry, in addition to a spectrophotometric detector with a DAD detector (Diode Array) - 2D-Grad-HPLC-UV-VISDAD / MS.
EN
Linaclotide, a first-in-class guanylate cyclase-C agonist, was recently approved by US Food and Drug Administration (FDA) as a promising pharmacotherapy for the management of constipation-predominant irritable bowel syndrome (IBS). In this communication, we present a novel stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of linaclotide along with its degradation products. During the International Conference on Harmonization (ICH) prescribed stress study, linaclotide was found susceptible to degrade under hydrolytic (acid and base) and oxidative (peroxide) conditions. The separation of the degradants from the analyte was achieved on a Zorbax Eclipse XDB C8 Column (250 mm × 4.6 mm, 5 μm) using 0.01 N potassium dihydrogen orthophosphate buffer and acetonitrile (80:20 v/v) as mobile phase at a flow rate of 1.00 mL min−1 at column temperature of 40 °C. The detection of the column effluents was realized on a photodiode array detector set at 220 nm. Under the above optimal condition, the method was validated with respect to specificity, linearity, range, precision, robustness, and sensitivity in compliance to the regulatory requirements.
EN
A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of doxazosin mesylate (DOX) and finasteride (FIN) in bulk powders and pharmaceutical formulations. The compounds were separated on a Pinnacle II C18 column (250 × 4.6 mm i.d.; particle size, 5 μm) with an isocratic mobile phase at a flow rate of 1.0 mL min−1. The mobile phase was a mixture of 25 mM ammonium acetate and acetonitrile in the ratio of 50:50 %v/v. The pH of the buffer was adjusted to 4.0 ± 0.05 with glacial acetic acid. The detection was performed at 230 nm. The total chromatographic analysis time per sample was 15 min with DOX and FIN eluting at 3.9 and 7.2 min, respectively. The accuracy, precision, specificity, linearity, and sensitivity of the method were validated according to the International Conference on Harmonization (ICH) guidelines. The calibration plots were linear (r2 > 0.999) over the concentration range 24.25–291.0 μg mL−1 and 122.5–1470.0 μg mL−1 for DOX and FIN, respectively. The method was used for the simultaneous determination of DOX and FIN in capsules.
10
EN
Three independent reversed phase high-performance liquid chromatography (HPLC) procedures with diode array detection (DAD) for the analysis of carbamazepine (CBZ), topiramate (TPM), and valproic acid (VPA) have been developed in order to determine drug penetration of the blood—brain barrier. Determination of CBZ was performed on C18 column with mobile phases containing methanol (55%, v/v), acetate buffer at pH 3.5 (20%, v/v), double distilled water (25%, v/v), and 0.025 M L−1 diethylamine (DEA). The mobile phase containing acetonitrile and water (8:2, v/v) or acetonitrile and phosphate—citrate buffer at pH 2.6 (1:1), respectively, for analysis of VPA and TPM was applied. Quantification of carbamazepine was performed at 285 nm without extraction procedure before the analysis. Determination of topiramate and valproic acid was performed using precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). FMOC-Cl is a suitable agent, which reacts with both primary and secondary amines and also with acidic groups. Topiramate was determined at 263 nm and valproic acid at 300 nm. The proposed procedures are simple, not time-consuming, and suitable for the determination of investigated compounds in mouse brain homogenates.
EN
In order to assess the contribution of adenosine triphosphate and its metabolites to the cellular metabolism process in Saccharomyces cerevisiae, it is very important to simultaneously determine the relative concentrations of ATP and its metabolites. In this study, a fast, simple reversed-phase high-performance liquid chromatography with high selectivity was developed to simultaneously measure adenosine triphosphate and its metabolites (adenosine diphosphate, adenosine monophosphate, and cyclic adenosine monophosphate) in yeast. The method was performed under the gradient grogram, and the detection was monitored at 254 nm. Analysis was achieved within 25 min. The four components can be detected with linear response over the concentration range from 1 to 100 mg L−1 with excellent correlation coefficients (r2) > 0.999. The recovery of the four analytes was 92.9%, 90.4%, 99.1%, and 105.1%, respectively. To demonstrate the good analysis of yeast samples, changes in the four adenine nucleotides levels caused by caloric restriction in yeast were determined. It is expected that the current method may contribute to further metabolomics and system biology investigations of yeast.
