simple and specific stability- indicating RP-HPLC method for routine assay of adefovir dipivoxil in bulk and tablet dosage form

A simple and reliable stability-indicating RP-HPLC method was developed and validated for analysis of adefovir dipivoxil [ADV].The chromatographic separation was performed on a C[18] column using a mixture of acetonitrile-citrate buffer [10 mM at pH 5.2] 36:64 [%v/v] as mobile phase, at a flow rate of 1.5 mL/min. Detection was carried out at 260 nm and a sharp peak was obtained for ADV at a retention time of 5.8 +/- 0.01 min. No interferences were observed from its stress degradation products. The method was validated according to the international guidelines. Linear regression analysis of data for the calibration plot showed a linear relationship between peak area and concentration over the range of 0.5-16 micro g/mL; the regression coefficient was 0.9999 and the linear regression equation was y = 24844x-2941.3. The detection [LOD] and quantification [LOQ] limits were 0.12 and 0.35 micro g/mL, respectively. The results proved the method was fast [analysis time less than 7 min], precise, reproducible, and accurate for analysis of ADV over a wide range of concentration. The proposed specific method was used for routine quantification of ADV in pharmaceutical bulk and a tablet dosage form

Formulation development and toxicity assessment of triacetin mediated nanoemulsions as novel delivery systems for rapamycin

The aim of this investigation was to design and develop nanoemulsions [NEs] as novel delivery systems for rapamycin. Phase behavior of quaternary systems composed of Traicetin [as oil], various surfactants and co-surfactants and water at different surfactant/co-surfactant weight ratios was investigated by the construction of phase diagrams. Formulations were taken from the o/w NE region of the phase diagrams, depending upon the extent of NE domain. The spontaneous emulsification method was used to prepare various formulations containing 1 mg/mL of the drug. The NEs were characterized and subjected to stability tests at various temperatures over 9-12 months. Cumulative drug release from the selected formulations was determined for a period of 48 h using a dialysis sac. The assay of rapamycin was carried out using an HPLC technique. The effect of NEs on the viability of SKBR-3 cells was evaluated by MTT assay. The integrity of Caco-2 cell monolayers was measured by Transepithelial Electrical Resistance [TEER] and the transport of rapamycin-loaded NEs across Caco-2 cell monolayers was then assessed. The uptake of NEs by SKBR-3 cells was also investigated using florescence microscopy. Maximum drug release was observed in case of 4 formulations prepared with Tween 80 and Tween 20. MTT test results revealed different toxicity of NEs for SKBR-3 cell line and TEER demonstrated that formulations containing Tween 20 caused a more considerable decrease in cell integrity in comparison with those prepared with Tween 80. The results obtained from cellular uptake experiments were in consistent with those obtained from TEER and cytotoxicity experiments

Study on the effect of solution conditions on heat induced-aggregation of human alpha interferon

A major problem in the formulation of therapeutic proteins is the irreversible protein aggregation. Recombinant human interferon alpha2b [rhIFN alpha 2b] has poor stability and undergoes physical degradation. The aim of this study was to investigate the effect of solution conditions on the heat-induced aggregation of rhIFN alpha 2b. The protein was incubated for 1 h at 40-70 [degree sign]C and for up to 240 h at 50 [degree sign]C and its aggregation tendency was then studied using optical density [at 350 nm], SE-HPLC, dynamic light scattering and SDS-PAGE methods. The effect of various pH [5, 6 and 7] and buffer concentrations [10, 55 and 100 mM] on the aggregation of protein following incubation at 50 [degree sign]C for 72 h was also evaluated. The results obtained for samples incubated at 50 [degree sign]C for up to 240 h showed that OD[350] and the amount of higher molecular weight aggregates [HMW] increased and the monomer content decreased significantly [p<0.05] as the incubation time increased. Following incubation at various temperatures, a significant increase in OD[350], drop in monomer content and increase in the amount of HMW aggregates were observed [p<0.05]. Data obtained from incubation of samples at 50 [degree sign]C for 72 h confirmed that regardless of the buffer concentration, the percentage of monomer at pH 6 was significantly higher than that at pH 7 and pH 5 [p<0.05]. At constant pH, although not significant, the same trend was observed when the buffer concentration increased to 100 mM. In conclusion, the change in solution conditions can influence the aggregation extent of rhIFN alpha 2b

Occurrence of deoxynivalenol in foods for human consumption from Tehran, Iran

The occurrence of deoxynivalenol [DON] in retail foods in Tehran [Iran] was determined using high-performance liquid chromatography technique and immunoaffinity column as the clean-up step. A method was validated for analysis of DON in rice, bread, puffed corn snack and wheat flour. The average recoveries and precision [RSD] for DON in different foods ranged 84.2-93.1% and 2.9-12.0%, respectively. A survey of DON was performed on the 72 samples of rice, bread, puffed corn snack, and wheat flour collected from Tehran retail market. The data showed that 10 samples [13.9%] out of 72 samples were contaminated with DON with the maximum level of 368.7 ng/g. The samples had contamination level lower than the maximum tolerated level of DON in foods in Iran. The total intake of DON was under the provisional maximum tolerable daily intake set for DON by the JECFA

