Final irrigation protocols can be used to promote stable long-term bond strength of AH Plus to dentin.

Irrigation solutions might affect dentin surface characteristics and, consequently, endodontic sealers adhesion. This study analyzed the effect of different final irrigation protocols on push-out bond strength (BS) of AH Plus to dentin seven days and 20 months after obturation. Scanning electron micrographs were obtained from the dentin surface of one sample/group after final irrigation. Canals of bovine incisors were instrumented and received final irrigation with (n=21): G1 - 2.5% sodium hypochlorite (NaOCl) + distilled water; G2 - 2.5% NaOCl + 17% EDTA; G3 - 2.5% NaOCl + 17% EDTA + 2.5% NaOCl; G4 - 2.5% NaOCl + 17% EDTA + 2% chlorhexidine (CHX); G5 - mixture 5% NaOCl + 18% etidronate (HEDP); and G6 - mixture 5% NaOCl + 10% tetrasodium EDTA (Na4EDTA). After irrigation, one root/group was split and images were obtained by scanning electron microscopy (SEM). The other 20 roots/group were filled with only AH Plus sealer. Three slices/root were used for push-out assessment seven days and 20 months after obturation. One-way analysis of variance and Tukey (α<0.05) were used to compare the results among experimental groups, and unpaired t-test (α<0.05) was used to compare the results of the same group over time. The photomicrographs showed that, excepting G1, all groups completely removed the smear layer from the samples. In G2 and G4, the opening of the dentin tubules enlarged. In G3, erosion was observed in the peritubular and intertubular dentin. Values of the BS in the seven days were G2=G3=G4=G5>G6=G1 and in the 20 months were G3=G5>G6=G4>G1=G2. G3, G5, and G6 presented values of BS in 20 months similar to the values of seven days (P>0.05). The final irrigation protocols tested produced dentin surfaces with different characteristics. Only G3 and G5 presented high BS values that were stable over time.

An IC-MS/MS Method for the Determination of 1-Hydroxyethylidene-1,1-diphosphonic Acid on Uncooked Foods Treated with Peracetic Acid-Based Sanitizers.

A rapid, sensitive, and specific analytical method for the determination of 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) on uncooked foods after treatment with a peracetic acid-based sanitizer (PAS) was developed. The method involves simple sample preparation steps and analysis using ion chromatography (IC) coupled with tandem mass spectrometry (MS/MS). The quantification limits of HEDP on uncooked foods are 0.007 mg/kg for vegetables and fruits and 0.2 mg/kg for meats. The recovery and relative standard deviation (RSD) of HEDP analyses of uncooked foods ranged from 73.9 to 103.8% and 1.9 to 12.6%, respectively. The method's accuracy and precision were evaluated by inter-day recovery tests. The recovery for all samples ranged from 93.6 to 101.2%, and the within-laboratory repeatability and reproducibility were evaluated based on RSD values, which were less than 6.9 and 11.5%, respectively. Analyses of PAS-treated fruits and vegetables using the developed method indicated levels of HEDP ranging from 0.008 to 0.351 mg/kg. Therefore, the results of the present study suggest that the proposed method is an accurate, precise, and reliable way to determine residual HEDP levels on PAS-treated uncooked foods.

A novel automated hydrophilic interaction liquid chromatography method using diode-array detector/electrospray ionization tandem mass spectrometry for analysis of sodium risedronate and related degradation products in pharmaceuticals.