EN
In the present study, the degradation behavior of Fenofibrate under different International Conference on Harmonization (ICH) suggested conditions was studied. Characterization of degradation products by liquid chromatography–tandem mass spectrometry (LC–MS/MS) studies in solution form was done, and the possible mechanism for the formation of degradants is discussed. Fenofibrate was subjected to different hydrolytic stress conditions and thermal stress condition (in solid form). Successful separation of drug from degradants was achieved on a C18 column using water–acetonitrile (25:75 v/v) as the mobile phase. Other high-performance liquid chromatography (HPLC) parameters were: flow rate, 1 mL min−1; detection wavelength, 286 nm; column temperature, 25 °C; and injection volume, 20 μL. The method was validated for linearity, precision, accuracy, robustness, and specificity and was stability-indicating one, based on the specificity studies. The drug degraded under acidic, basic, and oxidative hydrolytic stress while it was relatively stable towards neutral hydrolysis and thermal stress. The stressed samples were subjected to LC–MS/MS analysis. On the basis of spectral data, the structures of four degradation products and one interaction product were suggested. Degradation products were characterized to be isopropyl acetate, 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl propanoic acid, 4-hydroxy benzoic acid, and benzoic acid. The structure of one interaction product was proposed as methyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate.
EN
A fast, simple, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and fully validated for the determination of moxifloxacin (MXF) in rat plasma. MXF and gatifloxacin (internal standard, I.S.) were extracted from plasma by single-step protein precipitation with acidified acetonitrile. Chromatographic separation was accomplished in less than 8 min on an Atlantis ® T3 column with 0.4% aqueous triethylamine–methanol–acetonitrile (60:35:5, v/v/v) solution as mobile phase. Detection was achieved by fluorescence (λexcitation = 295 nm, λemission = 500 nm), and the calibration curves were found to be linear over the plasma concentration range of 10–2,500 ng mL−1 with a mean correlation coefficient (r) of 0.9946 (n = 6). The intra- and inter-assay imprecision (% CV) was less than 2.4 and 3.3%, respectively, and the accuracy was >90%. The mean extraction recoveries for MXF and I.S. from plasma were 77 and 82%, respectively. The method was also validated for specificity, sensitivity, and stability; all the results were within the acceptable range. The proposed method was then successfully applied to the quantitative analysis of MXF in rat plasma samples, being a valuable and high-throughput assay to support ongoing pharmacokinetic studies on this promising anti-infective agent.
