Correlated quantification using microbiological and electrochemical assays for roxithromycin determination in biological and pharmaceutical samples.

Microbiological and electrochemical assays, applying the cylinder-plate and differential pulse voltammetry as techniques, are reported for the quantitative determination of roxithromycin in serum and solid pharmaceutical form. The microbiological assay is based upon the inhibitory effect of this drug on the strain Bacillus subtilis ATCC 9372 used as the test microorganism. Linearity of the calibration curve was observed over the concentration range of 8.37-83.70 µg mL-1, with relative standard deviation values less than 5.0%. The electrochemical behavior of roxithromycin was studied at a graphite screen-printed electrode modified with graphene by using cyclic voltammetry and differential pulse voltammetry. The current value of the oxidative peak obtained for roxithromycin at 0.65 V vs. Ag/AgCl in 0.03 mol L-1 phosphate buffer solution (pH 7.0) with a scan rate of 0.1 V-1 is a linear function of the concentration in a range of 4.19-83.70 µg mL-1 (5-100 µmol L-1). A comparative study was carried out and both methods were applied for the determination of roxithromycin in solid dosage forms and spiked serum. The bioassay results of human serum samples were in accordance with the electrochemical ones (R2 = 0.988, P < 0.001), and the Bland-Altman method also showed good agreement between the values obtained by both procedures. Moreover, the statistical comparison indicated that there was no significant difference between the proposed techniques regarding both accuracy and precision.

Synthesis of 99m Tc-roxithromycin: A novel diagnostic agent to discriminate between septic and aseptic inflammation.

Roxithromycin is a second-generation macrolide antibiotic derived from erythromycin. In the current study, roxithromycin (ROX) was successfully labeled with technetium-99m for early diagnosis of bacterial infection and discrimination between septic and aseptic inflammation. The highest radiochemical purity of ≥95% was achieved by investigating different labeling parameters such as pH, ligand/reducing agent concentration, temperature, and amount of stabilizing agent. For this purpose, 0.3-0.5 mg ligand, 2-6 µg SnCl2 ·2H2 O as a reducing agent at basic pH (8-10 pH) and 2 mg mannitol used as a stabilizing agent, in the end, 370 MBq 99m Tc added into the reaction vials and incubated for a wide range of temperature (-4 to 65°C). The percent radiochemical purity of 99m Tc-roxithromycin was assessed with the help of the radio-thin-layer chromatography technique. The characterization studies were carried out using electrophoresis and Radio-HPLC techniques as well as saline stability and serum stability studies were also performed. Furthermore, biodistribution study was also performed in an inflamed animal model to discriminate between septic (heat-killed Staphylococcus aureus) and aseptic (turpentine oil) inflammatory lesions. The results were elaborated that 99m Tc-roxithromycin (99m Tc-ROX) was clearly bounded at the septic inflammation site (T/NT ratio of 7.08 ± 1.14) at 30 min postadministration, and maximum accumulation was seen in heart, lungs, liver, stomach, kidneys, and intestine. The results were suggested that 99m Tc-ROX might be used to discriminate between septic and aseptic inflammatory lesions at an early stage.

Determination of roxithromycin from human plasma samples based on magnetic surface molecularly imprinted polymers followed by liquid chromatography-tandem mass spectromer.

In this paper, a simple and reproducible method for the determination of roxithromycin in human plasma samples is proposed. The surface magnetic molecularly imprinted polymers (MMIPs) were utilized as sorbent. Roxithromycin was used as imprinted compound. The experimental results showed that the MMIPs had high affinity and selectivity toward roxithromycin. The extraction process was carried out in a single step by mixing the extraction solvent, MMIPs and human plasma samples by vortex. When the extraction process was completed, the MMIPs adsorbed the analyte were separated from the sample matrix by an external magnet due to the magnetism. The analyte eluted from the MMIPs was analysed by liquid chromatography-tandem mass spectrometry. Some main factors affecting the extraction of roxithromycin such as the amount of MMIPs, extraction solvent, extraction time, washing and elution conditions were optimized in this study. The calibration curve obtained by analyzing matrix-matched standards showed excellent linear relationship (r(2)=0.9997) in the concentration range of 10-1000ngmL(-1). The limit of detection and quantification obtained were 3.8 and 9.8ngmL(-1), respectively. The relative standard deviations of intra- and inter-day obtained were in the range of 3.9 %-5.5 % and 2.9 %-4.6 % with the recoveries ranging from 86.5 % to 91.5 %.

