Method For Determination Of Azithromycin Research Articles

A LC-MS/MS validated method for determination of azithromycin in human tears and its application to an ocular pharmacokinetic study.

A rapid and sensitive method for the quantitative analysis of azithromycin in human tears by LC-MS/MS was developed and validated. Following extraction from collected Schirmer tear strips by methanol-water (4:1, v/v), the analyte and IS (azithromycin-d3) were separated on a Waters Atlantis™ dC18 column (2.1 mm × 30 mm, 3 μm) by gradient elution with 0.1% (v/v) formic acid in methanol-water (1:9) and methanol-acetonitrile (9:1) as the mobile phase. Electrospray ionization in positive ion mode and MRM were used to monitor the ion transitions at m/z 749.6 → 591.6 (azithromycin) and 752.4 → 594.4 (azithromycin-d3). The results indicated that the method had excellent sensitivity and specificity. The analyte appeared to have good linearity in the range of 5-1000 ng/ mL. Both the intra-batch and inter-batch precisions (in terms of RSD) were <10%, and the accuracies (in terms of RE) were within ±15%. The lower limit of quantification, matrix effect, extraction recovery, stability and dilution integrity were also evaluated and satisfied the validation criteria. Artificial tears served as the surrogate matrix, and no matrix difference was found when compared with that of real human tears. Finally, this method was successfully applied in an ocular pharmacokinetic study in healthy volunteers following instillation of azithromycin eyedrops.

A rapid, developed and validated RP-HPLC method for determination of azithromycin

Azithromycin (Azr) is a member of the macrolide antibiotic and it used on a wide scale in prescribed antibiotic drugs as anti-gram-positive and anti-gram-negative microorganisms. The present study aimed to develop an HPLC method of Azr analysis enjoyed highly linearity, repeatability, robust, rugged, selective and rapid to use. The chromatographic method uses a reversed phase column ODS-3 (250 mm × 4.6 mm x 5 μm). The mobile phase was prepared by mixing Methanol: Phosphate buffer (9:1, v/v) at flow rate 1.2 ml/min with PDA detector 210 nm, column oven adjusted to 40° C with injection volume 50 μL. The method revealed satisfied linearity regression R2 (0.9996) with repeatability (0.66%), LOD and LOQ 28.7 µg/ml and 86.9 µg/ml respectively. The method showed a successful application for Azr determination in bulk and pharmaceutical formulations.

Development of Rapid, Simple and Stability-Indicating Method for Determination of Azithromycin Using RP-HPLC

An accurate, sensitive, precise and stability indicating (in accordance with ICH guidelines) reversed phase high performance liquid chromatographic (RP-HPLC) method of analysis of Azithromycin was developed and validated. The chromatographic conditions comprised of a reversible phase C8 column (250 × 4.6 mm, 5μ) with a mobile phase consisting of a mixture of dipotassium hydrogen Phosphate and Acetonitrile in the ratio of 65:35 at pH 6.5 adjusted with phosphoric acid. Flow rate was 1.5 ml/min, the detection was carried out at 200 nm and the retention time of Azithromycin was 2.99 min. Azithromycin was subjected to acid and alkali hydrolysis, oxidation and photochemical degradation. The method was validated for accuracy, precision and robustness. The results indicate that the drugs are susceptible to degradation in different conditions. All the peaks of degraded products were resolved from the active pharmaceutical ingredient with significant different retention times. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.

Stability indicating HPLC method development and validation for the simultaneous determination of Azithromycin & Ofloxacin in bulk and its dosage forms

A simple, precise, sensitive and reproducible stability indicating Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method for determination of Azithromycin and Ofloxacin in tablet dosage form was developed. Chromatographic separation was achieved on Hypersil-Keystone RP C18 (250 4.6 mm, 5m) column maintained at 30 C eluted with mobile phase at flow rate of 1.2 ml/min. The mobile phase consists of Buffer (0.02 M potassium dihydrogenphosphate): methanol: acetonitrile in the ratio 65:25:10 v/v at pH maintained at 3.2 with OPA was used and the determination was carried out at 285 nm. The retention time for Azithromycin and Ofloxacin were 9.7 min and 5.01 respectively. The linearity was found in the range of 5?50 ?g/ml for Azithromycin and 4?40 ?g/ml for Ofloxacin. In stability studies the drugs were well separated from degradation products. The degradation was studied in the individual standard drugs, their mixture and formulation which gave the idea about the orgin of the degradant products. The analytical method was validated as per ICH guideline for linearity, accuracy, precision, and specificity, limit of detection, limit of quantification, stability in analytical solution etc. and method can be extended to the analysis of Azithromycin and Ofloxacin in tablet formulations.