PL
Chromatografia cieczowa jest jedną z technik najczęściej wykorzystywanych w badaniach składu mieszanin, a także do wydzielania czystych składników z mieszanin w postaci frakcji eluatu. Z punktu widzenia współczesnej chemii, farmakologii, a także medycyny, czy biotechnologii, chromatografia pełni ważną rolę, w zakresie analityki jakościowej i ilościowej. Dzięki możliwości otrzymywania naturalnych składników biologicznie czynnych w czystej postaci z ekstraktów pochodzenia naturalnego, jak i składników mieszanin po syntezie organicznej, ma też ważne znaczenie w skali preparatywnej, i coraz większe, w skali procesowej. W celu zapewnienia efektywności procesu konieczne jest dobranie optymalnych warunków realizacji operacji rozdzielania, detekcji oraz kolekcji frakcji, w zależności od właściwości fizykochemicznych składników rozdzielanej mieszaniny. Niniejsza praca dotyczy badań nad optymalizacją warunków stosowanych przy rozdzielaniu składników jednej z grup związków chemicznych stanowiących metabolity roślinne – polifenole. W pracy zbadano wpływ na retencję, selektywność i sprawność rozdzielania, w tym także na symetrię pików, poprzez zastosowanie dodatków do eluentu w postaci kwasów (HCl, H3PO4, H2SO4, CH3COOH, TFA), do rozdzielania wybranych, naturalnie występujących materiale roślinnym polifenoli w warunkach RP HPLC. W rozdzielaniu peptydów w układach RP, w tym, w proteomice, najczęściej używanym modyfikatorem fazy ruchomej, jest kwas trifluorooctowy (TFA). Spełnia on wówczas dwie funkcje:- ogranicza kwaśną dysocjację peptydu (na tzw. „C-końcu”), co zwiększa poziom hydrofobowości molekuł; - solwatuje sprotonowane lub spolaryzowane, dodatnio naładowane fragmenty cząsteczki peptydu lub białka ujemnymi jonami zdysocjowanego kwasu, powodując wyraźne dodatkowe podwyższenie hydrofobowości rozdzielanych cząsteczek. W przypadku polifenoli i warunków RP, obecny w eluencie kwas, powoduje jedynie cofnięcie dysocjacji elektrolitycznej rozdzielanych mniej lub bardziej kwaśnych związków chemicznych. Wpływa to na wyraźny wzrost hydrofobowości cząsteczek. Zwiększenie hydrofobowości skutkuje wzrostem współczynnika retencji, poprawą symetrii pików i lepszym rozdzieleniem składników mieszaniny w stosunków do warunków rozdzielania bez dodatku kwasu do eluentu. Badania tej pracy potwierdzają konieczność dodania kwasu do eluentu, w przypadku stosowania warunków faz odwróconych (RP). Pokazują, że każdy z badanych kwasów powoduje korzystne efekty, jednak, najbardziej korzystne - zwłaszcza dla celów analityki - okazuje się stosowanie niewielkiego dodatku kwasu siarkowego (VI).
EN
Liquid chromatography is the one of commonly used technique in research of mixture composition, and also for separation of pure components form of eluate fractions. From the standpoint of modern chemistry, pharmacology, medicine or biotechnology, chromatography plays an significant role in the field of qualitative and quantitative analysis. Additionally, through the possibility of isolation from natural origin extracts the biologically active components in its pure form, as well as the components of the mixture after organic synthesis, chromatography is important technique, which can be used in preparative and also in process scale. In order to provide the effectiveness of the process, it is necessary to select the optimum conditions of the separation, detection and collection of fractions, which depend on the physiochemical properties of separated components constituting the mixture. This paper, include the research related to optimization of chromatographic conditions, applied for the separation of the one group of chemical compounds, namely plant metabolites – polyphenols. In this research, effects of retention, separation selectivity and efficiency, including the symmetry of peaks, by applying the additive to the eluent in the form of acids (HCl, H3PO4, H2SO4, CH3COOH, TFA) for separation naturally occurring in plant polyphenols under RP-HPLC conditions was investigated. In the separation of peptides under RP conditions, including proteomics, the most commonly used acidic modifier is trifluoroacetic acid (TFA). It meets two functions: - reduces the acid dissociation of the peptide (on the so-called “C-end”), which results in an increase hydrophobicity of the molecules; - causes the salvation of the protonated or positively polarized fragments of the peptide or protein molecule by negative ions dissociated acid, resulting in a noticeable increase hyrophobicity of separated molecules. In the case of polyphenols and RP conditions, the presence of acid in the eluent, cause only the withdrawal of electrolytic dissociation of separated acidic compounds. This can affect the significant increase in the hydrophobicity of the molecules. In addition, increasing hydrophobicity resulting in increase the retention rate, improvement of peak symmetry and better separation of mixture components, regarding to separation without the addition of acid to the eluent. Research, carried out in this study, confirm that it is necessary to use acid additive to the eluent, in the case of reversed phase (RP) conditions. Indicate that each of tested acids results in beneficial effects, however, the most preferred - especially for the purpose of analysis – turns out a small addition of sulfuric acid (VI).
