Isocratic System Research Articles

Development and Standardization of Chromatographic Technique for Quantification of Bicalutamide in Pure and Pharmaceutical Dosage Forms

Background Bicalutamide BTM is a non-steroidal antiandrogen found to be available in various pharmaceutical dosage forms. Quantitative estimation of BTM is essential in the quality control laboratories of pharmaceutical industries.Objective The present research attempts to develop optimize and standardize chromatographic methodology for quantitative estimation of BTM in its pure and pharmaceutical dosage forms. Methodology The analysis was resolved by using a mobile phase acetonitrile methanol in the ratio of 6040 vv at a flow rate of 1 mlmin on an isocratic High Performance Liquid Chromatography HPLC system consisting of SPD-20 A20AV UV-VIS detector using C18 column at a wavelength of 275 nm. The developed method was subjected for standardization as per ICH guidelines. Results The retention time was found to be 4.7 minutes. The drug showed linear response between the concentration range from 10-100 mugmL. This method was found to be selective specific and precise with RSD Relative Standard Deviation less than 2.nbsp Conclusion The reported method was found to be simple precise and accurate for quantitative estimation of BTM in bulk and pharmaceutical dosage forms and can be employed for routine quality control analysis of BTM.

Biogenic Amines as Freshness Indicator in Halal and Non-Halal Slaughtered Chicken Meat Using Chromatographic Approach

Biogenic amines are naturally occurring organic bases produced by bacterial decarboxylation of amino acids that have been associated with harmful toxicology effects to humans. The main objective of this work is to investigate the possible role of biogenic amines (BAs) as indicators of spoilage in fresh chicken meat stored at 4°C for 15 days. A reversed-phase high-performance liquid chromatographic method with isocratic elution system is used for the quantification of four biogenic amines (putrescine, histamine, tyramine, cadaverine and spermine as well as spermidine) in chicken meat. Amines were extracted with 5% of trichloroacetic acid (TCA) and derivatised using dansyl chloride. The variation storage time differentiated the chicken meat on the microbiological characteristics. The results obtained shows that histamine, spermidine, tyramine, cadaverine, and putrescine increased slowly while spermine decreased for both chicken breast in halal and non-halal chicken meat. Thus, this BAs could be used as a spoilage index of fresh chicken meat.

Novel Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Alfuzosin and Dutasteride in Pharmaceutical Dosage Form

Abstract: Aim: The present study was to develop a simple, accurate and stable reverse phase liquid chromatographic method and validate in bulk drug and pharmaceutical dosage form for the simultaneous determination of Alfuzosin Hydrochloride (ALF) and Dutasteride (DUT). Materials and Methods: Chromatographic separation has been accomplished using an XTerra C18 Column (150×4.6mm, 5μm particle size) as the stationary phase, with an isocratic system of mobile phase Ammonium dihydrogen phosphate buffer (pH 6.5) and methanol (25:75 proportions) at a flow rate of 1.0 mL/min, detection was executed at 246 nm using an UV detector. The optimized method was validated in accordance to International Conference on Harmonization guidelines. Results: The method developed was found to be linear as regression analysis showed good correlation (R2 = 0.999) with a linear curve at concentration range of 25-150 μg/mL for ALF and 1.25-7.5 μg/mL for DUT. The approach was unique as it was free of degradants in spite of subjecting the drugs to forced degradation. The percentage recovery was in the range of 99.55 and 99.23 % for ALF and DUT respectively from the pharmaceutical dosage form. The developed method showed accurate, precise, robust results with an LOD and LOQ of 0.41 and 0.71 μg/mL and 4.27 and 2.14 μg/mL respectively. Conclusion: Due to the flexibility, accuracy and high precision, the developed method can be employed in routine analysis of bulk and dosage forms. Key words: Alfuzosin Hydrochloride, Dutasteride, RP-HPLC, Method development, Validation, Degradation.

New liquid chromatography assays for simultaneous quantification of antihypertensives atenolol and valsartan in their dosage forms.