A simple, sensitive and fast hydrophilic interaction liquid chromatography (HILIC) method using ultraviolet diode-array detector (UV-DAD)/electrospray ionization tandem mass spectrometry was developed for the automated high performance liquid chromatography (HPLC) determination of sodium risedronate (SR) and its degradation products in new pharmaceuticals. The chromatographic separations were performed on Ascentis Express HILIC 2.7µm (150mm×2.1mm, i.d.) stainless steel column (fused core). The mobile phase consisted of formate buffer solution (pH 3.4; 0.03M)/acetonitrile 42:58 and 45:55 (v/v) for granules for oral solution and effervescent tablet analysis, respectively, at a flow-rate of 0.2mL/min, setting the wavelength at 262nm. Stability characteristics of SR were evaluated by performing stress test studies. The main degradation product formed under oxidation conditions corresponding to sodium hydrogen (1-hydroxy-2-(1-oxidopyridin-3-yl)-1-phosphonoethyl)phosphonate was characterized by high performance liquid chromatography-electrospray ionization-mass tandem mass spectrometry (HPLC-ESI-MS/MS). The validation parameters such as linearity, sensitivity, accuracy, precision and selectivity were found to be highly satisfactory. Linear responses were observed in standard and in fortified placebo solutions. Intra-day precision (relative standard deviation, RSD) was ≤1.1% for peak area and ≤0.2% for retention times (tR) without significant differences between intra- and inter-day data. Recovery studies showed good results for all the examined compounds (from 98.7 to 101.0%) with RSD ranging from 0.6 to 0.7%. The limits of detection (LOD) and quantitation (LOQ) were 1 and 3ng/mL, respectively. The high stability of standard and sample solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of many samples and consecutive chromatographic analyses by using an autosampler. The developed stability indicating method is suitable for the quality control of SR in new and commercial pharmaceutical formulations.

Development of an ion-pair reversed-phase HPLC method with indirect UV detection for determination of phosphates and phosphites as impurities in sodium risedronate.

A method based on RP-HPLC with indirect UV detection was developed for the determination of phosphates and phosphites as impurities in sodium risedronate. RP separation of the phosphates and phosphites was achieved by adding tetrabutylammonium hydroxide as an ion-pairing agent in the mobile phase. Potassium hydrogen phthalate was added to the mobile phase as an ionic chromophore in order to obtain high background absorption of the mobile phase. Separation was performed on a C18 column using a mixture of pH 8.2 buffer (containing 0.5 mM tetrabutylammonium hydroxide and 1 mM phthalate) and acetonitrile (95 + 5, v/v) as the mobile phase, with indirect UV detection at 248 nm. The validation of the method included determination of specificity/selectivity, linearity, LOD, LOQ, accuracy, precision, and robustness. The LOD was 0.86 microg/mL for phosphates and 0.76 microg/mL for phosphites. The LOQ was 2.60 microg/mL for phosphates and 2.29 microg/mL for phosphites. The developed method is suitable for quantitative determination of phosphates and phosphites as impurities in QC of sodium risedronate.

Development and validation of a micellar high-performance liquid chromatographic method for determination of risedronate in raw material and in a pharmaceutical formulation: application to stability studies.

A micellar HPLC method was developed for analysis of the antiosteoporosis drug risedronate. The analysis was carried out using a 250 x 4.6 mm id, 5 microm particle size C18 Waters Symmetry column. The mobile phase consisted of 0.02 M sodium dodecyl sulfate + 0.3% triethylamine + 10% n-propanol, prepared in 0.02 M orthophosphoric acid. The pH of the mobile phase was adjusted to pH 6.0, and it was pumped at a flow rate of 0.7 mL/min with UV detection at 262 nm. The method showed good linearity in the range of 2-80 microg/mL, with an LOD of 0.40 microg/mL (1.31 x 10(-6) M) and an LOQ of 1.21 microg/mL. The suggested method was successfully applied for the analysis of risedronate in raw material and a tablet formulation, with average recoveries of 99.91 +/- 1.30 and 101.52 +/- 0.30%, respectively. The stability-indicating capability of the proposed method was proved using forced degradation. By changing the pH of the mobile phase to 4.0, the oxidative degradation product could be separated from risedronate.

Analysis of risedronate and related substances by ion-pair reversed-phase high-performance liquid chromatography with evaporative light-scattering detection.

