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1

Simultaneous reverse-phase HPLC determination of major antioxidant phenolics in Commelina benghalensis L. tubers

Misra A., Srivastava A., Srivastava S., Rawat A. K. S.

Acta Chromatographica

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2016

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Vol. 28, no. 4

541--554

EN

Commelina benghalensis (Commelinaceae) is widely used as traditional and folklore medicine in India. In the present study, a reverse-phase high-performance liquid chromatography—photodiode array detection (RP-HPLC—PDA) method was developed for the separation, identification, and quantification of bioactive phenolics. Antioxidant potential was also accessed to validate the presence of identified markers. Method was developed on C18 column with 1% formic acid (in water) and acetonitrile as solvent system, and data acquisitions were achieved at wavelength of 285 nm. The developed method was also validated for accuracy, precision, robustness, limit of detection and quantification (LOD and LOQ), repeatability, and recovery according to International Conference on Harmonization (ICH) guidelines. In this method, five phenolics, viz., protocatechuic acid (0.033%), vanillic acid (0.262%), ferulic acid (0.365%), apigenin (0.126%), and kaempferol (0.544%), were quantified in linearity range of 0.2–1.0 μg with correlation coefficient of more than 0.9949. Relative standard deviation (RSD) (%), LOD, LOQ, and recovery (%) are within the acceptable limit. Besides that, methanolic extract shows the inhibition (%) range from 24.45 to 68.75% at 0.02–0.12 mg Ml-1. IC50 of extract was observed at 46.75 μg Ml-1, suggesting the promising activity in methanol extract. Hence, the proposed method for simultaneous quantification of five bioactive phenolics in the tuber of C. benghalensis using HPLC–PDA detection under the specified conditions is specific and accurate, and validation proves its selectivity and reproducibility.

2

Synthesis of three strong UV-absorbing Naproxen-based chiral derivatizing agents and their application for enantioseparation of Baclofen by RP-HPLC

Batra S., Bhushan R.

Acta Chromatographica

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2015

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Vol. 27, no. 2

267--280

EN

A sensitive and stereoselective high-performance liquid chromatography (HPLC) method based on diastereomer formation was developed and fully validated for the simultaneous determination of the two enantiomers of Baclofen. (S)-Naproxen was used to prepare three chiral derivatizing reagents (CDRs) which were used for synthesizing diastereomers of Baclofen. The diastereomers so synthesized were separated on C18 column under reversed phase conditions using a binary mixture of acetonitrile and triethylammonium phosphate, with UV detection at 220 nm. The results obtained for enantioseparation of Baclofen using the three CDRs have been compiled and compared. The conditions for derivatization and chromatographic separation have been optimized. The method was validated for linearity, repeatability, limits of detection, and limit of quantification.

3

Validated stability-indicating RP-HPLC UV method for simultaneous determination of metformin and repaglinide

Joshi S. S, Nahire R. R, Shastri N. R, Surendranath K. V, Satish J

Acta Chromatographica

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2012

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Vol. 24, no. 3

419--432

EN

A simple, rapid, precise, and accurate, stability-indicating reversed phase high performance liquid chromatographic method was developed and validated for simultaneous determination of metformin HCl and repaglinide. The chromatographic separation was achieved on YMC Pack AM ODS (5 μm, 250 mm length × 4.6 mm i.d.) column at a detector wavelength of 210 nm, using an isocratic mobile phase consisting of methanol and 10 mM potassium dihydrogen phosphate buffer (pH 2.5) in a ratio of 70:30 v/v at a flow rate of 1 mL min-1. The retention times for metformin and repaglinide were found to be 2.6 and 11.3 min, respectively. The drugs were exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. Validation of the method was carried out as per International Conference on Harmonization (ICH) guidelines. Linearity was established for metformin and repaglinide in the range of 5–200 μg mL-1 and 1–200 μg m-1. L, respectively. The limits of detection were 0.3 μg mL-1 and 0.13 μg mL-1 for metformin and repaglinide, respectively. The method was found to be specific and stability-indicating as no interfering peaks of degradants and excipients were observed. The proposed method is hence suitable for application in quality-control laboratories for quantitative analysis of both the drugs individually and in combination, since it is simple and rapid with good accuracy and precision.