Nowadays, various single-pill combinations are used as the best choice in hypertension management. However, these pills made a high challenge to analysts in terms of quality control assays. We have developed three sensitive, selective, fast, simple, green, accurate, precise, and robust isocratic high-performance liquid chromatography methods for simultaneous determination of valsartan and atenolol in dosage forms. To find the appropriate high-performance liquid chromatography conditions for the separation of the examined drugs, various columns, isocratic mobile phase systems were tried, and successful attempts were performed. The used columns proved to be indispensably applicable and gave a shorter analysis time and peak symmetries. This reduction in total run time leads to low solvent consumption and makes all methods more economical. The linearity, accuracy, and precision remained within the acceptable limits. Therefore, all developed methods are suitable for the routine quality control analysis of any pharmaceutical preparation containing the two tested drugs with the proposed chromatographic methods advantages for checking quality during stability studies of their pharmaceutical preparations.

Development and validation of HPLC method for simultaneous quantification of alpha-tocopherol (free or encapsulated) and cholesterol in semen cryopreservation media.

The HPLC method was developed and validated for assaying alpha-tocopherol and cholesterol in cryopreservation media. Chromatographic separation was performed on an isocratic system, using a C-18 column. The mobile phase was composed of a mixture of methanol:acetonitrile:water 68:28:4 (v/v/v), using a flow rate of 1.5 mL/min and 20 μL injection volume, at a wavelength of 208 nm. The method was validated according to International Conference on Harmonization guidelines. The method proved to be specific, accurate, precise, and linear with correlation coefficients greater than 0.996 over a wide concentration range of both analytes. Vitamin E and cholesterol presented limits of detection of 0.002 mg/mL, 0.026 mg/mL and limits of quantitation of 0.006 mg/mL, 0.086 mg/mL, respectively. This method is simple and rapid, shows high precision and accuracy, and offers the advantage of simultaneous assaying of vitamin E and cholesterol (alone, in cyclodextrins complexes or in liposome loaded) on semen cryopreservation media.

Systematic Development and Validation of a RP-HPLC Method for Estimation of Abiraterone Acetate and its Degradation Products.

The present study described the development of a reversed-phase liquid chromatographic method for the estimation of abiraterone acetate by Quality by Design (QbD) approach. Using an isocratic solvent system for the mobile phase, the chromatographic estimation of analyte was performed on a Hypersil BDS C18 column using mobile phase mixture containing acetonitrile and water with pH adjusted with 0.1% v/v orthophosphoric acid (15:85%v/v ratio), flow rate 1.0mL.min-1 and detection at 250nm using photodiode array detector. Systematic development of the chromatographic method was carried out by factor screening using a half-factorial design which suggested organic modifier (%), flow rate (mL.min-1) and autosampler temperature (°C) as influential variables. Further, the method was optimized by Box-Behnken design and trials performed were evaluated for the area under peak, retention time, theoretical plate count and tailing factor as the responses. Validation of the developed method showed good linearity, accuracy, precision and sensitivity. Evaluation of the stability-indicating profile of the method using forced degradation studies revealed the formation of a possible degradation product under acidic and alkaline conditions, while no such degradation product peaks were observed under the oxidative environment. Overall, the study construed the successful development of HPLC assay method for pharmaceutical applications.

Thermoanalytical and phytochemical study of the cupuassu (Theobroma grandiflorum Schum.) seed by-product in different processing stages

