Developing a High-Performance Liquid Chromatography (HPLC) Method for Simultaneous Determination of Oxytetracycline, Tinidazole and Esomeprazole in Human Plasma.

A high performance liquid chromatography method had been developed and validated for rapid simultaneous separation and determination of three anti-helicobacter drugs, oxytetracycline (OXY), tinidazole (TIN) and esomeprazole (ESM) in human plasma within 6 minutes. Drugs extraction method from plasma was based on protein precipitation technique. Separation was carried out on a Equisil BDS C18 column (5 µm, 150 × 4.60 mm) using a mobile phase of acetonitrile: 0.025 M KH2PO4 (25: 75, v/v) adjusted to pH 3.50 with ortho-phosphoric acid at ambient temperature. The flow rate was 1 mL/min and maximum absorption was measured using Diode Array (DAD) detector at 285 nm. The retention times of OXY, TIN and ESM were recorded to be 2.68, 3.52 and 5.17 minutes, respectively, indicating a shorter analysis time. Limits of detection were also reported to be 0.10, 0.07 and 0.04 µg/mL for OXY, TIN and ESM, respectively, showing a high degree of the method sensitivity. The method was then validated according to FDA guidelines for the determination of the drugs clinically in human plasma specially regarding pharmacokinetic and bioequivalence studies.

Sensitive inexpensive chromatographic determination of an antimicrobial combination in human plasma and its pharmacokinetic application.

This study represents simple inexpensive chromatographic determination of ciprofloxacin (CIP) and tinidazole (TIN) simultaneously in human plasma using HPLC-DAD followed by a pharmacokinetic application. C18 column was used as stationary phase with isocratic elution of a mobile phase composed of acetic acid solution (2%) and acetonitrile (85: 15, v/v) and ornidazole as internal standard (IS) with UV detection at 318 nm. The two drugs and the IS were separated at 6.55, 7.91 and 11.07 min for CIP, TIN and IS, respectively, with good selectivity and sensitivity for their analysis in presence of plasma matrix components and the drugs' metabolites. Sample preparation involved only protein precipitation without any complicated extraction procedures decreasing analysis time. For method validation, FDA regulations for analysis in biological fluids were followed. Pharmacokinetic (PK) study on six healthy volunteers was conducted after single oral dose administration of 500 and 600 mg of CIP and TIN, respectively. Dugs' plasma levels were followed for 12 or 72 h post dosing for CIP and TIN, respectively, and different PK data for the two drugs were calculated and they were comparable to the reported values demonstrating successful future application of the presented method in PK, bioequivalence and bioavailability studies.

Preparation and application of a carbon paste electrode modified with multi-walled carbon nanotubes and boron-embedded molecularly imprinted composite membranes.

An innovative electrochemical sensor was fabricated for the sensitive and selective determination of tinidazole (TNZ), based on a carbon paste electrode (CPE) modified with multi-walled carbon nanotubes (MWCNTs) and boron-embedded molecularly imprinted composite membranes (B-MICMs). Density functional theory (DFT) calculations were carried out to investigate the utility of template-monomer interactions to screen appropriate monomers for the rational design of B-MICMs. The distinct synergic effect of MWCNTs and B-MICMs was evidenced by the positive shift of the reduction peak potential of TNZ at B-MICMs/MWCNTs modified CPE (B-MICMs/MWCNTs/CPE) by about 200 mV, and the 12-fold amplification of the peak current, compared with a bare carbon paste electrode (CPE). Moreover, the coordinate interactions between trisubstituted boron atoms embedded in B-MICMs matrix and nitrogen atoms of TNZ endow the sensor with advanced affinity and specific directionality. Thereafter, a highly sensitive electrochemical analytical method for TNZ was established by different pulse voltammetry (DPV) at B-MICMs/MWCNTs/CPE with a lower detection limit (1.25 × 10-12 mol L-1) (S/N = 3). The practical application of the sensor was demonstrated by determining TNZ in pharmaceutical and biological samples with good precision (RSD 1.36% to 3.85%) and acceptable recoveries (82.40%-104.0%).

