quantification of levofloxacin in human plasma by ...

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Nov 11, 2015 - Levofloxacin has low concentration in plasma, thus it requires ... This study was focused on analyzing levofloxacin in human plasma with three ...

Journal of International Research in Medical and Pharmaceutical Sciences 7(2): 54-62, 2016 ISSN: 2395-4477 (P), ISSN: 2395-4485 (O)

International Knowledge Press www.ikpress.org


Bioavailability/Bioequivalence Laboratory, Faculty of Pharmacy, Universitas Indonesia, Depok, 16424, Indonesia.

AUTHORS’ CONTRIBUTIONS This work was carried out in collaboration between all authors. Author YH designed the study, wrote the protocol and interpreted the data. Author AAIB did the sample analysis. Author Harmita supervised author AAIB. Author SPS did the subject management and blood sampling. Authors YH and AAIB managed the literature searches and produced the initial draft. All authors read and approved the final manuscript.

Received: 26th August 2015 Accepted: 15th September 2015 Original Research Article Published: 11th November 2015 __________________________________________________________________________________ ABSTRACT Levofloxacin has low concentration in plasma, thus it requires sensitive and selective analysis method. Many kinds of anticoagulant are often used to obtain plasma as analytical matrix from whole blood. Citrate, heparin, and ethylenediaminetetraacetic acid (EDTA) are anticoagulant commonly used in analyzing drug in human plasma. This study was focused on analyzing levofloxacin in human plasma with three types of anticoagulants. The analysis was performed using High Performance Liquid Chromatography (HPLC) – photodiode array with column C18 SunfireTM (250 x 4.6 mm), 5 µm; temperature of 45°C, mobile phase consisting of 0.5% triethylamine pH 3.0 and acetonitrile (88:12 v/v); flow rate of 1.25 mL/minute, and ciprofloxacin HCl was used as internal standard. The method was linear at concentration range of 50.0 – 10.000.0 ng/mL with r>0.9994. Accuracy and precision for citrate, heparin, and EDTA plasma fulfilled the acceptance criteria of both intrabatch and inter-batch. There was no significant difference for stability and recovery of levofloxacin in citrate, heparin, and EDTA plasma (p>0.05; ANOVA), but it showed significant difference for peak area ratio (p 0.05 showed that there was no significant differences, P value < 0.05 showed significant differences among each parameter.

The comparison of some parameter analysis, there was no significant difference for citrate, heparin, and EDTA in stability and recovery of levofloxacin in plasma (p > 0.05; ANOVA). However for levofloxacin area ratio in citrate, heparin, and EDTA plasma show significant difference (p < 0.05) between citrate-EDTA plasma and heparin-EDTA plasma for low concentration and between heparin-citrate plasma and EDTA-citrate plasma for medium and high concentrations.

Comparison of the three types of anticoagulant on levofloxacin analysis in plasma can be seen by the presence of interference that appeared in each plasma. Interference appeared at retention time of less than 4 minutes for citrate and heparin plasma, while interference for EDTA plasma appeared in retention time of less than 8 minutes (Fig 2). The interference can affected the analyte or internal standard that appeared in the retention time. Therefore, it is important to consider the anticoagulant type in the optimization of chromatography condition so that the retention time can be set for both analyte and internal standard. In overlay, there was no significant difference between citrate and heparin plasma however there was significant interference for EDTA blank plasma.

The method has been successfully applied for pilot bioequivalence study with six Indonesian healthy subjects. This bioequivalency study was conducted to compare the quality of generic levofloxacin with the comparator drug, thus it gave result of pharmacokinetic parameter ratio such as AUCο-t, AUCο-∞, and Cmax. The ratio value fulfilled the criteria of 80 – 125% with CI 90%.

All validation parameters fulfill the test EMEA 2011 criteria, as well as the partial validation. Based on the stability data, it showed that levofloxacin in the three plasma from different anticoagulant was stable for 3 freeze-thaw cycles, 24 hours in room temperature, 24 hours on auto sampler, and 31 days at -20°C.

5. CONCLUSION The method has been validated and can be used to analyze levofloxacin with citrate, heparin, and EDTA


Harahap et al.; JIRMEPS, 7(2): 54-62, 2016

as anticoagulant. Based on the comparison of some analysis parameter, there is no significant difference for the three plasma in stability and recovery of levofloxacin in plasma but for peak response ratio of levofloxacin in citrate, heparin, and EDTA plasma showed significant difference. The method has been successfully applied for pilot bioequivalence study with six Indonesian healthy subjects.

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