Roxithromycin Pharmacokinetics in Hospitalized Geriatric Patients: Oral Administration of Whole Versus Crushed Tablets.

BACKGROUND: Drug administration as tablets to debilitated elderly patients in crushed form can modify the pharmacokinetic characteristics of the active components. Only scarce information is available on the pharmacokinetics when administered in such form. The aim of this study was to evaluate the pharmacokinetics of roxithromycin administered in crushed form and to compare it with the pharmacokinetics of a group of geriatric patients receiving it in the conventional tablet form. METHODS: Twenty patients from the acute ward of the Shmuel Harofeh Geriatric Medical Center in stable, clinical, and hemodynamic condition were studied. Patients in group 1 (n = 10) received medications orally in tablet form. Group 2 (n = 10) included age- and disease-matched patients from the same department, who received oral roxithromycin in crushed tablet form. The mean daily dose was the same in both groups: 300 mg (150 mg twice daily). The patients received the drug for 3 days before the initiation of the study. Blood samples for determination of the roxithromycin concentration were taken at the baseline, 1 hour before the drug administration, and at 1, 3, 4, 6, 8, and 10 hours after drug administration. Roxithromycin concentration was measured by a liquid chromatography-tandem mass spectrometry method. RESULTS: Pharmacokinetic parameters of roxithromycin were significantly different between the 2 groups: the Cmin and Cmax were significantly higher, the tmax significantly longer, AUC0-10 larger, and CL/F smaller in group 2. CONCLUSIONS: Roxithromycin pharmacokinetic parameters were significantly different between the 2 patient groups resulting in higher drug serum concentrations in the crushed tablets group. The impact of the increased drug exposure is unclear.

Study of pharmacokinetic interaction of paroxetine and roxithromycin on bencycloquidium bromide in healthy subjects.

PURPOSE: The aim of this study was to investigate the potential drug-drug interaction between Bencycloquidium bromide (BCQB) and paroxetine, and between BCQB and roxithromycin. METHODS: Two studies were conducted on healthy male Chinese volunteers. Study A was an open-label, two-period, one-sequence crossover study (n=21). Each participant received a single nasal spray dose of BCQB 180µg on day 1. After a 7-day wash-out period, subjects received 20mg of paroxetine from day 8 to 17, and were co-administered 20mg of paroxetine and BCQB 180µg on day 18. In study B, participants (n=12) were randomly assigned to two groups. In period I, group A received BCQB 180µg on day 1, followed by the same dose four times daily from day 4 to 10, then, on day 11 a single dose of 150mg roxithromycin with BCQB 180µg were co-administered. In parallel, group B received a single dose of roxithromycin 150mg on day 1, followed by 300mg of roxithromycin from day 4 to 10, then, on day 11 a single dose of BCQB 180µg with roxithromycin 300mg were co-administered. After a wash-out time of 7days the respective treatments of each group (A and B) were swapped in period II. Blood samples were collected for pharmacokinetic analysis. Statistical comparison of pharmacokinetic parameters was performed to identify a possible drug interaction between treatments. Tolerability was evaluated by recording adverse events. RESULTS: Study A: Geometric mean AUC0-36 for BCQB alone and co-administered with paroxetine were 474.3 and 631.3pgh/ml, respectively. The geometric mean ratio (GMR) of AUC0-36 was 1.33 (1.13-1.46), 90% C.Is, and was out the predefined bioequivalence interval (90% C.Is, 0.80-1.25). Geometric mean Cmax were 187.0 and 181.2pg/ml. Study B: The GMR of AUC0-36 was 0.98 (0.90-1.07), 90% C.Is for BCQB, and the GMR of AUC0-72 was 0.98 (0.87-1.11), 90% C.Is for roxithromycin. Both GMRs were within the predefined bioequivalence interval (90% C.Is, 0.80-1.25). Other pharmacokinetic parameters were within the predefined interval. No serious adverse events were reported and no significant clinical changes were observed in laboratory test results, vital signs and ECGs in any of the studies. All treatments were well tolerated. CONCLUSION: The co-administration of BCQB with paroxetine showed a moderate increase in BCQB exposure, but was not clinically relevant. Also, no drug interaction was found between BCQB and roxithromycin.