Multiobjective optimization strategy based on desirability functions used for the microemulsion liquid chromatographic separation and quantification of norfloxacin and tinidazole in plasma and formulations.

The aim of the present study was to optimize a microemulsion liquid chromatography method for the simultaneous determination of norfloxacin and tinidazole binary mixture using a chemometric protocol. Optimization experiments were conducted through a process of screening and optimization. A 2(7-4) fractional factorial design was used as screening design. While the location of optimum conditions was established by applying Derringer's desirability function. The optimal mobile phase composition was predicted to be: 3.5% w/v SDS, 10.03% v/v 1-propanol, 0.5% v/v 1-octanol, and 0.3% triethylamine in 0.02 M phosphoric acid at pH 6.5. The mobile phase was delivered isocratically at a flow rate of 1 mL/min with UV detection at 290 nm. Tinidazole and norfloxacin were eluted with retention times of 1.8 and 5.8 min, respectively. The calibration plots displayed good linear relationships in the concentration ranges of 0.5-50 and 0.75-75 µg/mL for norfloxacin and tinidazole, respectively. The method was successfully applied for determination of both drugs in pharmaceutical dosage forms and real human plasma. Where the accuracy was proved by the low values of % error and high values of recovery, also the relative standard deviation for the results did not exceed 1.5%, proving the precision of the method.

Simultaneous determination of triple therapy for Helicobacter pylori in human plasma by reversed phase chromatography with online wavelength switching.

The infection of gastric mucosa by Helicobacter pylori (HP) is an essential cofactor in the aetiology of gastroduodenal ulcer and gastric carcinoma. Because of the bacterial resistance, combination therapy containing omeprazole (OME), tinidazole (TNZ) and clarithromycin (CLA) is commonly used for eradication of HP. However, the simultaneous determination of the triple therapy in human plasma was not reported. A simple, reproducible, and selective HPLC method was developed for the simultaneous determination of the triple therapy mixture used for management of HP infections in human plasma. An HPLC procedure based on a liquid-liquid extraction, enrichment of the analytes and subsequent reversed-phase chromatography with UV detection was used. To enable sensitive and selective detection, the method involved the use of online wavelength switching detection, with two different detection wavelengths; 280nm for detection of OME and TNZ and 210nm for detection of CLA. Separations were performed on C18 analytical column with acetonitrile-10mM phosphate buffer of pH=3.0 at flow rate of 1.0mLmin(-1). The linear ranges in human plasma were 0.05-10µgmL(-1) with correlation coefficients >0.9990. The detection limits in human plasma were 0.02-0.07µgmL(-1). Validation parameters were assessed in compliance with US-FDA guidelines. The method proved to be valuable for the therapeutic drug monitoring after oral administration of triple therapy tablets.

Micellar HPLC and derivative spectrophotometric methods for the simultaneous determination of fluconazole and tinidazole in pharmaceuticals and biological fluids.

Micellar high-performance liquid chromatography (HPLC) and first-derivative ultraviolet spectrophotometry were used to simultaneously determine fluconazole (FLZ) and tinidazole (TNZ) in combined pharmaceutical dosage forms. The derivative procedure is based on the linear relationship between the drug concentration and the first derivative amplitudes at 220 and 288 nm for FLZ and TNZ, respectively. The calibration graphs were linear in the range of 1.5-9.0 µg/mL for FLZ and 10.0-60.0 µg/mL for TNZ. Furthermore, an HPLC procedure with ultraviolet detection at 210 nm was developed. For the HPLC procedure, good chromatographic separation was achieved using an ODS C18 column (250 × 4.6 mm i.d.). The mobile phase containing 0.15M sodium dodecyl sulphate, 0.3% triethylamine and 12% n-propanol in 0.02M orthophosphoric acid at pH 5.5 was pumped at a flow rate of 1 mL/min. Indapamide was used as an internal standard. The method showed good linearity over the concentration ranges of 1.5-30.0 and 10.0-200.0 µg/mL, with limits of detection of 0.36 and 2.70 µg/mL and limits of quantification of 1.1 and 8.2 µg/mL for FLZ and TNZ, respectively. The suggested methods were successfully applied for the simultaneous analysis of the drugs in their laboratory prepared mixture, co-formulated tablet and single dosage forms. Moreover the second method was also extended to the determination of the drugs in biological fluids.