4

Analysis of the enantiomers of serine by reversed-phase high-performance liquid chromatography with OPA-Boc as pre-column derivatization agent. Critical evaluation of the analysis of d-serine in rat brain

Oláh E, Berky R, Fekete J

Acta Chromatographica

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2011

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Vol. 23, no. 1

59--68

EN

The enantiomers of serine have been separated after pre-column derivatization with ortho-phthaldialdehyde (OPA) and N-tert-butyloxycarbonyl-L-cysteine (Boc). Analysis of the enantiomers of amino acids after pre-column derivatization with OPA-Boc has been reported in the literature but no critical evaluation has been carried out. In this study, decomposition of the diastereomers, effect of the sample pH, and amount of derivatization agent were studied; the half life of the decomposition was also determined. The method was used for analysis of D and L-serine from three different areas of rat brain (the striatum, prefrontal cortex, and cerebellum).

5

Direct Liquid Chromatography with Photodiode and Fluorescence Detection for Simultaneous Quantification of Gonadotropin-Releasing Hormone (GnRH), Its Complex with Cu2+, and Catabolites

Michaluk A., Gajewska A., Kulon K., Kozłowski H., Kochman K., Kowalczyk J., Czauderna M.

Polish Journal of Chemistry

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2009

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Vol. 83, nr 3

383-390

EN

Gonadotropin-releasing hormone (GnRH) and its complex with Cu2+(Cu-GnRH) were separated on a Nova Pak C18 column (4 m, 150 3.9 mm I.D., Wa ters). Analyses of underivatized GnRH and Cu-GnRH were per formed by a gradient elution program (HPLC method I), UV detection at 280 nm and fluorescence detection (gammexcitation = 280 nm/gammaemis sion = 360 nm). The mobile phases used were: acetonitrile with 0.08% trichloroacetic acid (w/w) and water with 0.1% trichloroacetic acid (w/w). Elutions were carried out in a binary gradient mode with a flow-rate of 1 mL/min and column temperature of 28°C. The proposed gradient elution program with UV and fluorescence detection allowed satisfactory fraction ation of GnRH (at 31.5 plus minus 0.2 min) from Cu-GnRH (at 30.3 plus-minus 0.2 min) and endogenous species present in samples of cytosol and subcellular organelles from the hypothalamus. The proposed reversed-phase HPLC method I with fluorescence detection provides a more sensitive analytical tool for routine and simultaneous quantification of GnRH, Cu-GnRH and their enzymatic degradation products (catabolites) in all biological samples as compared with HPLC method I with UV detection. To avoid problems due to overlap ping peaks corresponding to GnRH, Cu-GnRH, and the respective enzymatic degradation products in samples of cytosol and subcellular organelles from the hypothalamus, we propose a very shallow binary gradient elution program (method I). The separation efficiency of GnRH and Cu-GnRH peaks in standards and biological samples was assessed based on purity analysis of UV spectra (250-300 nm) and on the values of ratios of the fluorescence response to UV response at 280 nm. Our second reversed-phase liquid chromatographic method (HPLC method II) with pre-column derivatization of aminoacids in catabolites of GnRH and Cu-GnRH enabled investigations of the degradation pattern as well as of the yield of enzymatic degradation of GnRH and its Complex with Cu2+ in pituitary cytosol and sub-cellular organelles.

6

Development and validation of a stability-indicating LC-UV method for rapid analysis of buspirone in pharmaceutical dosage forms

Azeem A., Rizwan M., Ahmad F. J., Iqbal Z., Khar R. K., Aqil M, Talegaonkar S.

Acta Chromatographica

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2009

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Vol. 21, no. 2

283--297

EN

An accurate, sensitive, precise, rapid and isocratic reversed-phase HPLC (RPHPLC) method for analysis of buspirone in the bulk drug and in solid dosage formulations has been developed and validated. The best separation was achieved on a 250 mm × 4.6 mm i.d., 5-μm particle, RP C 18 column with 70:30 ( υ/υ ) methanol-0.01 M sodium dihydrogen phosphate buffer (pH 3.5) as mobile phase at a flow rate of 0.8 mL min -1. UV detection was at 244 nm. Response was a linear function of concentration over the range 0.05–20 μg mL -1 ( r = 0.9998) and the limits of detection and quantitation were 3.7 and 11.3 ng mL -1, respectively. The method was validated in accordance with ICH guidelines. The drug was subjected to oxidative, hydrolytic, photolytic, and thermal stress. Degradation products produced as a result of this stress did not interfere with detection of buspirone and the assay can thus be regarded as stability-indicating. The method was used for quantification of buspirone in commercial buspirone tablets and to check content uniformity. The excipients present in the formulation did not interfere with the assay. The method is suitable for application in quality-control laboratories, because it is simple and rapid with good accuracy and precision.