Decrease in gastrointestinal absorption of roxithromycin in bile duct cannulated rats due to depletion of bile salts.

The purpose of this study was to investigate the effect of bile salts on gastrointestinal absorption and the plasma second peak phenomenon of roxithromycin in rats. The pharmacokinetic parameters of roxithromycin were calculated after single oral administration at a dose of 20 mg/kg in sham-operated (control), bile duct cannulated (BDC) and bile salt co-administered bile duct cannulated (BSBDC) rats. In BDC rats, the total area under the plasma concentration-time curve from time zero to time infinity (AUC0-∞) and the peak plasma concentration (Cmax) were significantly smaller (0.572-fold) and lower (0.412-fold), respectively, than those in the control rats. These values were recovered by co-administration of bile salt (0.831- and 0.828-fold for AUC0-∞ and Cmax compared with the control, respectively). Thus, the decreased absorption of roxithromycin in BDC rats could be due to a depletion of bile. The solubility of roxithromycin was 3.09-fold increased at 30 mm of sodium taurocholate. The oral dosage regimen of roxithromycin could be changed in patients with bile deficiency or when the drug is administered to individuals on a high-fat diet, if the present rat data can be extrapolated to humans.

Continuous, quantitative monitoring of roxithromycin in human saliva by flow injection chemiluminescence analysis.

Human saliva quantitative monitoring of roxithromycin (ROX) at picomolar-level by flow injection (FI) chemiluminescence (CL) analysis is described for the first time, to our knowledge. Monitoring was based on the CL intensity from luminol-BSA reaction, which can be quenched in the presence of ROX, with the decreasing CL intensity linearly proportional to the logarithm of the ROX concentration, ranging from 0.6 to 1000 pmol·L(-1). The detection limit of the proposed method for the determination of ROX was as low as 0.2 pmol·L(-1) (3σ), and the relative standard deviations were less than 4.0% (n = 7). A complete analytical process, including sampling and washing for ROX determination, conducted at a flow rate of 2.0 mL·min(-1), was performed completely within 30 s, yielding a sample efficiency of 120 h(-1). The proposed method was successfully applied to the determination of ROX in human saliva and serum samples with recoveries from 90.9% to 110.1%. The continuous monitoring of ROX in human saliva after oral intake showed that the total elimination ratio was 87.1% during 24 h, and the pharmacokinetic parameters were 0.97 ± 0.18 h(-1) for the absorption rate constant K(a), 0.082 ± 0.010 h(-1) for the elimination rate constant K(e), and 8.56 ± 1.11 h for the elimination half-life time t(1/2). It was also found that ROX in human saliva and urine simultaneously reached the maximum at 2 h with the concentration correlate ratio of 0.97.

Pharmacokinetics of tulathromycin and its metabolite in swine administered with an intravenous bolus injection and a single gavage.

Tulathromycin is a macrolide antimicrobial agent proposed for therapeutic use in treatment of porcine and bovine respiratory disease. In this study, the absolute bioavailability of tulathromycin solution was investigated in pigs. Eight pigs, with body weight of 20.5 ± 1.6 kg, were given a single dose of tulathromycin at 2.5 mg/kg oral (p.o.) and intravenous (i.v.) in a crossover design. The plasma concentrations of tulathromycin and its metabolite were determined by LC-MS/MS method, and the pharmacokinetic parameters of tulathromycin were calculated by noncompartmental analysis. After p.o. administration, the maximum plasma concentration (C(max) ) was 0.20 ± 0.05 µg/mL at 3.75 ± 0.71 h. The terminal half-life (t(1/2λz) ) in plasma was 78.7 ± 6.75 h, and plasma clearance (Cl/F) was 1.14 ± 0.28 L/h/kg. After i.v. injection, plasma clearance (Cl) was 0.580 ± 0.170 L/h/kg, the volume of distribution (Vz) was 64.3 ± 21.2 L/kg, and the t(1/2λz) was 76.5 ± 13.4 h. In conclusion, an analytical method for the quantification of tulathromycin and its metabolite in plasma in swine was developed and validated. Following p.o. administration to pigs at 2.5 mg/kg b.w., tulathromycin was rapidly absorbed and the systemic bioavailability was 51.1 ± 10.2.