Simultaneous quantification of linezolid, tinidazole, norfloxacin, moxifloxacin, levofloxacin, and gatifloxacin in human plasma for therapeutic drug monitoring and pharmacokinetic studies in human volunteers.

BACKGROUND: Linezolid may be administered in combination with norfloxacin, gatifloxacin, levofloxacin, moxifloxacin, and tinidazole for the treatment of various infections, such as urinary and respiratory tract infections, to improve the efficacy of the treatment or to reduce the duration of therapy. Knowledge of the antibiotic plasma concentrations combined with bacterial susceptibility evaluated in terms of minimum inhibitory concentration would optimize treatment efficacy while limiting the risk of dose-related adverse effects and avoiding suboptimal concentrations. METHODS: A new high-performance liquid chromatography assay method was developed and validated for determination of the above-mentioned drugs in small samples of human plasma. After protein precipitation with acetonitrile:methanol (1:1, vol/vol), satisfactory separation was achieved on a Hypersil BDS C18 column (250 × 4.6 mm, 5 µm) using a mobile phase comprising 20 mM sodium dihydrogen phosphate-2 hydrate (pH = 3.2) and acetonitrile at a ratio of 75:25, vol/vol; the elution was isocratic at ambient temperature with a flow rate of 1.5 mL/min. The ultraviolet detector was set at 260 nm. The validated method was applied to assay real plasma samples used for pharmacokinetic studies and therapeutic drug monitoring of the selected drugs. RESULTS: The assay method described was found to be rapid, sensitive, reproducible, precise, and accurate. Linearity was demonstrated over the concentration ranges as follows: 0.1-30 µg/mL for linezolid and tinidazole; 0.05-5 µg/mL for norfloxacin; and 0.1-10 µg/mL for moxifloxacin, levofloxacin, and gatifloxacin (mean r = 0.9999, n = 12). The observed within- and between-day assay precisions were within 12.5%, whereas accuracy ranged between 92.0% and 112% for all the analytes. The lower limit of quantification was 0.1 µg/mL for all the analytes except norfloxacin which was 0.05 µg/mL. CONCLUSIONS: This assay method was valid within a wide range of plasma concentrations and may be proposed as a suitable method for pharmacokinetic studies, therapeutic drug monitoring implementation, and routine clinical applications, especially for some populations of patients who receive a combination of these drugs.

Development of a HPLC/MS/MS method for simultaneous determination of tinidazole, dyclonine and chlorhexidine in rat plasma and its application in the pharmacokinetic research of a film-forming solution.

A rapid and sensitive HPLC/MS/MS method was developed and validated for simultaneous determination of tinidazole, dyclonine and chlorhexidine, the three main components of a film-forming solution, in rat plasma. The plasma samples were pretreated by solid phase extraction (SPE) method. Separation was achieved on a Phenomenex Gemini C(18) column (50 mm × 2.0 mm, 5 µm) using an isocratic mobile phase system composed of methanol-ammonium formate (10 mM)-formic acid (56:44:0.2, v/v/v) (pH 3.5) at a flow rate of 0.2 mL/min. Analytes were determined by tandem mass spectrometry with electrospray positive ionization and multiple-reaction monitoring (MRM) mode. The monitoring ions were (m/z) 247.4 → (m/z) 81.9 for tinidazole, (m/z) 290.1 → (m/z) 97.8 for dyclonine, (m/z) 505.0 → (m/z) 335.3 for chlorhexidine and (m/z) 282.1 → (m/z) 212.0 for phentolamine (internal standard). The calibration curves were linear in the range of 2-1000 ng/mL for the three components. The precision and accuracy of the method were well within the generally accepted criteria for biomedical analysis. It has been successfully applied to the pharmacokinetic research of a film-forming solution in rat.

