Nickel ion release and surface analyses on instrument fragments fractured beyond the apex: a laboratory investigation.

BACKGROUND: To analyse the changes in surface and nickel ion release characteristics of fractured root canal shaping instruments in a simulated body fluid environment. METHODS: A total of 54 new instruments were studied. The instrument groups consisted of five different NiTi alloys and a stainless-steel alloy. To standardize instrument fracture, a torsional type of failure was created on each instrument. The fractured specimens of each instrument group were randomly divided into three static immersion subgroups of 1 h, 7-day, and 30-day (n = 3). Simulated body fluid (SBF) was prepared to mimic human blood plasma by Kokubo&Takadama protocol for ex situ static immersions at 37ºC. The surfaces were examined via scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy. To determine the quantitative ion release, the retrieved SBFs were analyzed using inductively coupled plasma mass spectrometry. Two-way ANOVA and Tukey post hoc tests sought the statistical significance of the nickel ion values(p < 0.05). RESULTS: In 1 h of immersion, the newly formed structures, exhibiting mostly oxygen signals, were widespread and evident on NiTi surfaces. In contrast, fewer structures were detected on the SS surface in that subgroup. In 7 days of immersion, a tendency for a decrease in the density of the new structures was revealed in NiTi groups. The oxygen signals on NiTi group surfaces significantly increased, contrary to their decrease in SS. Signals of sodium, chlorine, and calcium were detected, indicating salt precipitates in groups. In 30 days of immersion, salt precipitates continued to form. The Ni-ion release values in all instrument groups presented significant differences in comparison to the SBF control in all immersion periods(p < 0.001). No significant differences were observed in immersion time periods or instrument groups(p > 0.05). CONCLUSIONS: Within the limitations of the presented study, it was concluded that the fractured SS and NiTi root canal instruments release Ni ions in contact with body fluid. However, the Ni ion release values determined during the observation periods are lower than the critical toxic or allergic thresholds defined for the human body. This was due to the ionic dissolution cycle reaching a stable state from 1-hour to 30-day exposure to the body fluid of fractured instruments.

Development and Validation of Two New Methods for the Determination of Rilmenidine in Bulk and Pharmaceutical Preparation.

In the present study, two new methods were developed and validated for the determination of rilmenidine in bulk and pharmaceutical preparation. Both methods are based on a derivatization reaction using 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) as a fluorogenic substance. The drug reagent derivatives were formed by the reaction of rilmenidine with NBD-Cl at pH 9.0 at 70°C for 40 min. The reaction mixtures were analyzed by spectrofluorimetry in the first method and high performance liquid chromatography (HPLC) in the second method. Derivatives were determined at λex 493 nm and at λem 536 nm in the spectrofluorimetric method. The separation was performed place on a Phenomenex, C18 column (250 × 4.6 mm, 5 µm i.d) using a mobile phase comprising 0.2% formic acid and acetonitrile gradient elution mode in the HPLC method. Analytes were detected by a fluorescence detector at the same wavelength. The methods were validated for limit of quantitation, linearity, robustness, recovery, limit of detection, precision and accuracy. Calibration curves for the first and second methods were found to be linear in the range of 2.0-12.0 and 250-2000 ng/mL, respectively. Detection limits for the spectrofluorimetric and HPLC methods were calculated as 0.16 and 18.28 ng/mL, respectively. The validated methods were applied successfully to the determination of rilmenidine in bulk and pharmaceutical preparation.

Analytical method development and validation of mucopolysaccharide polysulfate in topical formulations by size exclusion chromatography.

