20 resultados para Hplc Method

em QUB Research Portal - Research Directory and Institutional Repository for Queen's University Belfast


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Abstract An HPLC method has been developed and validated for the determination of spironolactone, 7a-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 µl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30–1000 ng/ml for spironolactone and 25–1000 ng/ml for 7a-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7a-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7a-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.

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An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45 min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95 pmol/8 x 101 RBCs and 2, 5, 2, 3, 20 ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200 mu l of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients. (C) 2008 Elsevier B.V. All rights reserved.

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Bovine serum albumin (BSA) is a commonly used model protein in the development of pharmaceutical formulations. In order to assay its release from various dosage forms, either the bicinchoninic acid (BCA) assay or a more specific size-exclusion high performance liquid chromatography (SE-HPLC) method are commonly employed. However, these can give erroneous results in the presence of some commonly-used pharmaceutical excipients. We therefore investigated the ability of these methods to accurately determine BSA concentrations in pharmaceutical formulations that also contained various polymers and compared them with a new and compared with a new reverse-phase (RP)–HPLC technique. We found that the RP-HPLC technique was the most suitable method. It gave a linear response in the range of 0.5 -100 µg/ml with a correlation coefficient of 0.9999, a limit of detection of 0.11 µg/ml and quantification of 0.33 µg/ml. The performed ‘t’ test for the estimated and theoretical concentration indicated no significant difference between them providing the accuracy. Low % relative standard deviation values (0.8-1.39%) indicate the precision of the method. Furthermore, the method was used to quantify in vitro BSA release from polymeric freeze-dried formulations.

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A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbamyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 microm ODS (C18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min-1 and the column temperature was maintained at 30 degrees C. Galactosamine hydrochloride (Gal-HCl) was used as an internal standard. Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32+/-1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15+/-0.1 cm2. The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1% v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 microg ml-1. The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) <12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <or=-5.60 and <or=-8.00, respectively. Using this assay, it was found that GL-HCl permeates through human skin with a flux 1.497+/-0.42 microg cm-2 h-1, a permeability coefficient of 5.66+/-1.6x10(-6) cm h-1 and with a lag time of 10.9+/-4.6 h.

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Clinical use of the imidazoquinoline immunomodulator imiquimod for the topical treatment of dysplastic and neoplastic lesions has increased markedly in recent years. However, despite guidance from the manufacturer of the proprietary imiquimod cream, there seems to be little consensus between clinicians as to the topically applied dose. Given that patients often apply the cream themselves at home, further dosing variability is expected and, consequently, accurate comparison of the results of different published studies is dif?cult. This paper describes, for the ?rst time, the formulation and physicochemical characterisation of a bioadhesive patch for dose-controlled topical delivery of imiquimod as well as a new HPLC method for sensitive ?uorescence determination of imiquimod released from such systems. Patches containing imiquimod loadings of 4.75, 9.50 and 12.50 mg cm-2 all released signi?cantly more drug across a model membrane than the proprietary cream over a period of 6 h. Inclusion of imiquimod in patches did not adversely affect their physicochemical properties. Of major importance, patches contained de?ned drug loadings per unit area; therefore, their use could reduce inter-clinician variability. This would make critical comparison of clinical studies and determination of an appropriate imiquimod dose for successful treatment much simpler. Since bioadhesive formulations are capable of adhering to body tissues in moist environments, the use of a bioadhesive patch system may allow extension of the clinical uses of imiquimod to the treatment of neoplastic conditions of the oral cavity and cervix, as well as the vulva. © 2005 Elsevier B.V. All rights reserved.

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Micellar electrokinetic. chromatography (MEKC) was used to separate components of hop extracts. The separation of a sample of iso-alpha -acids by MEKC was better and faster than by an established HPLC method, giving <0.8 % RSD on migration times and 5-10% RSD on peak areas. MEKC was also successfully used to separate the oxidation products of the - and beta -acids and thus to monitor the stability of hop products containing them. Furthermore, MEKC distinguished among samples of reduced iso-alpha -acids (rho-, tetrahydro- and hexahydro- derivatives). (C) 2001 Elsevier Science Ltd. All rights reserved.