Chemometric Approach for RP-HPLC Determination of Azithromycin, Secnidazole, and Fluconazole Using Response Surface Methodology

This paper demonstrates a chemometric-assisted RP-HPLC method for the simultaneous determination of azithromycin, secnidazole, and fluconazole using water:methanol (63:37% v/v) as mobile phase at a flow rate of 1.2 mL · min−1. The separation of the three drugs was achieved on an Xterra RP-18 column (250 × 4.6 mm ID, 5 µM) and the detection of the eluents was realized on a diode array detector at 210 nm wavelength. The combined effects of % methanol (organic phase) and flow rate, each at three levels influencing the chromatographic efficiency were investigated and optimized employing central composite design. Under the optimal condition, the percent relative standard deviations for precision study were in the range of 0.2–0.7% and 0.5–1.2% for standard and formulation, respectively. The limits of detection were 12.5, 0.62, and 1.7 µg · mL−1 and limits of quantification were found 37.5, 1.86, and 5.1 µg · mL−1 for azithromycin, secnidazole, and fluconazole, respectively. Consequently, the method has been effectively applied to their direct determination in the commercial formulation (combi-kit).

An optimal LC-MS/MS method for determination of azithromycin in white blood cells: application to pediatric samples.

Studies suggest that particular antimicrobial and anti-inflammatory properties of azithromycin (AZM) can be related to its extensive accumulation in white blood cells (WBCs). However, available methods for determination of AZM in WBCs require large blood volumes unsuited to a pediatric context. Therefore, an LC-MS/MS method was developed for determination of AZM in WBCs. WBCs were isolated from 500 µl of whole blood by lysing red blood cells. Then, lysis of WBCs was performed with methanol/buffer containing AZM-d3-(13)C as internal standard. After reversed phase LC, detection was performed under multiple reaction monitoring conditions in positive electrospray mode. Linearity ranged from 0.5 to 200 ng per WBC sample. Within-run and overall accuracy and precision ranged from 95.3 to 101.1% and from 1.6 to 4.7%, respectively. All validation parameters fulfilled international requirements. This method can be easily performed on small samples and provides reliable data, including in children and neonates.

Development of a Simple RP-HPLC-UV Method for Determination of Azithromycin in Bulk and Pharmaceutical Dosage forms as an Alternative to the USP Method.

The present study was designed to develop a simple, validated liquid chromatographic method for the analysis of azithromycin in bulk and pharmaceutical dosage forms using ultraviolet detector. The best stationary phase was determined as C18 column, 5 μm, 250 mm × 4.6 mm. Mobile phase was optimized to obtain a fast and selective separation of the drug. Flow rate was 1.5 mL/min, Wavelength was set at 210 nm and the volume of each injection was 500 μL. An isocratic methanol/buffer mobile phase at the ratio of 90:10 v/v gave the best separation and resolution. The proposed method was accurate, precise, sensitive, and linear over a wide range of concentration of azithromycin. The developed method has the advantage of using UV detector compared to the USP method in which electrochemical detector has been used. The validated method was successfully applied to the determination of azithromycin in bulk and pharmaceutical dosage forms.

Stress Degradation Studies on Azithromycin and Development of a Validated Stability-Indicating TLC-Densitometric Method with HPLC/Electrospray Ionization-MS Analysis of Degradation Products

The purpose of this work was to develop a sensitive stability-indicating TLC-densitometric method for determination of azithromycin (AZ) in the presence of its impurities E, I, and L, and to study the stability of AZ under different stress conditions. The method was developed on TLC aluminum plates precoated with silica gel F254 using the mobile phase methanol-acetone-ammonia 25% (2+13+0.1, v/v/v), which gives compact zones for AZ (Rf= 0.39) and impurities E, I, and L (Rf = 0.54, 0.20, and 0.05, respectively). Densitometric analysis of AZ and its impurities was carried out at 483 nm after spraying with sulfuric acid-ethanol (1 + 4, v/v) and heating at 100 degrees C for 5 min. The linear regression analysis data for the calibration plots showed good linear relationships with r = 0.9941 for AZ, 0.9987 for impurity E, 0.9989 for impurity I, and 0.9984 for impurity L. AZ was subjected to acidic and alkaline hydrolysis, oxidation, and reduction stress. The drug underwent degradation under these conditions. The degradation products were well-resolved from the pure drug, with significantly different Rf values. A plausible degradation pathway of AZ was established by HPLC/electrospray ionization-MS analysis of the products.