In vivo and real time determination of ornidazole and tinidazole and pharmacokinetic study by capillary electrophoresis with microdialysis.

The aim of this study was to develop a rapid and sensitive method for in vivo and real time monitoring unbound ornidazole (ONZ) and tinidazole (TNZ) in rabbit blood using capillary electrophoresis coupled with microdialysis. The UV wavelength was set at 214 nm and all separations were performed in 20 mM Tris-H3PO4 (pH 1.5) buffer. Microdialysis probes were perfused at 4 microl/min resulting in relative recoveries of 33.1+/-3.6% and 34.8+/-3.3% (n=3) for ONZ and TNZ, respectively. The linearity was studied in the concentration range of 1.0-412 microg/ml for ONZ and 1.0-520 microg/ml for TNZ. The detection limits were 0.7 microg/ml for ONZ and 0.6 microg/ml for TNZ (S/N=3). All separation could be achieved within 15 min. This method has been successfully applied to the pharmacokinetic study of ONZ and TNZ in rabbit blood.

Validated HPLC method for the determination of tinidazole in human serum and its application in a clinical pharmacokinetic study.

A high performance liquid chromatographic (HPLC) method for the determination of tinidazole in human serum using metronidazole as internal standard (IS) is described. Protein precipitation is used for the preparation of sample. Mobile phase consisting of 0.002 M phosphate buffer, methanol and acetonitrile mixture (85:7.5:7.5/v/v/v) was used at a flow rate of 1 ml/min on a C18 column. The eluate was monitored using an UV/Vis detector set at 320 nm. Ratio of peak area of analyte to IS was used for quantification of serum samples. The absolute recovery was greater than 95% over a concentration range of 0.5 to 30 micrograms/ml and the limit of quantitation was 0.05 microgram/ml. The intra-day relative standard deviation (RSD) measured at 0.5, 5, 15 and 30 micrograms/ml ranged from 0.36 to 6.14%. The inter-day RSD ranged from 1.14 to 4.21%. The method is simple, sensitive and has been successfully used in a pharmacokinetic study conducted in healthy human volunteers.

Assay of tinidazole in human serum by high-performance thin-layer chromatography--comparison with high-performance liquid chromatography.

Determination of tinidazole in human serum by high-performance thin-layer chromatography (HPTLC) is presented. It includes use of 10 x 10 cm plates coated with silica gel 60 and chloroform-acetonitrile-acetic acid (60 + 40 + 2) as mobile phase. Quantitation was performed by densitometry at 320 nm. The linearity (1-10 ng), precision (6%), reproducibility (5%), recovery (96%), and detection limit (1 mg/L) of tinidazole determination by HPTLC were comparable with corresponding method parameters by reversed-phase HPLC. A satisfactory correlation was found between the 2 analytical methods. The procedure was used to quantitate tinidazole in patient sera.

Direct assay of tinidazole in human serum by micellar liquid chromatography.

A liquid chromatographic procedure for the direct determination of tinidazole in human serum is presented. It includes the use of a micellar mobile phase consisting of SDS (5.10(-2) M): propan-1-ol; (94:6, v/v) and a mu Bondapak CN column with UV detection at 320 nm. No solvent extraction or deproteinization are necessary. The linearity (0.1-10 mg L), the precision (3%), the reproducibility (1.3%), the recovery (99%), and the detection limit (0.1 mg L) in the tinidazole determination are comparable and sometimes greater than the corresponding tinidazole parameters when deproteinization and conventional reversed-phase HPLC are used. One hundred injections of serum samples do not affect the column life. The procedure is applied to ascertain the pharmacokinetics of 10 mg/kg of tinidazole.