Cupuassu (Theobroma grandiflorum Schum.) is a typical Amazonian fruit, whose seed is used as raw material to produce cupulate. The by-product of its seeds, cupuassu seed by-product (CSB), is often discarded by the cosmetic and food industry, but studies showed that it has a high content of phenolic compounds with exploitable antioxidant activity. This study aimed to identify the classes of chemical compounds present in the extract of CSB by HPTLC, to confirm them by RMN 1H spectroscopy and to analyze the extract thermal analytical behavior in different processing stages. After elution of the extract in an isocratic system, TLC plates were developed with selective reagents for several classes of chemical compounds and antioxidant activity by the DPPH· method. Butter, CSB and crude extract presented mass losses characteristic of lipid decomposition reactions (92.63, 14.80, and 15.42%, respectively). Thermal degradation of bioactive and nutritional compounds (mainly polyunsaturated fatty acids) present in both CSB and crude extract has occurred at temperatures above 189 °C. Microencapsulation of the CBS extract by spray drying (MECoC) using maltodextrin as coating agent was shown to effectively protect the phenolic compounds. RMN 1H spectra confirmed HPTLC and FTIR results, which allowed us to conclude that the crude extract of the cupuassu by-product was mainly composed of terpenes, steroids and phenolic compounds, mainly flavonoids. These analytical techniques have proven to be effective tools for assessing the bioactive compounds incorporation into the polymeric matrix expected in the pre-formulation stage.

A Validated RP-HPLC Method for Quantification of Steviol Glycoside: Rebaudioside A in Extracts of Stevia Rebaudiana Leaf

RP-HPLC-PDA method has been developed and validated for the quantification of Steviol glycoside: Rebaudioside A in aqueous extract of Stevia rebaudiana plant leaf. Rebaudioside A was separated on a Phenomenex Luna C18 analytical column with column temperature 40 °C using an isocratic solvent system consisting (70:30 v/v) mixture of 10 mmol/L sodium phosphate buffer and acetonitrile having pH-2.6 at 1.0 mL/min flow rate with detection at 210 nm wavelength. The method has been validated for linearity, precision, accuracy, detection limit, quantitation limit and robustness. The proposed method linearity for Rebaudioside A with a regression coefficient of 0.9999 was obtained within the range of 0.0005–1.5 mg/mL. The relative standard deviation was less than 2.0% in intra-day and inter-day precision. The proposed method’s accuracy was determined by a three different level recovery study. The recovery average was between 95.20 and 96.77 percent. Rebaudioside A levels of LOD and LOQ were 0.01 and 0.05 mg/mL respectively. The leaves from eight different Indian territories were extracted by sonication extraction method. Rebaudioside A content obtained in the dried leaves was within the range of 2.39–7.53 percent w/w. The method proposed is simple, rapid and reproducible; therefore, it is appropriate for routine analysis of Rebaudioside A in plant material as well as finished products which are used commercially as high-intensity sweeteners across the globe to replace sugar.

LC-MS/MS Estimation of the Anti-Cancer Agent Tandutinib Levels in Human Liver Microsomes: Metabolic Stability Evaluation Assay.

PurposeTandutinib (MLN518 or CT 53518) (TND) is a novel, oral, small-molecule inhibitor of type III receptor tyrosine kinases utilized for the treatment of acute myeloid leukemia (AML).Materials and MethodsIn silico prediction of hepatic drug metabolism for TND was determined using the StarDrop® WhichP450™ module to confirm its metabolic liability. Second, an efficient and accurate LC-MS/MS method was established for TND quantification to evaluate metabolic stability. TND and entrectinib (ENC) (internal standard; IS) were resolved using an isocratic elution system with a reversed stationary phase (C8 column).ResultsThe established LC-MS/MS method exhibited linearity (5–500 ng/mL) with r2 ≥0.9999 in the human liver microsomes matrix. The method sensitivity was indicated by the limit of quantification (3.8 ng/mL), and reproducibility was revealed by inter- and intraday precision and accuracy (below 10.5%). TND metabolic stability estimation was calculated using intrinsic clearance (22.03 µL/min/mg) and in vitro half-life (29.0 min) values.ConclusionTND exhibited a moderate extraction ratio indicative of good bioavailability. According to the literature, the approach developed in the present study is the first established LC-MS/MS method for assessing TND metabolic stability.