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Previous studies have shown that in vitro adenosine enhances histamine release from activated human lung mast cells obtained by enzymic dispersion of lung parenchyma. However, adenosine alone has no effect on histamine release from these cells. Given the evidence for direct activation of mast cells after endobronchial challenge with adenosine and previous studies indicating that mast cells obtained at bronchoalveolar lavage are a better model for asthma studies than those obtained by enzymic dispersion of lung tissue, the histamine-releasing effect of adenosine was examined on lavage mast cells. Bronchoalveolar lavage fluid was obtained from patients attending hospital for routine bronchoscopy (n = 54). Lavage cells were challenged with adenosine or adenosine receptor agonists (20 min, 37 degrees C) and histamine release determined using an automated fluorometric assay. Endogenous adenosine levels were also measured in lavage fluid (n = 9) via an HPLC method. Adenosine alone caused histamine release from ravage mast cells in 37 of 54 patients with a maximal histamine release of 20.56 +/- 2.52% (range 5.2-61 %). The adenosine receptor agonists (R)-N-6-(2-phenylisopropyl)adenosine, 5'-N-ethylcarboxamido-adenosine and CGS21680 also induced histamine release from lavage mast cells. Preincubation of lavage mast cells with the adenosine receptor antagonist xanthine amine congener caused significant inhibition of the response to adenosine (P = 0.007). There was an inverse correlation between endogenous adenosine levels in the lavage fluid and the maximal response to in vitro adenosine challenge of the lavage cells. The findings of the present study indicate a means by which adenosine challenge of the airways can induce bronchoconstriction and support a role for adenosine in the pathophysiology of asthma. The results also suggest that cells obtained from bronchoalveolar ravage fluid may provide the ideal model for the testing of novel, adenosine receptor, targeted therapies for asthma.

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In the present study the extraction of paralytic shellfish poisoning (PSP) toxins from a toxic strain of the marine dinoflagellate Alexandrium tamarense CCMP-1493 using various mechanical and/or physical procedures was investigated. PBS buffer was investigated as the extraction solvent in order for these procedures to be used directly with immuno-magnetic Ferrospheres-N. The extraction was performed following the determination of when toxin content by the algae was at its highest during batch culture. The methods used for cell lysis and toxin extraction included freeze-thawing, freeze-boiling, steel ball bearing beating, glass bead beating, and sonication. The steel ball bearing beating was determined to release a similar amount of toxin when compared to a modified standard extraction method which was reported to release 100% of toxins from the algal cells and was therefore used in the next phase of the study. This next phase was to determine the feasibility of utilising an antibody coupled to novel magnetic microspheres (Ferrospheres-N) as a simple, rapid immune-capture procedure for PSP toxins extracted from the algae. The effects of increasing mass of Ferrospheres-N on the immuno-capture of the PSP toxins from the toxic algal strain extracts were investigated. Toxin recovery was found to increase when an increasing mass of Ferrospheres-N was used until 96.2% (+/- 1.3 SD) of the toxin extracted from the cells was captured and eluted. Toxin recovery was determined by comparison to an appropriate PSP toxin standard curve following analysis by the AOAC HPLC method. (C) 2011 Elsevier B.V. All rights reserved.

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A rapid, sensitive reversed-phase high-performance liquid chromatographic method has been developed for the determination of in vitro release of 17 beta-estradiol and its ester prodrug, 17 beta-estradiol-3-acetate, from silicone intravaginal rings. Partial hydrolysis of the acetate under the aqueous conditions provided by the 1% benzalkonium chloride release medium necessitates its conversion to 17 beta-estradiol prior to HPLC analysis. Both steroid peaks have been fully resolved from the benzalkonium chloride peaks by the reported chromatographic method,which employs a C-18 bonded reversed-phase column, an acetonitrile-water (50:50, v/v) mobile phase and a UV detection wavelength of 281 nm. The peak area versus 17 beta-estradiol concentration was found to be linear over the range of 0.0137-1347 mu g ml(-1) The HPLC method has also been used to determine the silicone solubilities and diffusion coefficients of the two related steroids. The almost 100-fold increase in 17 beta-estradiol-3-acetate release from the silicone core-type intravaginal rings compared to 17 beta-estradiol is shown to be due to a 60-fold increase in silicone solubility and a one and a half-fold increase in diffusitivity. The results demonstrate that an effective estrogen replacement therapy dose of 17 beta-estradiol may be administered from a silicone intravaginal reservoir device containing the labile 17 beta-estradiol-3-acetate prodrug. (C) 2000 Elsevier Science B.V. All rights reserved.