Concentrations of tinidazole in gingival crevicular fluid and plasma in dogs after multiple dose administration.

Tinidazole 15 mg/kg was administered to eight Beagle dogs with gingivitis or periodontitis twice daily for 3 days. Tinidazole concentrations in blood and gingival crevicular fluid (GCF) were measured 1, 3, 6 and 9 h after the morning dose each day. The concentration of tinidazole was determined by high performance liquid chromatography (HPLC). The mean concentration of tinidazole in GCF for each dog ranged from 6.05 to 9.32 micrograms/mL at different time points after the first dose, and on the first day the highest concentration was observed 6 h after the drug administration. Tinidazole concentrations were 34 +/- 4%-72 +/- 9% (mean +/- SEM) of simultaneous plasma concentration. At steady-state, on the third treatment day, the mean tinidazole concentrations in GCF ranged from 6.68 to 13.1 micrograms/mL, i.e. 44 +/- 6%-75 +/- 25% of the corresponding concentrations in plasma. Tinidazole concentration in GCF exceeded the MIC values for putative path-ogenic periodontal bacteria and it is concluded that, when indicated, tinidazole could be used for chemotherapy of periodontitis in dogs.

Rapid and selective high-performance liquid chromatographic method for the determination of metronidazole and its active metabolite in human plasma, saliva and gastric juice.

A rapid and selective HPLC method has been developed for the separation and quantitation of metronidazole and its hydroxylated metabolite in human plasma, saliva and gastric juice. The assay requires a simple protein precipitation step prior to analysis and is selective, sensitive and reproducible. The limits of quantitation (0.5-ml sample) were at least 0.25 microgram/ml for metronidazole and 0.20 micrograms/ml for its hydroxy metabolite. A Hypersil ODS 5 micron (150 x 4.5 mm I.D.) column was used with a mobile phase of acetonitrile-aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1 % triethylamine (10:90) delivered at a flow-rate of 1.0 ml/min.

Pharmacokinetics of single-dose administration of tinidazole in unweaned calves.

In a crossover trial, 7 healthy, 7- to 29-day-old, unweaned Finnish Ayrshire calves were given a single dose of 20 mg of tinidazole/kg of body weight, IV, and a single dose of 25 mg of tinidazole/kg orally. Blood samples were collected serially, and serum concentration of tinidazole was measured by use of high-performance liquid chromatography. Serum concentration vs time data were analyzed by use of the statistical moment theory. Terminal half-life was 394 minutes after IV administration and 524 minutes (harmonic mean) after oral administration. The corresponding system moment mean residence times were 542 +/- 61.8 minutes and 812 +/- 117 minutes (arithmetic mean +/- SD), respectively. Estimated volume of distribution at steady state and total body clearance were 0.74 +/- 0.05 L/kg and 1.37 +/- 0.13 ml/min/kg, respectively. Tinidazole was rapidly and totally absorbed from the gastrointestinal tract. Mean absorption time was 270 +/- 160 minutes, and the observed peak serum concentration was detected at 240 minutes. Bioavailability was 99.5 +/- 3.9%.

Penetration of tinidazole into skin blister fluid following its oral administration.