A simple method has been developed for the analysis of risedronate and related substances by ion-pair reversed-phase high-performance liquid chromatography (RPLC) with evaporative light-scattering detection (ELSD). After optimization of the chromatographic conditions, satisfactory separation of the compounds was achieved on an Intersil C(8) column with an isocratic mobile phase: 8:4:88 (v/v) acetonitrile-methanol-12 mM ammonium acetate buffer containing 35 mM n-amylamine (pH 7.0). The mobile-phase flow rate was 1.0 mL min(-1). The calibration plot was linear in the range of 352 to 1760 microg mL(-1) for risedronate. The precision and reproducibility were 0.3 and 0.5%, respectively. The average recovery of risedronate was 100.4% and the RSD was 0.6%. The method was validated and shown to be precise, accurate, and specific for the assay of risedronate in both bulk material and dosage forms. The proposed liquid-chromatographic method can be satisfactorily used for the quality control of risedronate.

Compatibility of risedronate sodium tablets with food thickeners.

PURPOSE: The chemical compatibility of commercially available food thickeners used with risedronate sodium tablets is examined. METHODS: Plastic 250-mL disposable beakers were used to prepare solutions for compatibility testing. Two ounces of purified water at room temperature was added to each beaker. One risedronate sodium 35-mg tablet was also added to each beaker and allowed to disintegrate without agitation. After two minutes, the water was stirred with a plastic spoon and an additional 4 oz of water was added to each beaker and stirred briskly for 30 seconds. The recommended amount of each food thickener was then added to each beaker. The control solution contained a tablet in water but no food thickener. Immediately after preparation, a portion of each solution was filtered through a 0.2-microm filter and assayed by high-performance liquid chromatography (HPLC). The solutions sat for 1 hour at room temperature and then were stirred briskly for 30 seconds, filtered, and assayed by HPLC. They then sat for 24 hours at room temperature and then were stirred briskly again for 30 seconds, filtered, and assayed by HPLC. RESULTS: The risedronate tablets alone showed a mean initial concentration of 0.194 mg/mL and a mean recovery of 99.8% after 1 hour and 99.2% after 24 hours. The mean initial concentration for risedronate sodium in each of the five food thickeners ranged from 0.193 to 0.195 mg/mL. All samples ranged from 98.8% to 99.9% of the initial concentration after 1 hour and from 96.7% to 99.2% after 24 hours. CONCLUSION: Risedronate sodium tablets were compatible for 24 hours with five food thickeners.

Application of UV-derivative spectrophotometry for determination of some bisphosphonates drugs in pharmaceutical formulations.

A method for determination of alendronate sodium salt, clodronate disodium salt and etidronate disodium salt in pharmaceutical formulation by direct UV-spectrophotometry and/or first and second derivative UV-spectrophotometry via complex formation with Cu (II) ions is described. The calibration graphs are linear in the range 25-600 mmol/L for all the investigated compounds. No interference was found from tablet excipients at the selected wavelength and assay procedure. The developed method was validated and found to be sufficiently precise and reproducible, at least for established conditions.

Direct stability-indicating method development and validation for analysis of etidronate disodium using a mixed-mode column and charged aerosol detector.

This paper describes the development and validation of a rapid, direct, and stability-indicating method for analysis of etidronate, a bisphosphonate compound without a UV chromophore. A mixed-mode column was used to separate etidronate from its impurities in an 8-min gradient method and a charged aerosol detector (CAD) was used for detection. The developed HPLC method was validated with respect to specificity, linearity, accuracy, precision, sensitivity, and stability. The method can be used for release and stability testing of etidronate and has applicability to other similar bisphosphonate compounds.

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations.

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C(18) analytical column (250 mm x 4.6 mm i.d., 5 microm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min(-1). The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 microg mL(-1), while detection and quantitation limits were found to be 0.48 and 1.61 microg mL(-1), respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, %E(r), was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.

Evaluating SPIO-labelled cell MR efficiency by three-dimensional quantitative T2* MRI.

