UHPLC-MS/MS method for the determination of azithromycin in human plasma of pediatric patients
Ningfang Cai1, Xiuping Wu1, Jiancheng Lin1
1Department of Pharmacy, Xiamen Children's Hospital, Xiamen, Fujian, P. R. China.
Abstract:
The presence of azithromycin in the human plasma of pediatric patients was determined with a UHPLC-MS/MS assay. Sample preparation was done by protein precipitation, and the separation was achieved on a C18 column by the gradient mixture of the mobile phase A (0.1% acetic acid and 3 mM ammonium acetate in water) and the mobile phase B (0.1% acetic acid and 3 mM ammonium acetate in the solution of acetonitrile, methanol, and water, 47.5/47.5/5, V/V/V). The multiple reaction monitoring mode was adopted to monitor the precursor-to-product ion transitions of m/z 749.6 → m/z 591.5 for azithromycin and m/z 753.6 → m/z 595.5 for azithromycin-13 C-d3 (the internal standard) at the positive ionization mode. The calibration curve ranged from 0.5 to 500.0 ng·mL-1 , and the correlation coefficient was greater than 0.99. The intra- and inter-batch precision was less than 13.7%. Accuracy determined at four concentrations ranged from 99.5 to 110.8%. The extraction recoveries were more than 95%, and the matrix effects were 98-100%. The stability under various conditions was acceptable with the accuracy deviation within 9.2%. In conclusion, our method was simple, sensitive, and reliable for quantifying azithromycin in plasma among pediatric patients.
Insights
This study developed a reliable UHPLC-MS/MS assay to accurately quantify azithromycin in pediatric plasma. The validated method ensures precise and sensitive measurement of this important antibiotic in young patients.
Area of Science:
- Pharmacology and Toxicology
- Analytical Chemistry
- Pediatric Medicine
Background:
- Azithromycin is a widely used antibiotic in pediatric populations.
- Accurate quantification of azithromycin in plasma is crucial for therapeutic drug monitoring and pharmacokinetic studies in children.
- Existing methods may require improvement in terms of sensitivity, speed, or applicability to pediatric matrices.
Purpose of the Study:
- To develop and validate a sensitive and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method.
- To quantify azithromycin concentrations in human plasma samples from pediatric patients.
- To establish a robust assay for pharmacokinetic and clinical studies involving pediatric populations.
Main Methods:
- Sample preparation involved protein precipitation.
- Chromatographic separation was performed on a C18 column using a gradient elution with aqueous acetic acid/ammonium acetate and an organic solvent mixture.
- Detection was achieved via UHPLC-MS/MS in positive ionization mode using multiple reaction monitoring (MRM).
Main Results:
- The assay demonstrated excellent linearity over a range of 0.5–500.0 ng·mL⁻¹, with a correlation coefficient > 0.99.
- Intra- and inter-batch precision were below 13.7%, and accuracy ranged from 99.5% to 110.8%.
- Extraction recoveries exceeded 95%, matrix effects were between 98–100%, and method stability was confirmed.
Conclusions:
- The developed UHPLC-MS/MS method is simple, sensitive, and reliable for azithromycin quantification in pediatric plasma.
- This validated assay provides a valuable tool for clinical and research applications in pediatric pharmacotherapy.
- The method's robustness supports its routine use in monitoring azithromycin levels in children.
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