IDENTIFIKASI DAN STUDI POLA FRAGMENTASI JAMU TERKONFIRMASI FENILBUTAZON MENGGUNAKAN LIQUID CHROMATOGRAPHY MASS SPECTROSCOPY (LCMS)

The Jamu is one type of traditional herbal medicine that still popular among the community until now. In twenty years often found the jamu containing synthetic drugs. Drug chemical which is commonly added to this traditional herbal medicine include the class of drugs of NAISD’s namely phenylbutazone. This study was conducted to determine the possibility of presence of phenylbutazone in the Jamu. The beginning of this study by initial screening using Thin Layer Chromatography (TLC) method. The results showed that 4 of the 6 samples of the positive contain phenylbutazone based TLC method. These 4 samples were then analyzed using the Liquid Chromatography Mass Spectroscopy (LCMS) method. The stationary phase is Octadecyl Silica (C18) and the Mobile Phase are ultrapure water motion: acetonitrile (90 : 10% v/v) with isocratic elution system and positive ion mode. Based on LCMS chromatogram data, the retention time (Rf) of phenylbutazone is 1.89 minutes. The retention time of samples same with the retention time of phenylbutazone. The next approach, based on the fragmentation pattern of Mass Spectroscopy. Based on mass spectrum data, there are 2 formed fragmentation paths. The first pattern is the fragmentation process of phenylbutazone (309 m / z), forming 2-butylmalonaldehyde (129 m / z) and 1,2-diphenylhydrazine (184 m / z). The second fragmentation pattern was obtained by the Indol 3-H pathway, producing 3-butylidene-3H-indol-2-olate fragments (188 m / z) and 3H-indole-2,3-diide (114 m / z). Based on the analysis using the LCMS method, four samples containing phenylbutazone, Samples A, C, D and E. The four samples should not be allowed to be traded and consumed for violating the Regulation of the Minister of Health of the Republic of Indonesia No 006 of 2012chapter 33 and chapter 37 on the industry and traditional medicine business that traditional medicine is prohibited to contain chemicals from the isolation or synthetic drug efficacious.

Box-Behnken Design-Desirability Function Approach in Optimization of HPLC Method for Simultaneous Determination of Ibuprofen Along with Additives in Syrup Formulation.

The advantages of simultaneous analyses are a decrease in analysis time and a more economical use of solvents and reagents. It is desirable that HPLC analyses possess both short term and good resolution. The aim of the current study is to develop an HPLC method for the simultaneous determination of ibuprofen, sodium benzoate, methyl paraben and propyl paraben as preservatives, and sunset yellow as a colorant, in syrup formulation. To optimize chromatographic separation conditions, multi-response optimization using the Derringer's desirability function was employed for the development of a rapid and efficient HPLC method. The ranges of independent variables used for the optimization process were 50-60% (v/v) for acetonitrile, 5.0-7.0 for pH, and 1.0-2.0 mL/min for flow rate of the mobile phase. The effects of these variables on the output responses, such as critical resolution between sunset yellow and sodium benzoate and retention time of the last peak indicating analysis time of the method, were evaluated by statistical experimental design. Optimum conditions fixed for the simultaneous analyses were acetonitrile:phosphate buffer (60:40, v/v), pH 5.0, and a flow rate of 1.8 mL/min. The eluate was monitored using a photo diode detector set at 220 nm. Total chromatographic analysis time was approximately 3 min. The developed method validated as per International Conference on Harmonization guidelines was successfully applied for the determination of five compounds in their pharmaceutical formulation. This efficient method has isocratic elution system and can be used for routine analyses of these compounds in similar pharmaceutical products.

Study of forced-acid/heat degradation and degradant/impurity profile of phenazopyridine hydrochloride through HPLC and spectrofluorimetric analyses