In this study, a new size exclusion chromatographic method has been developed and validated for the analysis of mucopolysaccharide polysulfate used as an anti-inflammatory and antithrombotic agent in topical formulations. Mucopolysaccharide polysulfate was analyzed in Repromer OH-4000 (10 µm, 8.0 × 300 mm) and Repromer OH-5000 (10 µm, 8.0 × 300 mm) columns using a 0.05 M sodium sulfate isocratic elution mobile phase system at 40 °C with a flow rate of 1 mL min-1 and detected by using refractive index detection. The method was validated by means of the limit of quantification, limit of detection, linearity, robustness, recovery, precision and accuracy using the Bioanalytical Method Validation Guidance. The calibration curve showed linearity in the 0.090-1.575 mg mL-1 range. The limits of detection and quantification were found to be 45.000 and 90.000 µg mL-1, respectively. Assay recovery and precision of mucopolysaccharide polysulfate from topical formulations at 0.450, 0.900 and 1.350 mg mL-1 concentrations were evaluated. Intra-day and inter-day relative standard deviation values were calculated to be less than 2.46%. The mean recovery was calculated as 96.64%. The validated method was successfully applied to the determination of mucopolysaccharide polysulfate in cream and gel formulations.

Determination of levofloxacin by HPLC with fluorescence detection in human breast milk.

Aim: A new HPLC method with fluorescence detection has been developed and validated for the determination of levofloxacin, one of the fluoroquinolone class antibiotics, in breast milk. Materials & methods: Chromatographic separation was carried out on a reversed phase C18 column with acetonitrile and 10 mM o-phosphoric acid (25:75, v/v) mobile phase composition. Moxifloxacin was used as internal standard and the peaks were detected by fluorescence detection. Results & conclusion: Calibration graph was found linearly within the range of 2.5-500 ng/ml. Limit of detection and limit of quantification were found to be 0.63 and 2.11 ng/ml, respectively. Mean absolute recovery was 96.18%. The developed method has been successfully applied to the determination of levofloxacin in human breast milk taken from two healthy volunteers.

Development of sensitive spectrofluorimetric and spectrophotometric methods for the determination of duloxetine in capsule and spiked human plasma.

A new, sensitive and selective spectrofluorimetric method has been developed for the determination of duloxetine (DLX) in capsule and spiked human plasma. DLX, as a secondary amine compound, reacts with 7-chloro-4-nitrobenzofurazon (NBD-Cl), a highly sensitive fluorogenic and chromogenic reagent used in many investigations. The method is based on the reaction between the drug and NBD-Cl in borate buffer at pH 8.5 to yield a highly fluorescent derivative that is measured at 523 nm after excitation at 478 nm. The fluorescence intensity was directly proportional to the concentration over the range 50-250 ng/mL. The reaction product was also measured spectrophotometrically. The relation between the absorbance at 478 nm and the concentration is rectilinear over the range 1.0-12.0 µg/mL. The methods were successfully applied for the determination of this drug in pharmaceutical dosage form. The spectrofluorimetric method was also successfully applied to the determination of duloxetine in spiked human plasma. The suggested procedures could be used for the determination of DLX in pure form, capsules and human plasma being sensitive, simple and selective.

A new HPLC method with fluorescence detection for the determination of memantine in human plasma.

A sensitive and selective HPLC method with fluorometric detection was developed for the determination of memantine in human plasma and applied to a pharmacokinetic study. Memantine was precolumn derivatized with 9-fluorenylmethyl chloroformate, and the fluorescent derivative was separated on an RP C18 column using a mobile phase composed of acetonitrile-10 mM orthophosphoric acid containing 1 mL/L triethylamine with gradient elution. The method was based on the measurement of the derivative using fluorescence detection at 310 nm with excitation at 260 nm. The calibration curve was linear over the range 1.0-50.0 ng/mL. LOD and LOQ were found to be 0.3 and 1.0 ng/mL, respectively. Intraday and interday RSD values were less than 3.39%.

Determination of rosuvastatin at picogram level in serum by fluorimetric derivatization with 9-anthryldiazomethane using HPLC.

For the first time, a carboxyl group derivatization assay has been developed and validated for the determination of the cholesterol-lowering drug rosuvastatin in human serum at picogram level by high-performance liquid chromatography with fluorescence detection. The assay procedure involved a simple one-step liquid-liquid extraction of rosuvastatin with lovastatin as internal standard from serum with an ethyl acetate-methyl tertiary buthyl ether (1:1) mixture. After pre-column derivatization with 9-anthryldiazomethane at room temperature for one hour, the reaction mixture was injected onto a Phenomenex, Synergi C18 column (250 × 4.6 mm, 4 µ i.d.). The analytes were separated with a mobile phase composed of acetonitrile-water in gradient elution mode and detected at λ(em) = 410 nm, exciting at 366 nm. Calibration curves were constructed in concentration range of 0.01-20.0 ng/mL and limit of detection and limit of quantification values were found to be 0.68 and 2.30 pg/mL, respectively. To test suitability of the developed methods for clinic use, the pharmacokinetics of rosuvastatin were investigated after oral administration of a 20 mg rosuvastatin film tablet to a healthy volunteer and maximum plasma concentration, time to reach that concentration and elimination half life were found to be 17.5 ng/mL, 3.5 h and 18.09 h, respectively.