An in vitro MR-assay for superparamagnetic iron oxide (SPIO) particle cell labelling assessment via three-dimensional quantitative T(2) (*) MR microscopy was proposed. On high-resolution images, and due to the high susceptibility difference between the particles and the surrounding medium, SPIO internalized in cells induces signal loss which may be counted and measured on T(2) (*) maps. The increase in both labelled cell percentage and the average perturbation volume with an added amount of iron in the incubation medium proved that intracellular iron uptake is dependent upon the initial concentration of incubation iron. It also proved that the observed increases in total cellular iron uptake measured by inductively coupled plasma optical emission spectroscopy are due to both an increase in the iron mass per cell and also an increase in labelled cell concentration. MR results were compared with Prussian blue staining histology. The sensitivity of the MR methodology was then used to distinguish labelling differences for two different types of particle coating. The MRI-assay we proposed is a compulsory tool to optimize labelling efficiency in order to improve in vivo cell detection. Key parameters for detection, such as the percentage of cell labelling, the effect on the image for a given amount of internalized iron and labelling distribution among a cell population, are easily obtained. The comparison of different contrast agents for labelling one cell type, the assessment of one type of contrast agent for labelling different cell types and/or the evaluation of labelling strategies, are possible without having recourse to classical methods, and provide improved accuracy, since the principle is based on intracellular relaxivity.

A general approach for the quantitative analysis of bisphosphonates in human serum and urine by high-performance liquid chromatography/tandem mass spectrometry.

Bisphosphonates are extremely hydrophilic and structurally similar to many endogenous phosphorylated compounds, making their selective extraction from serum or urine very challenging. Many bisphosphonates lack strong chromophores for sensitive UV or fluorescence detection. We report here the first general approach to enable sensitive and selective quantitation of N-containing bisphosphonates by liquid chromatography/tandem mass spectrometry (LC/MS/MS) following derivatization with diazomethane. The novelty of the strategy lies in performing the derivatization on silica-based anion-exchange sorbents as an integrated step in the sample purification by solid-phase extraction (SPE). The 'on-cartridge' reaction with diazomethane not only led to higher efficiency of derivatization, but also enabled a more discriminatory recovery of the drug's derivatives. The derivatized bisphosphonates demonstrated improved chromatographic separation and increased sensitivity of the detection. The general applicability of the approach was demonstrated by validation of bioanalytical methods for risedronate and alendronate in human serum and urine. Sensitivity was achieved at the pg/mL level with merely 100-200 microL of sample.

Adsorption of Me(II), HEDP, and Me(II)-HEDP onto boehmite at nonstoichiometric Me(II)-HEDP concentrations.

In aqueous environments, certain heavy metals are toxic even at very low concentrations. The main pathway of metal removal in the aquatic systems is via adsorption onto surfaces. These are desired processes that help decrease the dissolved fraction of metals in natural water. The presence of organic ligands as mono- and polyphosphonates may produce drastic changes in the mobility of the heavy metals. 1-Hydroxyethane-(1,1-diphosphonic acid) (HEDP) is a very strong chelating agent widely used in industrial applications. This study examines the effect of HEDP on the adsorption of Cu(II), Zn(II), and Cd(II) onto boehmite in nonstoichiometric conditions, with the HEDP concentration higher than the corresponding Me(II) cations. At high surface loading and low pH, HEDP removes Zn(II) and Cd(II) from solution to an appreciable extent. The data are modeled assuming an anionic-ternary complex formation. In the same conditions, Cu(II) adsorption is significantly suppressed at intermediate values of pH, and this behavior is linked to Cu-HEDP complex formation in solution. At low surface covering, the effects of HEDP on metal adsorption are either negligible or slight. This behavior suggests that both ligand and metal are mainly adsorbed in separate form. All experimental data indicate that no changes are observed in the pH edges for phosphonate adsorption. The surface constants to fit the experimental data were calculated by applying the 2-K model constant capacitance (CCM).

Spectrophotometric determination of some drugs for osteoporosis.