Validation of a fully automated high throughput liquid chromatographic/tandem mass spectrometric method for roxithromycin quantification in human plasma. Application to a bioequivalence study.

A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the determination of roxithromycin in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 2.2 mL 96-deep-well plates. Roxithromycin and the internal standard clarithromycin were extracted from 100 microL of human plasma by LLE, using methyl t-butyl ether as the organic solvent. All liquid transfer steps were performed automatically using robotic liquid handling workstations. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted. Sample analysis was performed by reversed-phase LC-MS/MS, with positive ion electrospray ionization, using multiple-reaction monitoring. The method had a very short chromatographic run time of 1.6 min. The calibration curve was linear for the range of concentrations 50.0-20.0x10(3) ng mL(-1). The proposed method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of roxithromycin in human plasma. Therefore, it was applied to the rapid and reliable determination of roxithromycin in a bioequivalence study after per os administration of 300 mg tablet formulations of roxithromycin.

[Relative bioavailability of roxithromycin dispersive tablets in healthy volunteers].

The relative bioavailability of roxithromycin dispersive tablet in healthy volunteers was evaluated in this study. Its concentration in plasma was detected by high performance liquid chromatography (HPLC) after twenty healthy male volunteers were given each a single dose of 300 mg roxithromycin. The experiment data were obtained using DAS programme. The values of Cmax were 10.16+/-1.46 and 10.34+/-1.66 microg x ml(-1) at 2.33+/-0.61 and 2.28+/-0.62 h respectively; of t1/2 were 9.00+/-1.58 and 8.68+/-1.66 h respectively; of AUC0-->Tn were 143.32 +/-25. 80 and 138.93+/-22. 49 microg x h x ml(-1) respectively; of AUC0-->infinity were 158.63+/-26.86 and 153.77+/-24.75 microg x h x ml(-1) for test and reference drugs. Relative bioavailability of the tested roxithromycin was 103.63%+/-14.04%. The result showed that the two dispersive tablets are bioequivalent.

Simultaneous determination and pharmacokinetic study of roxithromycin and ambroxol hydrochloride in human plasma by LC-MS/MS.

BACKGROUND: Although roxithromycin and ambroxol HCl were often administered concomitantly for the treatment of respiratory infections, the pharmacokinetic interactions between them have not been reported. We investigated the interactions between these drugs in health male Chinese volunteers by LC-MS/MS in human plasma. METHODS: The pharmacokinetics were studied in 12 healthy male Chinese volunteers after an overnight fast by a single oral dose, 4-way crossover design with a period of 7-day washout. Each subjects was randomized to receive a single oral dose of 1 compound roxithromycin (150 mg) and ambroxol HCl (30 mg) dispersible tablet (test formulation, treatment A), one 150 mg roxithromycin dispersible tablet together with one 30 mg ambroxol HCl tablet (combined reference formulations, treatment B), one 150 mg roxithromycin dispersible tablet (reference formulation I, treatment C), or one 30 mg ambroxol HCl tablet (reference formulation II, treatment D) with 250 ml of water. Venous blood was collected at pre-dose (0 h) and 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 48, 72 h after dosing. The plasma concentrations of roxithromycin and ambroxol HCl were simultaneously determined by using a validated internal standard LC-MS/MS method. RESULTS: No significant differences were observed for the major pharmacokinetic parameters such as C(max), T(max), t(1/2) and AUC of both roxithromycin and ambroxol HCl between different treatments. CONCLUSION: The pharmacokinetics of both roxithromycin and ambroxol HCl are not affected by their concomitant oral administration. Therefore, there are no obvious pharmacokinetic interactions between roxithromycin and ambroxol HCl after oral administration. Roxithromycin and ambroxol HCl dispersible tablets were bioequivalent with reference to the roxithromycin dispersible tablets and ambroxol HCl tablets in combination usage.

Determination of roxithromycin in human plasma by HPLC with fluorescence and UV absorbance detection: application to a pharmacokinetic study.