Novel spectrophotometric method for the determination of azithromycin in pharmaceutical formulations based on its charge transfer reaction with quinalizarin

This paper proposes a new method for simple and fast spectrophotometric determination of azithromycin in pharmaceutical formulations. The method is based on the charge transfer reaction between the azithromycin and quinalizarin in methanol medium. In order to achieve maximum sensitivity the effect of some chemical variables such as the type of solvent, reagent concentration and reaction time were evaluated. The reaction was characterized in terms of stability of the product formed and its stoichiometry, and the apparent molar absorptivity and association constant were derived. Best conditions for the analytical determination of azithromycin were observed in methanol medium with a quinalizarin concentration of 50 mg L-1. At these conditions, the radical anion (absorbing specie) was formed in the medium immediately after mixing of the reagents and showed maximum absorption at 564 nm. The method presented a limit of detection of 0.35 mg L-1 and a limit of quantification of 1.2 mg L-1. It was successfully applied in the determination of azithromycin in three commercial pharmaceutical formulations of azithromycin and no matrix interferences were observed.

Quantitative determination of azithromycin in human plasma by liquid chromatography–mass spectrometry and its application in a bioequivalence study

A sensitive, rapid liquid chromatographic–electrospray ionization mass spectrometric method for determination of azithromycin in human plasma was developed and validated. Azithromycin in plasma (0.2 mL) was extracted with methyl tert-butyl ether–hexane (50:50, v/v), organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness at 40 °C and dissolved in mobile phase, samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm × 2.1 mm i.d., 5 μm), together with a mobile phase containing of 20 mM ammonium acetate (pH 5.2)–acetonitrile–methanol (50:40:10, v/v/v) and was isocratically eluted at a flow rate of 0.2 mL/min. Azithromycin and its internal standard, clarithromycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 2 to 1000 ng/mL with r = 0.9977. The limit of quantification for azithromycin in plasma was 2 ng/mL with good accuracy and precision. The higher mean extraction recovery, say 81.2% and 75.5% for azithromycin and internal standard (IS), respectively, was obtained in this work. The intra-day and inter-day precision ranged from 4.8% to 8.6% and 6.4% to 10.7% (R.S.D.), respectively. The established method has been successfully applied to bioequivalence study of 2 azithromycin formulations for 24 healthy volunteers.

Validated HPLC–MS–MS method for determination of azithromycin in human plasma

A validated, highly sensitive, and selective HPLC method with MS-MS detection has been developed for quantitative determination of azithromycin (AZI) in human Na2EDTA plasma. Roxithromycin (ROX) was used as internal standard. Human plasma containing AZI and internal standard was ultrafiltered through Centrifree Micropartition devices and the concentration of AZI was determined by isocratic HPLC-MS-MS. Multiple reaction monitoring mode (MRM) was used for MS-MS detection. The calibration plot was linear in the concentration range 2.55-551.43 ng mL(-1). Inter-day and Intra-day precision and accuracy of the proposed method were characterized by R.S.D and percentage deviation, respectively; both were less than 8%. Limit of quantification was 2.55 ng mL(-1). The proposed method was used to determine the pharmacokinetic profile of AZI (250-mg tablets).

High performance liquid chromatographic determination of azithromycin in serum using fluorescence detection and its application in human pharmacokinetic studies

A fast and sensitive high-performance liquid chromatographic method for determination of azithromycin in human serum using fluorescence detection was developed. The drug and an internal standard (clarithromycin) were extracted from serum using n-hexan and subjected to pre-column derivatization with 9-fluorenylmethyl chloroformate as labeling agent. Analysis was performed on a phenyl packing material column with sodium phosphate buffer containing 2 ml/l triethylamine (pH 5.9) and methanol (29:71, v/v) as the mobile phase. The standard curve was linear over the range of 10–500 ng/ml of azithromycin in human serum. The means between-days precision were from 13.3% (for 10 ng/ml) to 2% (500 ng/ml) and the within-day precision from 11.9 to 1.7% determined on spiked samples. The accuracy of the method was 100.7–107.2% (between days) and 100.3–107.8% (within day). The limit of quantification was 10 ng/ml. This method was applied in a bioequivalence study of four different azithromycin preparations in 12 healthy volunteers.