Three simple, accurate and sensitive spectrophotometric methods are developed for the determination of some new drugs for the treatment of osteoporosis: risedronate sodium (I), alendronate sodium (II) and etidronate disodium (III). The first method is based on the measurement of difference in absorbance (Delta A) of risedronate sodium in 0.01 mol l(-1) hydrochloric and 0.1 mol l(-1) sodium hydroxide at 262 nm. Beer's law is obeyed over a concentration range of 15-150 microg ml(-1) with mean recovery 99.75+/-1.22 and molar absorptivity (epsilon) 1.891 x 10(3). The second method is based on the reaction of the primary amino group of (II) with ninhydrin reagent in methanolic medium in the presence of 0.05 mol l(-1) sodium bicarbonate. The colored product is measured at 568 nm, and the linearity range is found to be 3.75-45 microg ml(-1) with mean recovery 99.77+/-0.73 and epsilon 9.425 x 10(3). The third method is based on oxidation of the three mentioned drugs with ceric (IV) sulphate in 0.5 mol l(-1) sulphuric acid at room temperature and subsequent measurement of the excess unreacted cerium (IV) sulphate at 320 nm. The method obeyed Beer's law over a concentration range of 2-24 microg ml(-1) for the three drugs with mean recovery 99.79+/-1.16, 99.73+/-1.38 and 99.86+/-1.13 and epsilon 14.427 x 10(3), 13.813 x 10(3) and 14.000 x 10(3) for drugs I, II, III respectively. The proposed methods were successfully applied for the determination of the studied drugs in bulk powder and in pharmaceutical formulations. The results were found to agree statistically with those obtained the reported methods. Furthermore, the methods were validated according to USP regulations and also assessed by applying the standard addition technique.

Preparation of 1-hydroxyethylidene-1,1-diphosphonic acid-intercalated layered double hydroxide and its physicochemical properties.

The intercalation of 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP), which is a drug for osteoporosis, in layered double hydroxide (LDH) was examined with the goal of developing a novel drug delivery system (DDS) of HEDP. To prevent side reactions, the intercalation reaction was carried out at 0 degrees C, and at pH 4-6. The uptake of HEDP was determined as 3.5 mmol x g(-1) of LDH, and the interlayer distance increased from 7.8 to 13 A. The HEDP-release profiles into K(2)CO(3) aqueous solution and into various buffer solutions were also examined.

Analysis of selected diphosphonic acid derivatives used in treatment of osteoporosis. Part I. Complexometric determination of diphosphonic acid derivatives.

The purpose of the study was to develop a simple method for determination of diphosphonic acid derivatives in pharmaceutical preparations used in treatment of osteoporosis: disodium etidronate, disodium clodronate, disodium tiludronate, disodium pamidronate, sodium alendronate. The analysis performed by the visual end point titration method with complexing reagent Th(DCTA) in presence of xylenol orange used the ability of these compounds to form complexes.

Determination of phosphonates in natural waters by ion-pair high-performance liquid chromatography.

The paper describes a new method for the determination of phosphonates by ion-pair high-performance liquid chromatography. The phosphonates are complexed with Fe(III) and separated on a reversed-phase polymer column. The eluent consists of a bicarbonate solution at pH 8.3, tetrabutylammoniumbromide as counter-ion and 14% acetonitrile. The complexes are detected at 260 nm. The four phosphonates HEDP (1-hydroxyethylene-1,1-diphosphonic acid). ATMP (aminotris [methylenephosphonic acid]), EDTMP [ethylenediaminetetra(methylenephosphonic acid)], and DTPMP [diethylenetriaminepenta (methylenephosphonic acid)] are well separated within 20 min. The limits of detection are 5.10(-8) M for ATMP and EDTMP, 1.10(-7) M for DTPMP and 5.10(-7) M for HEDP. The method is suitable for the determination of phosphonates in the influent and effluent of wastewater treatment plants. A preconcentration of 10 is easily achieved by adsorption of the phosphonates onto freshly precipitated calcium carbonate and subsequent dissolution of the solid-phase by HCl.

Identification of adenine nucleotide-containing metabolites of bisphosphonate drugs using ion-pair liquid chromatography-electrospray mass spectrometry.