15
Content available remote HPLC and UPLC Analyses of Acetoin in Bacterial Culture Fluid
EN
A simple and rapid ultra-performance liquid chromatographic (UPLC) method for analyzing acetoin in bacterial culture fluid was developed and validated for the first time. The samples were separated using an Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm particle size) and isocratic elution with 30 mM phosphoric acid—1% acetonitrile as the mobile phase. A photodiode array detector (PDA) was used. The run time was 6 min, and the detection limit was 2.11 × 10−4 mg mL−1. The UPLC method was compared with high-pressure liquid chromatography (HPLC) for acetoin analysis. The proposed UPLC method is highly sensitive and was successfully applied to the analysis of acetoin in bacterial culture fluid.
EN
A rapid, accurate, and sensitive reverse phase high-performance liquid chromatographic method was developed and validated for the simultaneous determination and quantification of glibenclamide and thymoquinone in rat plasma in the presence of internal standard (thymol). Chromatograms were developed with methanol, acetonitrile, and buffer (50:20:30, v/v/v) solvent system on a Symmetry® C18 (5 μm, 3.9 × 150 mm) column, and pH was adjusted to 4.5 with orthophosphoric acid. Mobile phase was pumped at a flow rate of 1.5 mL min-1 with 254 nm ultraviolet (UV) detection. Validation of the method was performed in order to demonstrate its selectivity, linearity, precision, accuracy, limits of detection, and quantification (LOD and LOQ). Standard curves were linear (r2 = 0.996 and 0.999 for glibenclamide and thymoquinone) over the concentration range 0.5–50 μg Ml-1. The coefficient of variation (CV) of < 6% and accurate recovery of 87.54–105.19% for glibenclamide and CV of <5% and accurate recovery of 86.08–103.19% for thymoquinone were found to be in the selected concentration range of 0.5–50 μg Ml-1. The lower limits of detection and quantitation of the method were 0.109 and 0.332 μg Ml-1 for glibenclamide and 0.119 and 0.361 μg Ml-1 for thymoquinone, respectively. The within and between-day coefficients of variation were less than 7%. The validated method has been successfully applied to measure the plasma concentrations in a drug interaction study of glibenclamide with thymoquinone in an animal model to illustrate the scope and application of the method.
EN
A new, rapid, and specific reversed phase high-performance liquid chromatographic (RP-HPLC) method involving precolumn derivatization with benzoyl chloride was developed and validated for the estimation of γ-aminobutyric acid (GABA) in rat brain tissue preparations. The derivatization product of GABA was identified by melting point, infrared, and proton nuclear magnetic resonance (1H NMR) spectroscopy to be n-benzoyl GABA. Various parameters which influenced derivatization and elusion were optimized. The chromatographic system consisted of C-18 column with ultraviolet (UV)—photodiode array detection ranging from 210 to 400 nm. Elution with an isocratic mobile phase consisting of 0.025 M disodium hydrogen phosphate buffer—methanol (65:35, v/v; pH 6) at a flow rate of 1 mL min-1 yielded sharp and specific peak of n-benzoyl GABA within 7 min. The method was validated with respect to the linearity, accuracy, precision, sensitivity, selectivity, and stability, wherein the benzoyl derivative of GABA showed stability for 2 months. The lower limit of detection was 0.5 nmol L-1. This novel derivatization procedure for the estimation of GABA with benzoyl chloride was also applied for rat brain tissue preparations that gave highly specific peak and good component recovery. The results show that the method for the determination of GABA by benzoylation using RP-HPLC has good linearity, accuracy, precision, sensitivity, and specificity and is simple and economical to perform.