Plasma and skin blister fluid concentrations of tinidazole following a single oral dose of 2 g drug, and after multiple doses of 0.25 g every 12 h, were determined. Skin blisters were produced by direct application of 0.25% cantharidin ointment to the skin. The maximum concentration in plasma of about 36 mg.l-1 was observed after about 2 h, whereas in skin blister fluid the peak occurred after about 6 h and was 30 mg.l-1. The half-life in plasma was slightly shorter than in blister fluid at 17 and 19 h, respectively, but the difference was not significant. The penetration of tinidazole into cantharidin-induced skin blister fluid, defined according to Wise as the ratio of the AUCs in blister fluid and plasma was 1.00. During routine treatment with tinidazole (0.25 g every 12 h), the concentrations in plasma and blister fluid collected before and 3 h after the morning dose exceeded the minimal inhibitory concentrations for susceptible pathogens. The results provide a pharmacokinetic basis for the proven efficacy of tinidazole in the treatment of protozoal and anaerobic infections.

Single-dose concentrations of tinidazole in gingival crevicular fluid, serum, and gingival tissue in adults with periodontitis.

Previous studies have shown that metronidazole is effective in the treatment of subgingival microflora associated with destructive periodontitis. The aim of this study was to determine whether tinidazole, a close analogue of metronidazole, would reach sufficient concentrations in serum, gingival crevicular fluid, and gingival tissue, to inhibit putative periodontopathic bacteria. Ten adult patients with moderate to advanced periodontitis took a single 2-g dose of tinidazole orally. Samples were assayed by high-performance liquid chromatography. The concentrations of tinidazole in serum and GCF were in a similar range (3.2-46.5 micrograms/mL). Tinidazole was not detected in the GCF in three of the patients. The drug was found in gingival tissue obtained at two h (0.17 +/- 0.14 micrograms/mg) and six h (0.15 +/- 0.18 micrograms/mg) after oral administration. The mean concentration of tinidazole in serum at 24 h (13 +/- 3.0 micrograms/mL) is greater than the minimum inhibitory concentration for anaerobic bacteria as reported by others. The present data suggest that a single 2-g oral dose of tinidazole may lead to the presence of potentially bactericidal levels of tinidazole for up to 24 h in the periodontal pockets of some patients with periodontitis.

N-oxidation: a possible route of tinidazole metabolism in man.

Treatment of tinidazole with a mixture of hydrogen peroxide and acetic acid, or liver homogenate preparations, yields the N-3 oxide. This was identified by thin-layer chromatographic analysis on silica gel G, Rf 0.6, using ethanol-chloroform-ammonia (50:49:1) as solvent, and by chemical reduction with sulphur dioxide. UV spectrophotometry and high performance liquid chromatography (HPLC) gave an RT of 0.55 min using Pye Unicam apparatus equipped with a UV detector at 330 nm, a reversed-phase RP 18 (10 microns) column which was 12.5 cm long, a mobile phase of methanol-0.005 M KH2PO4 (pH 4) (20:80, v/v) and a flow rate of 2 ml/min. In-vitro metabolic N-oxidation was achieved by incubating the parent drug, tinidazole, with rat liver homogenates fortified with cofactors at 37 degrees C. HPLC analysis of blood and urine samples from healthy volunteer subjects who took a single oral dose of tinidazole showed the presence of an in-vivo N-oxidation metabolite of the drug. The identical physico-chemical characteristics of the synthetic and biologically produced tinidazole N-oxide strongly suggest that tinidazole, a tertiary amine drug, undergoes metabolic N-oxidation.

Pharmacokinetics of tinidazole in the horse.

Serum tinidazole concentrations were monitored in five clinically healthy adult horses after intravenous (i.v.) and oral administration of the drug (15 mg/kg and 25 mg/kg, respectively). After i.v. administration, the mean residence time was 7.0 h, the elimination half-life 5.2 h and the body clearance rate 1.6 ml/min/kg. The distribution volume was found to be 660 ml/kg. After oral administration, the mean residence time was 8.5 h, the absorption half-life 1.1 h and the bioavailability essentially 100%. In view of the in-vitro sensitivities of various anaerobic bacteria, a dosage of 10-15 mg/kg of tinidazole, orally, at 12-h intervals, can be recommended for the treatment of anaerobic infections in horses.