The article handles two related subjects. First, it's the study of forced-acid/heat degradation of phenazopyridine HCl (PAP). The reaction pathway was evidenced with different criteria. A three wavelengths-based reversed phase HPLC method with an isocratic mobile phase system (acetonitrile:acetate buffer of pH 4, 1:1, v/v) was developed as a stability-indicating assay of particular application for simultaneous determination of PAP and its related degradation products/impurity. The analysis time was less than 8 min at a flow rate of 1 mL min-1. The detection and quantification limits of degradation products/impurity were in the ranges of 0.02-0.1 and 0.06-0.3 μg mL-1, respectively. The impurity profile of PAP in drug substance and tablets was investigated and could be determined down to a level between 0.03% and 0.005%. The second subject deals with establishment of spectrofluorimetric method for quantification of phenol (PH) and 2,6-diaminopyridine (DAP), as degradant/impurity in PAP, at trace concentrations. The method is based on measurement of the native fluorescence in acidic ethanol solution (DAP) or in aqueous acidic solution (PH). The method was applied to check the purity/stability of PAP in drug substance and in tablets dosage form.

Simultaneous identification of eight sunscreen compounds in cosmetic products using high-performance liquid chromatography and capillary electrophoresis

p-Aminobenzoates (PABA) and benzophenones in cosmetic products absorb UV radiation. We have developed two simple methods to simultaneously determine whether cosmetic products contain any of eight sunscreen compounds. The first method used high-performance liquid chromatography (HPLC) and a Cosmosil 5C18-MS column with an isocratic system consisting of acetonitrile-H2O solution (60/40, v/v) acidified by 0.1% phosphoric acid (v/v); the analysis was monitored by absorbance at 254 nm with a constant flow-rate 0.8 mL/min. This method could easily determine the presence of these compounds in non-pretreated sunscreen products in 30 min. The second method employed capillary electrophoresis (CE) in buffer solution (pH 10.0) containing 20 mM borate; the same analysis was made within 10 min. Reproducibility (relative standard deviation) of the proposed methods, on the basis of the peak-area ratios in six replicate injections, was good with only a slight deviation of 0.31-0.75% (for HPLC system) and 1.65-3.55% (for CE system). The detection limit (S/N=3) of the individual marker substances varied from 0.08 to 0.87 μg/mL (for HPLC system) and 0.23 to 1.86 μg/mL (for CE system). Although the HPLC method was superior to the CE method in both reproducibility and resolution, it required longer retention time. The HPLC and CE methods developed were both successfully applied to the assay of 8 UV-absorbing agents in 11 commercial sun protection products.

Liquid chromatographic and spectrophotometric determination of diflucortolone valerate and chlorquinaldol in creams

A new reversed phase high pressure liquid chromatography and a spectrophotometric method are proposed for the simultaneous determination of diflucortolone valerate (DIF) and chlorquinaldol (CHL) in creams. An isocratic system consisted of an ACE® C18 analytical column and a mobile phase composed of methanol/phosphate buffer (pH 5.5, 0.1 M) (95:5, v/v) at a flow rate 1.0 mL/min was used for the optimal chromatographic separation using UV detection at 220 nm. Ephedrine hydrochloride was used as internal standard. Principal component regression (PCR) was used as the chemometric technique in spectrophotometry. In this technique, the concentration data matrix was prepared by using the synthetic mixtures containing these drugs in methanol/water (3:1). The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbances at 60 wavelengths in the range of 230-348 nm with appropriate interval Δλ of 2 nm for DIF and CHL in the zero-order spectra for the binary combinations. The linear ranges were found 2.40-300 μg/mL for DIF, 0.72-240 μg/mL for CHL for LC method, and 0.88-3.0 μg/mL for DIF and 1.0-11.2 μg/mL for CHL for PCR method. The accuracy, precision and the linear ranges of the methods have been evaluated and they have been validated by analyzing synthetic mixtures containing the title drugs. These two methods were successfully applied to two pharmaceutical cream preparations and the results were compared with each other.

An improved LC-MS/MS method for determination of docetaxel and its application to population pharmacokinetic study in Chinese cancer patients.