A new HPLC method with fluorescence detection for the determination of memantine in human plasma.

A sensitive, selective, simple and fast HPLC method based on the formation of derivative with fluorescamine was developed for the determination of memantine (ME) in human plasma. Separation was achieved on a CN column (200 mm×4.6 mm) using acetonitrile-10 mM orthophosphoric acid containing 1 mL/L triethylamine (45:55, v/v) at a flow rate of 1 mL/min. Emission and excitation wavelengths were 480 and 380 nm, respectively. Amantadine was used as an internal standard. Calibration graphs were rectilinear over the range of 1.0-100.0 ng/mL. Limit of detection and limit of quantification were found to be 0.3 and 1.0 ng/mL, respectively. Intra-day and inter-day relative standard deviation values were found to be <2.03%. Average recovery was also found to be around 94%. Proposed method was applied for the pharmacokinetic study in a healthy volunteer after a single oral administration of 20 mg of ME.

Detection of methamphetamine, methylenedioxymethamphetamine, and 3,4-methylenedioxy-N-ethylamphetamine in spiked plasma by HPLC and TLC.

HPLC and TLC methods were developed for separation and detection of some amphetamine analogs: methamphetamine (MA); 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"); and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) in spiked plasma samples. The methods are based on purple chromogens formed by displacement reaction of these secondary aliphatic amine-bearing drugs with 7,7,8,8-tetracyanoquinodimethane at 80 degrees C for 25 min. For HPLC, both normal phase (silica gel) and RP (C18) columns were used. With the former, good detection limits in plasma were obtained with a 6 min run: 70, 100, and 500 ng/mL for MDMA, MA, and MDEA, respectively. For TLC, hexane-chloroform (1 + 9) and benzene-diethyl ether-petroleum ether (40-60 degrees)-acetonitrile-ethyl methyl ketone (2 + 3.5 + 3.5 + 0.5 + 0.5) were used as mobile phases for silica gel 60 TLC and cyano-bonded silica gel HPTLC plates, respectively. The former offered more sensitive results than the latter. Influence of evaporation steps on recovery and interferences for the HPLC and TLC methods were investigated. The developed methods are selective, simple, and easily applicable.

Ondansetron-loaded chitosan microspheres for nasal antiemetic drug delivery: an alternative approach to oral and parenteral routes.

BACKGROUND: The aim of this study was to develop chitosan microspheres for nasal delivery of ondansetron hydrochloride (OND). METHOD: Microspheres were prepared with spray-drying method using glutaraldehyde as the crosslinking agent. Microspheres were characterized in terms of morphology, particle size, zeta potential, production yield, drug content, encapsulation efficiency, and in vitro drug release. RESULTS: All microspheres were spherical in shape with smooth surface and positively charged. Microspheres had also high encapsulation efficiency and the suitable particle size for nasal administration. In vitro studies indicated that all crosslinked microspheres had a significant burst effect, and sustained drug release pattern was observed until 24 hours following burst drug release. Nasal absorption of OND from crosslinked chitosan microspheres was evaluated in rats, and pharmacokinetic parameters of OND calculated from nasal microsphere administration were compared with those of both nasal and parenteral administration of aqueous solutions of OND. In vivo data also supported that OND-loaded microspheres were also able to attain a sustained plasma profile and significantly larger area under the curve values with respect to nasal aqueous solution of OND. CONCLUSION: Based on in vitro and in vivo data, it could be concluded that crosslinked chitosan microspheres are considered as a nasal delivery system of OND.