EN
A novel simple, sensitive, and rapid isocratic reverse phase high-performance liquid chromatographic (RP-HPLC) method was developed for the quantitative determination of alosetron HCl, a 5-HT3 antagonist used in the treatment of severe irritable bowel syndrome (IBS) in females. The optimized chromatographic separation was achieved using a stationary phase of Phenomenex® kromasil C-18 (250 mm × 4.6 mm; 5 μm particle size) column, and mobile phase of 0.025 M disodium hydrogen orthophosphate buffer, pH adjusted to 3.0 with orthophosphoric acid, and acetonitrile in the ratio of 65:35 (v/v) with a flow rate of 1 mL min. -1 The UV detection was carried out at 217 nm. The developed method provided linear responses within the concentration range 100–2000 ng Ml -1, and regression analysis showed a correlation coefficient value (r2) of 0.997. The HPLC method was validated as per International Conference on Harmonization (ICH) guidelines with respect to selectivity, precision, linearity, and robustness. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 1 ng Ml -1 and 5 ng Ml -1, respectively.
EN
A simple, sensitive, specific, and cost effective method for simultaneous determination of Aspirin and Rosuvastatin calcium was developed and validated in single dosage formulation. The sample solution of ASP and RSTC was prepared using methanol as a solvent. Separation of ASP and RSTC was achieved with a mobile phase consisting of 20 mM KH2PO4 : Methanol (30:70 v/v) at a flow rate of 1.0 ml/min. Separations were performed on Merck hibar 250-4.6 RP18 (5 μm) column (150 mm X 3.0 mm), using a Shimadzu Prominence HPLC system equipped with a Shimadzu SPD-20A detector, Rhenodyne 7725i injector with 20 μL loop, LC-20 AD pump, CBM-20 Alite controller and LC Solution software. Retention times of ASP and RSTC were 3.747 and 5.969 minutes respectively. Absolute recovery of ASP and RSTC was 100.3 and 100.03 % respectively. The lower limit of quantification (LLOQ) of ASP and RSTC was 0.3097 and 0.1063 ppm and lower limit of detection (LLOD) of ASP and RSTC was 0.01535 and 0.01358 ppm respectively. Linearity was established for the range of concentrations 15.00-90.0 μg/ml and 2.0-12.0 μg/ml for ASP and RSTC respectively with the coefficient of determination (R2) of 0.994 and 0.999 for both the compounds. The inter- and intra-day precision in the measurement of ASP quality control (QC) sample 75 μg/ml, were in the range 0.1-0.2 % relative standard deviation (R.S.D.) and 0.2-0.3 % R.S.D., respectively. The inter- and intra-day precision in the measurement of RST quality control (QC) sample 10 μg/ml, were in the range 0.1-0.2 % R.S.D., and 0.0-0.3 % R.S.D., respectively. The developed method would be applicable for routine quality control of ASP And RSTC in bulk as well as in pharmaceutical formulations.
EN
A sensitive validated high-performance liquid-chromatographic method for analysis of cilostazol in human plasma (in vitro) has been developed, and it was applied to determine pharmacokinetics of cilostazol in male albino rabbit. Cilostazol was extracted from human plasma (in vitro) by acetonitrile, and efficient chromatographic elution was achieved on a C18 column (250 × 4.60 mm i.d., 0.5 μm particle size) with an isocratic mobile phase [acetonitrile-50 mM acetate buffer (pH 5.0, glacial acetic acid)-water (50:20:30)] at flow rate of 1.5 mL min−1. Quantification was carried out by photo-diode array (PDA) detection at 248 nm. The linearity of the method was excellent over the range 0.2–2 μg mL-1 with low limits of detection (0.005 μg mL-1) and quantification (0.05 μg mL-1). The extraction recovery of the drug from plasma was consistently good (73.45–78.64%), with low relative standard deviation (0.44–1.65%). Robustness studies confirmed that peak area was unaffected by small changes in temperature, mobile phase (composition and pH). The maximum concentration (Cmax) in rabbit (in vivo) was determined 1.620 μg mL-1 at tmax (0.51 h) with 0.63% RSD by validated bioanalytical method.
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