Because of its unpredictable side effects and efficacy, the anticancer drug docetaxel (DTX) requires improved characterisation of its pharmacokinetic profiles through population pharmacokinetic studies. A sensitive and rugged LC-MS/MS method for the detection of DTX in human plasma was developed and optimised using paclitaxel as an internal standard (IS). The plasma samples underwent rapid extraction using hybrid solid-phase extraction-protein precipitation. The analyte and IS were separated with an isocratic system on a Zorbax Eclipse Plus C18 column using water containing 0.05% acetic acid along with 20 μM of sodium acetate and methanol (30/70, v/v) as the mobile phase. Quantification was performed using a triple quadrupole mass spectrometer through multiple reaction monitoring in positive mode, using the m/z 830.3 → 548.8 and m/z 876.3 → 307.7 transitions for DTX and paclitaxel, respectively. The range of the calibration curve was 1-500 ng/mL for DTX, and the linear correlation coefficient was >0.99. The accuracies ranged from -4.6 to 4.2%, and the precision was no higher than 7.0% for the analytes. No significant matrix effect was observed. Both DTX and the IS showed considerable recovery. This method was finally applied to the establishment of a population pharmacokinetic model to optimise the clinical use of DTX.

Validated Stability Indicating Chromatographic Methods for Quantification of Imidocarb Dipropionate; Application for the Determination of Its Residues in Bovine Meat and Milk Samples.

Imidocarb dipropionate (IMD) is an immunomodulator agent commonly used for treatment of anaplasmosis in cattle. Thus, two sensitive, specific, and precise stability-indicating chromatographic methods have been developed, optimized, and validated for its determination in presence of its acid, alkaline, and oxidative stressed degradation products. The first method is based on separation of IMD and its forced induced degradation products on reversed phase cyano column using isocratic elution system consisted of sodium acetate buffer-methanol-acetonitrile (55: 30:15, v/v/v), pH 4.6 at a flow rate of 1.2 mL/min, and UV detection at 254 nm. The second method utilized TLC combined with densitometric determination of the separated bands at 254 nm. The separation was achieved using silica gel 60 F254 TLC plates with a mixture of ethyl acetate-methanol-ammonia-water (8.5:1:0.5:0.2, v/v/v/v) as a developing system. HPLC analysis was applied in range of 0.25-40 µg/mL with LOD of 0.073 µg/mL. While densitometric measurements showed linearity in the range of 0.1-1.8 µg/band with LOD of 0.02 µg/band. The suggested methods were validated in compliance with the ICH guidelines and were successfully applied for determination of IMD in its commercial veterinary formulations with good recoveries. Furthermore, the proposed HPLC method was extended to the determination of IMD residues in bovine meat and milk samples. Bovine meat, HPLC, Imidocarb dipropionate, Milk, TLC.

Determination of Pyridaphenthion in Aqueous and Food Samples by Reverse Phase High Performance Liquid Chromatography (HPLC) after QuEChERS Extraction and Degradation Studies under Ultraviolet (UV) Radiation

This study employed QuEChERS as an efficient sample clean-up and extraction method for the determination of pyridaphenthion in aqueous, fruit and vegetable samples by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection. All of the instrumental parameters were carefully optimized to obtain a sharp analyte signal eluted at an short retention time. The analyte was eluted on a C18 column using an isocratic mobile phase system that was composed of 40:60 (v/v) water:acetonitrile. The linear working range of pyridaphenthion was from 10 μg/L to 50 mg/L, and the respective limits of detection and quantification were determined to be 2.4 and 8.1 μg/L. The instrumental precision was very good with a relative standard deviation value of 1.8%. Spike recovery studies were used to validate the accuracy and applicability of the method for food and aqueous samples. Degradation studies for pyridaphenthion under UV radiation were also performed with a liquid chromatography quadrupole time of flight mass spectrometer.