Ondansetron-loaded biodegradable microspheres as a nasal sustained delivery system: in vitro/in vivo studies.

The aim of this study was to prepare ondansetron-loaded biodegradable microspheres as a nasal delivery system. Microspheres were prepared with emulsification/spray-drying technique using poly(d,l-lactide) (PLA) and two different types of poly(d,l-lactide-co-glycolide) (PLGA). The effect of the type of organic solvent (dichloromethane (DCM) or a mixture of DCM and ethyl acetate) on the microsphere characteristics was also examined. The prepared microspheres were evaluated with respect to the morphological properties, particle size, zeta potential, drug loading efficiency, and in vitro drug release. The mean particle size (d(50)) of microsphere formulations was ranged from 11.67-25.54 µm, indicating suitable particle size for nasal administration. All microspheres had low drug loading efficiency in the range of 12.28-21.04%. The results indicated that particle size of microspheres were affected by both type of polymer and organic solvent, however drug loading efficiency of microspheres were affected by only the type of organic solvent used. All microspheres were negatively charged due to the polymers (PLA or PLGA) used. A prolonged in vitro drug release profile was observed for 96 h. Based on in vitro data, the selected microsphere formulation has been applied via nasal route to rats in vivo. Following nasal administration of ondansetron-loaded microsphere to rats, ondansetron plasma levels were within a range of 30-48 ng/mL during 96 h, indicating a sustained drug delivery pattern and relatively a constant plasma drug concentration level. The results suggested that biodegradable microspheres prepared with emulsification/spray-drying technique could be considered to deliver ondansetron via nasal route to obtain a prolonged release.

Determination of linezolid in human breast milk by high-performance liquid chromatography with ultraviolet detection.

A rapid and simple HPLC method was developed for the determination of linezolid (LNZ) in human breast milk after a simple protein precipitation with methanol. The chromatographic separation was achieved on a C18 column (5 microm, 250 x 4.6 mm id) using a mobile phase of acetonitrile-10 mM acetic acid (25:75, v/v) at a flow rate of 1 mL/min. The LNZ peak was measured by photodiode array detection at 250 nm. The calibration graph was linear over the range of 0.5-20.0 microg/mL. The limits of detection and quantitation were found to be 0.1 and 0.5 microg/mL, respectively. The precision of the assay and the recovery of LNZ from breast milk at three different concentrations were assessed. The intraday and interday RSD values were found to be < 5%. The mean absolute recovery was 85.33%. The developed method was successfully applied to the determination of LNZ in breast milk obtained from the breastfeeding mother after oral administration of LNZ.

A selective high-performance liquid chromatography method for the determination of reboxetine in bulk drug and tablets.

Reboxetine is used as a selective noradrenaline reuptake inhibitor for the treatment of major depressive disorders. It is effective in the treatment of severe depression and safer to use than traditional tricyclic antidepressants. In this study, a novel, simple, and rapid stability-indicating high-performance liquid chromatography (HPLC) method for reboxetine methansulfonate was successfully developed and validated for the assay of tablets. The method was used to quantify reboxetine in tablets; it employed a C18 column (150 x 4.6 mm id) with an isocratic mobile phase consisting of methanol-phosphate buffer (pH 7, 0.02 M; 55 + 45, v/v) at a flow rate of 1.0 mL/min. Reboxetine was detected by an ultraviolet detector at 277 nm. The retention time of reboxetine was about 4.5 min. The developed HPLC method was validated with respect to linearity, precision, sensitivity, accuracy, and selectivity. The method was linear over the concentration range 1-50 microg/mL (r = 0.9999). The limits of detection and the quantitation of reboxetine were 0.1 and 0.3 microg/mL, respectively. The relative standard deviation values for intraday and interday precision were 0.78-1.01 and 1.08-1.37%, respectively. Selectivity was validated by subjecting a stock solution of reboxetine to neutral, acid, and alkali hydrolysis, as well as oxidation, dry heat treatment, and photodegradation. The peaks of the degradation products did not interfere with the peak of reboxetine. The results indicated that the proposed method could be used in a stability assay. The proposed method was successfully applied to the determination of reboxetine in tablets. Excipients present in the tablets did not interfere with the analysis.