Development of a Combined Solution Formulation of Atropine Sulfate and Obidoxime Chloride for Autoinjector and Evaluation of Its Stability

Atropine [AT] and oximes, alone or in combination, have been proven greatly valuable therapeutics in the treatment of organophosphates intoxications. An injectable mixture of AT andobidoxime [OB] was formulated for the administration by automatic self-injector. The aqueous single dose solution contained 275 mg obidoxime chloride and 2.5 mg atropine sulfate per 1 mL [220 mg and 2 mg per 0.8 effective doses, respectively]. The final solution was sterilized by filtration through a 0.22 µm pore size filter. This more concentrated solution allowed using a smaller size and lighter weight cartridge. Quality control tests, including assay of the two major compounds were performed separately, using reversed-phase HPLC methods. Besides, the stability test was carried out according to ICH guideline for the accelerated test. The obtained results showed that the proposed formulation is stable over a period of 2 years after preparation

Rapid high performance liquid chromatographic method for determination of clarithromycin in human plasma Using amperometric detection: application in pharmacokinetic and bioequivalence studies

A rapid, sensitive and reproducible HPLC method using amperometric detector was developed and validated for the analysis of clarithromycin in human plasma. The separation was achieved on a monolithic silica column [MZ- C8 125×4.0 mm] using acetonitrile- methanol-potassium dihydrogen phosphate buffer [40:6:54,v/v], with pH of 7.5, as the mobile phase at a flow rate of 1.5 mL/min. The assay enables the measurement of clarithromycin for therapeutic drug monitoring with a minimum quantification limit of 20 ng/mL. The method involves simple, protein precipitation procedure and analytical recovery was complete. The calibration curve was linear over the concentration range of 0.1-6 [micro]g/mL. The coefficients of variation for inter-day and intra-day assay were found to be less than 6%. This method was used in bioequivalency and pharmacokinetic studies of the test [generic] product 2 × 500 mg clarithromycin tablets, with respect to the reference product

Improvement of capillary electrophoretic enantioseparation of fluoxetine by a cationic additive

One of the problems encountered in CE separations of basic compounds is the adsorption of analytes in negatively charged capillary wall which could lead to poor repeatability of migration time and peak area. Additionally, separation of enantiomers of chiral of basic drugs is commonly carried out in low pH buffer which contributes to strong ionic interaction of the cationic drug ions with negatively charged chiral selectors. The two phenomena results in poor enantioseparations. To overcome the problems associated with chiral separations of basic drugs by CE, the effect of guanidine [GU] on the improvement of chiral separation of a model basic drug, fluoxetine [FLX], was investigated. In the present study, GU was used as a cationic additive to the running buffer containing a chiral selector, sulfated beta cyclodextrine. Better results obtained with GU as the buffer additive in enantioseparation of FLX

reversed phase high performance liquid chromatographic method for determination of rapamycin

Easily degradating and various isomeric forms of rapamycin [Sirolimus] face the determination of this compound to many challenges. In this study, we developed and validated the isocratic reversed phase high performance liquid chromatographic [RP-HPLC] method for rapamycin. Separation was performed on a C[8] column [MZ, 15 × 4.6 mm, 5 [micro]m particle size] using methanol:water [80:20 v/v] as the mobile phase with the flow rate of 1 mL/min. The column temperature was set at 57degreeC and the detection was carried out at the wavelength of 277 nm. The method was linear over a concentration range of 0.025-2 [micro]g/mL. The coefficient of variation of intra- and inter-day, assessed at three concentration levels of 0.075, 0.3 and 0.900 [micro]g/mL, was less than 2%. Limit of quantification [LOQ] was found 25 ng/mL. The method with high percent recovery and short retention time of rapamycin, was found to be simple, rapid and reproducible

Improvement of electrophoretic enantioseparation ofamlodipine by polybrene

In chiral and non-chiral electrophoretic resolution of basic drugs, adsorption of analytes to negatively charged capillary wall could lead to poor repeatability of migration time and peak area. In addition, chiral resolutions of basic drugs are commonly performed in low pH buffers. Therefore, longer analysis time due to suppression of electroosmotic flow [EOF] is another dilemma. In this work the improvement effect of polybrene [PB], a cationic polymer, on chiral separation of a model basic drug, amlodipine [AML], was investigated. PB both as a semi-permanent coating agent and as an additive in the running buffer was utilized. Better results were obtained with PB as a buffer additive. Compare to untreated bare silica without using PB in running buffer, addition of 0.0005% PB to buffer decreased analysis time downed to 3 folds; efficiency improved up to 5 folds; limit of detection [LOD] and limit of quantification [LOQ] downed to 8 folds and within-day migration time and peak area repeatabilities, in terms of relative standard deviations [RSD] downed to 5 and 20 folds, respectively