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Tetrodotoxin (TTX) is a potent neurotoxin emerging in European waters due to increasing ocean temperatures. Its detection in seafood is currently performed as a consequence of using the Association of Analytical Communities (AOAC) mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins, but TTX is not monitored routinely in Europe. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. An AOAC-accredited high-performance liquid chromatography (HPLC) method has now been accepted by the European Union as a first action screening method for PSP toxins to replace the MBA. However, this AOAC HPLC method is not capable of detecting TTX, so this potent toxin would be undetected; thereby, a separate method of analysis is required. Surface plasmon resonance (SPR) optical biosensor technology has been proven as a potential alternative screening method to detect PSP toxins in seafood. The addition of a similar SPR inhibition assay for TTX would complement the PSP assay in removing the MBA. The present report describes the development and single laboratory validation in accordance with AOAC and IUPAC guidelines of an SPR method to be used as a rapid screening tool to detect TTX in the sea snail Charonia lampas lampas, a species which has been implicated in 2008 in the first case of human TTX poisoning in Europe. As no current regulatory limits are set for TTX in Europe, single laboratory validation was undertaken using those for PSP toxins at 800 µg/kg. The decision limit (CCa) was 100 µg/kg, with the detection capability (CCß) found to be =200 µg/kg. Repeatability and reproducibility were assessed at 200, 400, and 800 µg/kg and showed relative standard deviations of 8.3, 3.8, and 5.4 % and 7.8, 8.3, and 3.7 % for both parameters at each level, respectively. At these three respective levels, the recovery of the assay was 112, 98, and 99 %.

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Abstract - This study investigates the effect of solid dispersions prepared from of polyethylene glycol (PEG) 3350 and 6000 Da alone or combined with the non-ionic surfactant Tween 80 on the solubility and dissolution rate of a poorly soluble drug eprosartan mesylate (ESM) in attempt to improve its bioavailability following its oral administration.

INTRODUCTION

ESM is a potent anti-hypertension [1]. It has low water solubility and is classified as a Class II drug as per the Biopharmaceutical Classification Systems (BCS) leading to low and variable oral bioavailability (approximately 13%). [2]. Thus, improving ESM solubility and/or dissolution rate would eventually improve the drug bioavailability. Solid dispersion is widely used technique to improve the water solubility of poorly water-soluble drugs employing various biocompatible polymers. In this study, we aimed to enhance the solubility and dissolution of EMS employing solid dispersion (SD) formulated from two grades of poly ethylene glycol (PEG) polymers (i.e. PEG 3350 & PEG 6000 Da) either individually or in combination with Tween 80.

MATERIALS AND METHODS

ESM SDs were prepared by solvent evaporation method using either PEG 3350 or PEG 6000 at various (drug: polymer, w/w) ratios 1:1, 1:2, 1:3, 1:4, 1:5 alone or combined with Tween 80 added at fixed percentage of 0.1 of drug by weight?. Physical mixtures (PMs) of drug and carriers were also prepared at same ratios. Drug solid dispersions and physical mixtures were characterized in terms of drug content, drug dissolution using dissolution apparatus USP II and assayed using HPLC method. Drug dissolution enhancement ratio (ER %) from SD in comparison to the plain drug was calculated. Drug-polymer interactions were evaluated using Differential Scanning Calorimetry (DSC) and FT-IR.

RESULTS AND DISCUSSION

The in vitro solubility and dissolution studies showed SDs prepared using both polymers produced a remarkable improvement (p<0.05) in comparison to the plain drug which reached around 32% (Fig. 1). The dissolution enhancement ratio was polymer type and concentration-dependent. Adding Tween 80 to the SD did not show further dissolution enhancement but reduced the required amount of the polymer to get the same dissolution enhancement. The DSC and FT-IR studies indicated that using SD resulted in transformation of drug from crystalline to amorphous form.

CONCLUSIONS

This study indicated that SDs prepared by using both polymers i.e. PEG 3350 and PEG 6000 improved the in-vitro solubility and dissolution of ESM remarkably which may result in improving the drug bioavailability in vivo.