7

Impurity profile study of capecitabine

Hiriyanna S. G., Basavaiah K.

Acta Chromatographica

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2008

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Vol. 20, no. 4

609-624

EN

Five new peaks of potential impurities, ranging from 0.05 to 0.12% on an area-percent basis, have been observed in reversed-phase high-performance liquid chromatography of capecitabine drug substance. On the basis of comparison of relative retention times and mass values two of the impurities were identified as intermediates in the process used for synthesis of capecitabine and were characterized as (2-(4-amino-5-fluoro-2-oxopyrimidin-1(2 H )-yl)-5-methyltetrahydrofuran-3,4-diyl diacetate and 2-(5-fluoro-2-oxo-4-(pentyloxycarbonylamino)pyrimidin-1(2 H )-yl)-5-methyl tetrahydrofuran-3,4-diyl diacetate. The area percentages of two of the other peaks increased substantially when the drug was treated with acid and base. The three compounds other than the two intermediates were isolated by preparative HPLC with gradient elution and were characterized by use of spectroscopic techniques, viz. MS-MS, FTIR, and NMR ( 1 H, 13 C and DEPT). On the basis of the spectral data, the structures 5?-deoxy-5-fluorocytidine, 5?-deoxy-5-fluorouridine, and 2-(4-(bis(pentyloxycarbonyl)amino)-5-fluoro-2-oxopyrimidin-(2 H )-yl)-5-methyltetrahydrofuran-3,4-diyl diacetate were proposed for the three impurities.

8

Application of derivatives of UV spectra and similarity indices to identification of milk proteins separated using reversed-phase high performance liquid chromatography

Minkiewicz P., Dziuba J., Nałęcz D.

Polimery

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2003

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T. 48, nr 2

106-110

EN

The method which may be applied as a tool for identification of proteins separated using reversed-phase high performance liquid chromatography, based on similarity indices of UV spectra derivatives, was proposed. Commercial milk protein preparations such as sodium and calcium caseinates, whey protein concentrate and milk protein coprecipitate as well as standards of milk proteins were used in the experiment. Statistical significance of difference between first and second similarity index, evaluated with Student's t test was used as a criterion to choose the mathematical treatment of spectra as well as to predict reliability of identification. First derivatives of spectra revealed the highest value of t parameter, The presence of statistically significant difference between first and second similarity indices always led to correct identification of proteins. The presented method enabled appropriate identification of caseins in commercial milk protein preparations, but failed ill the case of whey proteins.

PL

Zaproponowano metodę identyfikacji białek - rozdzielonych za pomocą wysokosprawnej chromatografii cieczowej w odwróconej fazie - na podstawie wskaźników podobieństwa (ST) pochodnych widm UV [równanie (1)]. Badano handlowe preparaty białek mleka, takie jak kazeinian wapnia, ka-zeinian sodu, koncentrat białek serwatkowych oraz koprecy-pitat białek mleka. Statystyczna istotność różnic między pierwszym a drugim wskaźnikiem podobieństwa, obliczona na podstawie testu t Studenta, posłużyła jako kryterium wyboru obróbki widm oraz do sprawdzenia wiarygodności identyfikacji białek. Pierwsze pochodne widm UV wykazały największą wartość parametru t (tabela 1). Jedynie występowanie statystycznie istotnych różnic między pierwszym a drugim wskaźnikiem podobieństwa zawsze prowadziło do poprawnej identyfikacji białek (tabela 2). Proponowana metoda umożliwiła prawidłową identyfikację poszczególnych frakcji kazeiny w handlowych preparatach białek mleka, ale nie pozwoliła na identyfikację białek serwatkowych.