A selective HPLC method with fluorescence detection for the determination of roxithromycin (ROX) in human plasma was described. After solid-phase extraction (SPE), ROX and erythromycin (internal standard, I.S.) were derivatized by treatment with 9-fluorenylmethyl chloroformate (FMOC-Cl). Optimal resolution of fluorescence derivatives of ROX and I.S. was obtained during one analytical run using reversed phase, C(18) column. The mobile phase was composed of potassium dihydrogenphosphate solution, pH 7.5 and acetonitrile. Fluorescence of the compounds was measured at the maximum excitation, 255 nm and emission, 313 nm, of ROX derivatives. Validation parameters of the method were also established. After SPE, differences in recoveries of ROX and erythromycin from human plasma were observed. The linear range of the standard curve of ROX in plasma was 0.5-10.0 mg/l. The validated method was successfully applied for pharmacokinetic studies of ROX after administration of a single tablet of ROX.

In vitro monitoring picogram roxithromycin in human urine using flow injection chemiluminescence procedure.

A sensitive chemiluminescence method, based on the enhancive effect of roxithromycin on the chemiluminescence reaction between luminol and hydrogen peroxide in a flow injection system, was proposed for the determination of roxithromycin. The increment of chemiluminescence intensity was linear with roxithromycin concentration in the range 1.0-1000 pg ml(-1) with the detection limit of 0.3 pg ml(-1) (3sigma). At a flow rate of 2.0 ml min(-1), a complete analytical process could be performed within 0.5 min, including sampling and washing, with a relative standard deviation of less than 5%. The proposed procedure was applied successfully in the monitoring of roxithromycin in human urine without any pre-treatment procedures and it was found that roxithromycin in urine reached its maximum after orally administrated for two hours, presenting an excretive ratio of 4.6% in 12 h. With urinary excretion rate method, the total elimination rate constant k and half-life time t(1/2) of roxithomycin was calculated, which was 0.1831, 3.785 h.

Stochastic resonance is applied to quantitative analysis for weak chromatographic signal of roxithromycin in beagle dog plasma.

Based on the theory of stochastic resonance, the signal to noise ratio (SNR) of HPLC/UV chromatographic signal of roxithromycin is enhanced by cooperation of signal, noise and nonlinear system. A simple new method for the determination of low concentration of roxithromycin in beagle dog plasma is presented. Using signal enhancement by stochastic resonance, this method extends the limit of quantitation from the reported 0.5 to 0.1 microg/ml. During validation of the new method, HPLC/MS was used as a comparison technique. The results indicate that the recovery and low concentrations of roxithromycin in beagle dog plasma were equivalent between the two methods (P>0.05). Stochastic resonance may be a promising tool for improving detection limits in trace analysis.

Determination of roxithromycin by liquid chromatography/mass spectrometry after multiple-dose oral administration in broilers.

A highly sensitive and specific method for the determination of roxithromycin in broiler tissues by LC/MS was developed and validated. A dichloromethane extract of the sample was separated on C18 reversed-phase column with acetonitrile-50 mM ammonium acetate (80:20, v/v) as the mobile phase and analyzed by LC/MS via atmospheric pressure ionization/electrospray ionization interface. The limit of detection and limit of quantitation were 1 ng/g and 5 ng/g. The method has been successfully applied to determine for roxithromycin in various tissues of broilers. Residue concentrations were associated with administered dose. At the termination of treatment, roxithromycin was found in all collected samples for both dose groups. Liver was detected to have the highest residual concentration of roxithromycin. Residue concentrations of roxithromycin were lower than its LOQ in all tissues from both dose groups 10 days after the treatment of roxithromycin mixed with drinking water at a dose rate of 15 mg/L or 60 mg/L to each broiler for 7 days.

Study on the electrochemical detection of the macrolide antibiotics clarithromycin and roxithromycin in reversed-phase high-performance liquid chromatography.

The optimal conditions of the amperometric detection of the macrolide antibiotics clarithromycin and roxithromycin were found by cyclic voltammetric studies and HPLC-electrochemical detection responses obtained in different temperatures (25.5-60 degrees C) and different but almost isoelutropic binary, ternary and quaternary mixtures of aqueous buffer (pH 7), methanol, acetonitrile and isopropanol. These conditions were also proved to be applicable for the quantitative detection of clarithromycin in human plasma using roxithromycin as an internal standard and vice versa. It was demonstrated that increased attention has to be paid to eluent composition and column temperature to ensure sensitive and reproducible electrochemical responses as well as regularly shaped peaks for both macrolides tested.