Simple determination and quantification of tofacitinib, a JAK inhibitor, in rat plasma, urine and tissue homogenates by HPLC and its application to a pharmacokinetic study

Tofacitinib, a janus kinase (JAK) inhibitor, was developed for the treatment of rheumatoid arthritis. To evaluate its pharmacokinetic characteristics, a simple method of quantifying tofacitinib by high-performance liquid chromatography (HPLC) was developed to estimate its concentrations in rat plasma, urine and tissue homogenates. Hydrocortisone was used as an internal standard. The mobile phase was an isocratic system of acetonitrile: 10 mM ammonium acetate, pH 5.0 (30.5:69.5, v/v), and the flow rate was 1.0 mL/min. Chromatograms were monitored by a UV detector at 287 nm. The retention times for tofacitinib and hydrocortisone were 7.21 and 11.3 min, respectively. The lower limits of quantification for tofacitinib in rat plasma and urine were 0.01 and 0.1 μg/mL, respectively. The intraday assay precisions (coefficients of variation) were generally low; 3.69–5.88% for rat plasma and 4.21–6.18% for rat urine. The corresponding values of interday assay precisions were 5.06% and 5.46%, respectively. Accuracies ranged from 92.9 to 107%, with no interference by endogenous substances. Tofacitinib has a short half-life (39.0 min) and was widely distributed in rat tissues. This HPLC method is very simple and sensitive and can be applied to future preclinical and clinical investigations of tofacitinib.

An HPLC tool for process monitoring: rare sugar D- psicose and D- fructose contents during the production through an enzymatic path

D-Psicose/allulose, a rare sugar, is an essential raw material in the pharmaceutical and food industries. It is scantly found in nature and to meet its demand in industries, D-Psicose is generated enzymatically using D-fructose as a substrate. In these conversations, it is important to monitor D-Psicose, in order to control the process, impurities, optimize the reaction time and reduce the process cost The available analytical methods have their limitations in quantifying D-psicose and D-fructose mixtures. Hence there is a need for the development of a routine, sensitive, quick and precise analytical method for D-psicose production on-line monitoring of reaction mixer. In the present work, a simplified reverse phase HPLC technique is developed and validated for the quick reaction monitoring of D-psicose from D-fructose, during enzymatic conversation procedures. The analysis is conducted at different concentrations ranging from 0.05 % to 0.5 % of the standard solutions of the D-psicose and D-fructose, by using water and Acetonitrile (at a ratio of 20:80) as eluent with a flow rate of 1.0 mL/min on isocratic HPLC-RID system with an aminopropyl silane stationary phase [ZORBAX SIL 4.6 x 150 mm, 5 µm particle size column (USP-L8)]. The applicability of this method is illustrated in reaction monitoring, where D-fructose (substrate) is converted to D-psicose (product) in the presence of the enzyme: D-Tagotose 3- epimerase. Separation of D-psicose and D-fructose is achieved within 8 minutes with a resolution ≥ 4 which is the key advantage for reaction monitoring and linearity is established with regression of ≥ 0.99. Additionally, the current method uses a simple mobile phase, without any buffers. It can be used routinely for reaction monitoring.

Reverse Phase High Performance Liquid Chromatographic-Diode Array Detection Method for Quantification of Rhein in Microsample of Rabbit Plasma

The present work describes a reversed phase high-performance liquid chromatography (RP-HPLC) assay with diode array detection (DAD) for the determination of rhein, an active metabolite of diacerein, in spiked rabbit plasma. The plasma samples were subjected to one-step liquid-liquid extraction using a protein precipitating agent. The separation of rhein was achieved on an RP C18 hypersil-ODS column (250×4.6mm, 5μm). The mobile phase consisted of a binary mixture of methanol and water (15: 85 v/v, pH 4.7) run under the isocratic system at a flow rate of 1 ml.min-1, the wavelength of DAD was set at 210 nm. The method was validated for linearity, accuracy, precision, and stability according to the International Council for Harmonization (ICH guidelines). The calibration curve was linear over the range of 0.015-5 µg. ml-1 for rhein with the coefficient of regression (r2) of value 0.9980. Extraction recovery for rhein from rabbit plasma was in the range of 95.0% to 98.60% to different sample concentrations. The retention time of rhein was observed at 5.34 min. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 5 ng.ml-1 and 15 ng.ml-1 respectively. The assay method was successfully applied to the pharmacokinetic study of diacerein plain aqueous dispersion in albino rabbits.