Bisphosphonates are synthetic pyrophosphate analogues, which are used as therapeutic drugs for the treatment of metabolic bone disorders. Some of these bisphosphonates can be metabolised in cells into non-hydrolysable nucleotide analogues. In this paper, we describe an ion-pairing high-performance liquid chromatography method that is compatible with negative ion electrospray mass spectrometry for the separation of these metabolites. Tandem mass spectrometry and collision-induced dissociation (CID) were used for identification of the metabolites. The CID mass spectra of bisphosphonate-adenine nucleotide adducts are very informative, because major fragment ions are formed by cleavage of the bisphosphonate moiety from the conjugate. The method was used for detection of the nucleotide metabolites of clodronate, tiludronate and etidronate in extracts from mammalian cells after treatment with bisphosphonates.

Comparison of the distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones.

Alendronate and etidronate are bisphosphonates used clinically to treat diseases associated with increased bone resorption. Etidronate is less potent and was reported to cause osteomalacia. This study examines if differences in distribution of alendronate and etidronate in the skeleton can explain differences in efficacy and in effects on mineralization between the two drugs. Eight-day old rat pups were injected s.c. with 3H-alendronate or 3H-etidronate both at either 1.3 mumol/kg or at their respective pharmacological effective doses in the growing rat of 0.12 mumol/kg for alendronate and 72.8 mumol/kg for etidronate. Twelve hours after administration at 1.3 mumol/kg both drugs showed a three- to fourfold higher localization on osteoclast vs. osteoblast surface. At the pharmacologically effective doses, 3H-alendronate labeled eightfold more osteoclast surface than osteoblast surface. In contrast, 3H-etidronate labeled approximately equal fractions of osteoclast and osteoblast surface. When similar doses of 3H-etidronate and 3H-alendronate (0.24 mumol/kg 3H-etidronate vs. 0.20 mumol/kg 3H-alendronate; 1.5 mumol/kg 3H-etidronate vs. 1.2 mumol/kg 3H-alendronate; and 14.6 mumol/kg 3H-etidronate vs. 12.0 mumol/kg 3 H-alendronate) were injected intravenously into adult mice at similar specific activities, 3H-etidronate labeled 1.5-2.5 times more osteoclast surface than 3-H-alendronate, but 3 to 15 times more osteoblast surface. Consequently, the ratio between the fraction of labeled osteoclast surface and the fraction of labeled osteoblast surface ranged for 3H-alendronate from 9 to 24, whereas for 3H-etidronate the range was from 4 to 7, due to more extensive labeling of osteoblast surface by 3H-etidronate. In a third experiment, we confirmed in adult mice the previous observation made in rat pups that normal bone formation occurs over alendronate-covered bone surfaces, and found that it occurred over etidronate-covered surfaces as well. Forty nine days after s.c. administration of alendronate at 0.12 mumol/kg or etidronate at 1.3 mumol/kg or 55.3 mumol/kg into adult mice bone formed over drug label. The distance from incorporated label to bone surface for both drugs (12.7 microns for alendronate and 8.7 and 9.2 microns for etidronate) was similar to wall width (defined by cement line) in controls (10.6 microns). In conclusion, alendronate, especially at pharmacologically active doses, shows higher uptake on resorption vs. formation surfaces than etidronate. The extent of bone formation on surfaces containing alendronate or etidronate is similar and is comparable to the "wall width" in controls.

Determination of bisphosphonate drugs in pharmaceutical dosage formulations by ion chromatography with indirect UV detection.

Application of ion chromatography (IC) to the analysis of non-chromophoric bisphosphonate drugs in pharmaceutical dosage formulations is described. The method is based on the use of single-column ion chromatography in conjunction with indirect UV detection that obviates the need for tedious chemical derivatization procedures. Diluted drug samples are chromatographed directly on a Waters IC-Pak HR anion-exchange column with dilute nitric acid (1.6-12 mM) as the mobile phase which exhibits a UV absorption maximum near 220 nm. Analyte detection is monitored by measuring the decrease in absorption of the mobile phase. The IC method has been validated and shown to be precise, accurate, specific and rugged for routine assay. Application of the method to the determination of alendronate sodium tablets, etidronate disodium injectable (which requires an eluent pH control for chromatographic resolution of active drug from chloride ions) and clodronate disodium injectable is presented. The performances of the Waters IC-Pak HR and several equivalent columns are also discussed.