[Determination of roxithromycin in serum by high-performance liquid chromatography with ultraviolet detection].

OBJECTIVE: To make better the RP-HPLC method for the determination of roxithromycin(RM) in human serum. METHODS: RM and clarithromycin (internal standard) were extracted from alkalinized serum sample (500 microliters) with methylene chloride. After evaporation of the organic layer, the residue was dissolved in 100 microliters of acetonitrile-ammonium phosphate (1:1, pH 6.0) and washed with n-hexane, then 20 microliters was injected onto a column (5 microns, 15 cm x 4.6 mm) of Penomenex luna C18. The mobile phase was acetonitrile-0.05 mol/L phosphoric acid (39:19:42, adjusted to pH 7.2 with ammonia water) pumped at 1.2 ml/min through the column. The variable wavelength UV detector operated at 0.01 aufs and the wavelength was set at 210 nm. RESULTS: The retention times for RM and clarithromycin were 4.4 min and 5.0 min, respectively. Standard curve was linear in the concentration range of 0.25 to 32 mg/L. The detection limit in serum was 0.06 mg/L; the average method recovery 97.4%; the inter-day RSD less than 3.0%. CONCLUSION: This method was found to be simple, rapid, sensitive and accurate for determination of RM in human serum.

Identification of the metabolites of roxithromycin in humans.

The semisynthetic antibiotic roxithromycin (RXM) exists in an (E)-configuration. Metabolites of RXM in the bile of four cholecystectomy patients with T-tube drainage and in the urine and plasma of four healthy volunteers after single oral doses of 150 mg of RXM were investigated. A total of 15 metabolites were found in bile, urine, and plasma by HPLC with ion trap mass spectrometric and electrochemical detection. These metabolites were identified as descladinose derivative of RXM (M1), erythromycin-oxime (M2), N-, O-, and N,O-di-demethylated derivatives of RXM (M3, M4, and M6), and N-mono- and N-di-demethylated derivatives of erythromycin-oxime (M5 and M7), as well as the (Z)-isomers (M8-M15) of RXM and metabolites M1 to M7, respectively. Structures of six major metabolites (M1-M4, M8, and M10) were established by chromatographic and mass spectrometric determination and comparison with synthesized standards. The stability of RXM and the six synthesized substances was investigated to exclude artifact products. These results, together with previous findings, suggest that biotransformation pathways elucidated for RXM include: 1) isomerization of RXM derivatives, from E-isomer to Z-isomer; 2) O-demethylation; 3) N-demethylation; 4) hydrolysis of the cladinose moiety; and 5) dealkylation of the oxime ether side chain. Secondary metabolism via these pathways was also evidenced. The O-demethylation and isomerization of RXM derivatives represent two novel biotransformation pathways recovered for RXM.

Lack of in vitro and in vivo selection of bacterial resistance by roxithromycin.

The in vitro and in vivo selective pressure exerted by roxithromycin on Lancefield group A beta-haemolytic streptococci (GABHS) was investigated. In vitro antimicrobial activity on fifty GABHS strains was determined by the microdilution method and by boundary concentration (BC) determination. Insorgence of resistance was evaluated by redetermining MIC and BC after exposure to 16 x MIC roxithromycin concentration. In vivo insorgence of resistance was evaluated by MIC and BC determinations on the GABHS strain recovered from infected mice peritoneum, after treatment with 20 mg/kg roxithromycin. The roxithromycin serum kinetics was established in healthy and infected mice. Neither significant changes in GABHS MIC or BC after in vitro or in vivo exposure to roxithromycin nor a difference in roxithromycin serum levels between healthy and infected mice were detected, suggesting that the roxithromycin selection of resistance in GABHS is low.

Determination of roxithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection.

A simple and reproducible method for the determination of roxithromycin in human plasma is presented. This method is based on liquid-liquid extraction with hexane-isoamylalcohol (98:2, v:v) and reversed-phase chromatography with spectrophotometric detection at 220 nm. The mobile phase consists of methanol-15 mM dihydrogen potassium phosphate (70:30, v:v), pH of the aqueous part of the mobile phase is 6.0. The column is operated at 60 degrees C. Clarithromycin is used as the internal standard. The limit of quantitation is 0.5 microg/ml and the calibration curve is linear up to 30 microg/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 9%. The assay was used for pharmacokinetic studies.