Acknowledgments

This work is a part of MSc thesis of O.M. Ali at the Faculty of Pharmacy, Aleppo University, Syria.

REFERENCES

[1] Ruilope L, Jager B: Eprosartan for the treatment of hypertension. Expert Opin Pharmacother 2003; 4(1):107-14

[2] Tenero D, Martin D, Wilson B, Jushchyshyn J, Boike S, Lundberg, D, et al. Pharmacokinetics of intravenously and orally administered Eprosartan in healthy males: absolute bioavailability and effect of food. Biopharm Drug Dispos 1998; 19(6): 351- 6.


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Mercury in plants or animal tissue is supposed to occur in the form of complexes formed with biologically relevant thiols (biothiols), rather than as free cation. We describe a technique for the separation and molecular identification of mercury and methylmercury complexes derived from their reactions with cysteine (Cys) and glutathione (GS): Hg(Cys)(2), Hg(GS)(2), MeHgCys, MeHgGS. Complexes were characterised by electrospray mass spectrometry (MS) equipped with an ion trap and the fragmentation pattern of MeHgCys was explained by using MP2 and B3LYP calculations, showing the importance of mercury-amine interactions in the gas phase. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a reversed-phase column. Detection was done by online simultaneous coupling of ES-MS and inductively coupled plasma MS. When the mercury complexes were spiked in real samples (plant extracts), no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting mercury biothiol complexes in plants.

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This article describes the development of SPE and HPLC methods for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma. Several extraction and HPLC methods have been described previously for the determination of each of these analytes in plasma separately. The simultaneous determination of these analytes is important for the routine monitoring of diabetic patients who take combination medications and for studying the pharmacokinetics of the combined dosage forms. In addition this developed method can serve as a standard method for the plasma determination of these analytes therefore saving time, effort and money. The recoveries of the developed methods were found to be between 76.3% and 101.9%. The limits of quantification were between 5 and 22.5 ng/ml. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error %) was always less than 12%. Stability analysis showed that all analytes are stable for at least 3 months when stored at -70degreesC. (C) 2004 Elsevier B.V. All rights reserved.

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A research element of the European Union (EU) sixth Framework project BioCop focused on the development of a surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins in shellfish as an alternative to the increasingly ethically unacceptable mouse bioassay. A biosensor assay was developed using both a saxitoxin binding protein and chip surface in tandem with a highly efficient simple extraction procedure. The present report describes the single laboratory validation of this immunological screening method, for this complex group of toxins with differing toxicities, according to the European Decision 2002/657/EC in conjunction with IUPAC and AOAC single laboratory validation guidelines. The different performance characteristics (detection capability CC beta, specificity/selectivity, repeatability, reproducibility, stability, and applicability) were determined in relation to the EU regulatory limit of 800 mu g of saxitoxin equivalents (STX eq) per kg of shellfish meat. The detection capability CC beta was calculated to be 120 mu g/kg. Intra-assay repeatability was found to be between 2.5 and 12.3% and interassay reproducibility was between 6.1 and 15.2% for different shellfish matrices. Natural samples were also evaluated and the resultant data displayed overall agreements of 96 and 92% with that of the existing AOAC approved methods of mouse bioassay (MBA) and high performance liquid chromatography (HPLC), respectively.

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A novel stir bar sorptive extraction (SBSE) method coupled with high performance liquid chromatography (HPLC) and UV detection for the extraction of diclofenac (DIC) from paediatric urine samples has been developed and validated. Selectivity and sensitivity being the prime objectives of the bioanalytical method for clinical samples, an optimised SBSE protocol was developed that selectively extracted DIC from various concurrently administered drugs. The validated assay was found to be linear (r=0.9999) over a concentration range of 100-2000 ng mL(-1). SBSE showed consistent recoveries (similar to 70%) of DIC across the validated linearity range. Overall, the method exhibited excellent accuracy and precision across all QC concentrations, tested over three days. Calculated LOD and LOQ were found to be 12.03 ng mL(-1) and 36.37 ng mL(-1), respectively, however, for the experimental purposes, 100 ngmL(-1) was considered as the validated LOQ(accuracy and precision at this LQC was