Exposure assessment of the Tehran population [Iran] to zearalenone mycotoxin

Zearalenone [ZEA] mycotoxin is a potent estrogenic metabolite. It is the primary toxin causing infertility, abortion or other breeding problems. A HPLC method was validated for ZEA in foods using a monolithic column with sample clean-up on an immunoaffinity column. A certified reference material [CRM] from FAPAS [UK] was analyzed. A survey of ZEA was performed on the 72 samples of rice, bread, puffed corn snack and wheat flour collected from Tehran retail market. The average recovery and coefficient of variation in different foods ranged 92.7-107.1 and 4.9-13.8%, respectively. The amount of ZEA in corn CRM was in the acceptable range of FAPAS. The limit of quantification was 3 ng/g for rice, bread and wheat flour and 2.7 ng/g for puffed corn snack. The retention time of zearalenone was 2.6 min. All samples had contamination level lower than the maximum tolerated level of ZEA in foods in Iran. The mean intake of ZEA from all samples was much lower than the tolerable daily intake estimated by JECFA. This is the first survey on ZEA contamination in bread and rice in Iran as well as the first study on exposure assessment of Tehran population to ZEA

Separation of tramadol enantiomers by capillary electrophoresis using highly sulfated cyclodextrins

In the pharmaceutical industry a continuing need for chiral resolution of drugs for various purposes and in diverse matrices exist. For these reasons, analysts may require a number of different separation systems capable of resolving a given pair of enantiomers. Highly sulfated cyclodextrins [HS-CDs] represent a relatively new class of chiral selectors in capillary electrophoresis [CE]. In this investigation the use of HS-CDs as chiral selectors in CE for enantioseparation of tramadol, a highly potent analgesic, as the model drug and the influence of the type of selector and its concentration on enantiomeric resolution were studied. All of the available HSCDs [alpha,beta and gamma] could resolve tramadol enantiomers, but HS-gamma-CD showed better resolution and a baseline resolution was achieved with this selector even at a concentration as low as 0.5% w/v. Additionally, effect of the buffer pH on the enantioresolution was studied. At low pH buffers, in which electroosmotic flow is low in CE, the negatively charged selector prevented the cationic tramadol to migrate out of the capillary even after a long analysis time of 60 minutes. However, at higher pH values [pH=7 or more], the electroosmotic flow is high enough to drag drug-selector complex toward the detector and a reasonable of the enantiomers of the drug was achieved

Effect of capillary rinsing proctol on the reproducibility of separation in capillary electrophoresis with indirect UV detection

Capillary electrophoresis [CE] with indirect UV detection is an interesting analytical method for the analysis of drugs and pharmaceuticals. Good and reproducible capillary quality is needed to develop robust methods and to facilitate method transfer in CE. It is widely accepted that preconditioning procedures are indispensable in capillary electrophoresis in order to achieve reproducibility of migration times and peak areas. In order to explore different aspects of this technique, a set of experiments were performed using vigabatrin as a model drug. The effects of capillary rinsing between each run was investigated using basic [NaOH 0.1 M] and acidic [phosphoric acid 0.1 M]-wash cycles. The results of 10 consecutive injection of the model drug after each of the two wash cycles, reveal that more reproducible results obtained when acid-wash cycle was performed as a capillary conditioning protocol. The higher pH changes during basic-wash cycle and its effects on the characteristics of the capillary inner surface were suggested as a source of greater variation between consecutive runs

Separation of somatropin and its degradation products by high-performance liquid chromatography using a reversed-phase polymeric column

The accurate prediction of protein stability is one of the most challenging goals in protein formulation and delivery. In this study, a gradient RP-HPLC method is described for the separation of human growth hormone [hGH] variants as deamidated and oxidized forms. The methodology employed a polymeric poly [styrene-co-divinylbenzene] column and a 1mL/min flow rate of a linear gradient of 0.1% v/v TFA/acetonitrile and TFA/Water [pH=2.0] mixture as the mobile phase. The overall run time of this method was 12 min and the average retention times were about 8.7 min for the native somatropin, 7.2 min for the deamidated form and 1.6 and 5.3 min for oxidized variants. The method was also validated in terms of selectivity, linearity, intra- and inter-day variations. In conclusion the method was found to have the potential for being applied as an initial and rapid evaluation method for assessing the quality and quantity of hGH during downstream processing, formulation and storage