Simultaneous determination of Trovafloxacin and Marbofloxacin in equine serum and human plasma samples using previously validated HPLC Method


Dr Adnan Hussein Mahmood

Clinical Pharmacology, PhD The Medical Technical Institute of Baghdad, The Middle Technical University, Iraq.

*Corresponding Author E-mail:,



A previously validated HPLC method was applied for the simultaneous determination of marbofloxacin (MBX) and trovafloxacin (TVX) in equine serum and human plasma. Samples have been precipitated with 20% HClO4 and drugs were separated using a gradient mobile phase system containing of 17.5mM of Monosodium phosphate (NaH2PO4) and 1.5mM aqueous solution of tetrabutylammonium hydroxide (C16H37NO), pH 3 (A) and 50% acetonitrile (C2H3N) and 50% methanol (B). Antibiotics were determined through UV detection at 293 and 270nm. The quantification limits for TVX and MBX were 10ng/ml and 2ng/ml respectively, and the retention times were 6.63 and 4.93 min respectively for both compounds. Over a concentration range of 10–50,000ng/ml for both antibiotics, the calibration curves were linear. No interacted signals were detected in both horse serum and human plasma. The linearity, precision and accuracy of the assay were estimated from spiked horse serum. The study suggested the use of this method in potential pharmacokinetics studies for selected matrices.


KEYWORDS: Human plasma; Horse serum; HPLC; Marbofloxacin; Trovafloxacin.




Many transferable diseases in horses such as pleuropneumonia and respiratory bacterial infections1-3 are susceptible to fluoroquinolones, where treatments with traditional antibiotic having inadequate effects. The quinolone antibiotics4, are wide spectrum, very effective specific antibacterial agents, used in human and animals for treatment of complicated bacterial infections. Marbofloxacin (MBX) is a 3rd generation fluoroquinolone antibiotic (Figure 1A), have been presently used only in veterinary medicine. Limited occurrences of resistant pathogens were found after long term treatment of MBX in cattle5, horses6-8 and pets9. Trovafloxacin (TVX), a 4th generation fluoronaphthyridone (Figure 1B) is no longer used in humans, after being withdrawn from pharmacies due to frequent occurrence of idiosyncratic hepatotoxicity in 199910,11.



Figure 1: Chemical structures of Marbofloxacin (MBX) and Trovafloxacin (TVX).


Consequently, MBX and TVX might be appropriate antibiotics for treatment of equine bacterial infections. In order to evaluate these candidates, a sequence of in vivo pharmacokinetic experiments will be needed, which necessitate the expansion of innocent and sensitive HPLC method for these antibiotics in biological fluids. In the literature, limited data for MBX assays in equine serum and no reports of determination of TVX levels in the same matrix and using HPLC with UV detection.


Since no adequate information about procedures that could be beneficial at the same time for variant animal species and for different tissues. The study defines the expansion and validation of an enhanced HPLC systematic method using UV detection for concurrent determination of MBX and TVX which is sensitive, fast, and necessitate very simple sample preparation.



2.1 Chemicals and materials:

Materials (Marbofloxacin and Trovafloxacin mesylate), were kindly provided by Pfizer Inc. (Eastern Point Rd., Groton CT 06340) as MBX and TVX respectively. The HPLC measurements were carried out using same Shimadzu chromatographic system described in previously published work12. Equine serum was purchased from Serum Australia, while human plasma samples were available in the lab from other experiments. The study was performed at the Therapeutic Research Centre (TRC), Translation research Institute (TRI), The University of Queensland, Brisbane, Australia. The mobile phase, gradient mixture flow and retention times were exactly similar to what are found in previously reported method for sheep plasma12.


2.2 Preparation of standard, quality control samples (QCs) and sample preparation procedure:

All stock solutions and samples were prepared and processed as same as previously described method sheep plasma.


2.3 Validation of method:

The selectivity, linearity, precision and accuracy detection limit and quantification limit were determined for equine serum. The method was also tested for human plasma. According to FDA guidelines, detection limit and quantitation limit were obtained based on the standard deviation of the response and the slope of the calibration curve13{, 1997 #14;U.S. Department of Health and Human Services, 1997 #287}. The precision and accuracy for both intra-day and inter-day of the control samples were assessed and measured as percentage variations from nominal concentrations of both compounds in horse serum.


50µL MBX and TVX stock solutions (1mg/ml) were spiked in 0.9ml of blank plasma, the calibration curves for both agents were formed to make a high concentration of 50,000ng/ml in plasma. Plasma solutions of 20,000, 10,000, 5,000, 2,000, 1,000, 500, 250, 100, 50, 20, and 10ng/ml were prepared by stepwise dilutions.  The Quantitation limit for each analyte was determined using pure standards that were analyzed based on peak areas in the HPLC method. The limit of detection was estimated from a low concentration reference standard through continuous dilution.


The method was validated using a standard statistical calculation was used for 3 groups of calibration curves plus 2 extra samples of lower limit of quantification (LLOQ) for each antibiotic.

The quality control samples (n = 6) were determined by extracting the spiked equine serum samples consisting of both MBX and TVX at 20,000, 4,000, 500 and 50ng/ml. The intra-assay precision was measured at 20,000, 4,000, 500 and 50ng/ml, while inter-assay precision was determined at 4,000, 500 and 50ng/ml (n = 4). The accuracy was measured by relating the calculated concentration to nominal concentrations.



Demonstrative chromatograms of equine serum and human plasma are shown in Figures 2 and 3 respectively. As seen, no endogenous constituent signals were interfered with either MBX or TVX peaks in the pre-treatment samples.



Figure 2: Chromatogram blank horse serum spiked with 4000 ng/ml of MBX and TV, blank horse serum spiked with 250 ng/ml of MBX and TVX, and a blank horse serum.



Figure 3: Chromatogram blank human plasma spiked with 4000 ng/ml, blank human plasma spiked with 500 ng/ml of MBX and TVX, and a blank human plasma.


The retention times of MBX and TVX were 4.9 and 6.7 min, respectively, with a calibration range of 10-50000 ng/ml for both antibiotics. The detection and quantitation limit (LLOQ) for MBX were (0.3,0.9), while for TVX were (3.0, 9.4) ng/ml, respectively. The precision of LLOQ was 4.4% for MBX and 9.4% for TVX.


Means of the control samples, precision and accuracy at intra-day and inter-day analysis are presented in Table 1.

This HPLC method allows simultaneous analysis for MBX and TVX, unlike the assays in14-16 and is shorter, simpler and the volume of plasma or serum required was slightly lower. than previously reported assays for MBX 17,18 and TVX19,20. The detection limit of MBX and TVX was slightly less than that stated by Garcia et al and Carretero et al15,18 and Liang et al20, respectively


Table 1:  Means, precision and accuracy ranges of the control samples in horse serum.

Concentration (ng/ml)



Mean± SD (ng/ml)

Precision (%)

Accuracy (%)

Mean± SD (ng/ml)

Precision (%)

Accuracy (%)























































The current study examined previously developed HPLC method for simultaneous analysis of both MBX and TVX on different matrices. The precision, accuracy and linearity are proposed to be appropriate clinical procedures for both antibiotics.


This method is simple, time and cost saving, and it was effectively tested in human plasma samples. Additional studies are needed in the future, in order to evaluate it this method in the other matrices.



The author wishes to thank The Therapeutic Research Centre (TRC) particularly Professor Michael Roberts for providing materials and helping in performing this study.



The present work has no conflict of interest.



No human or animals were participants in this study performed by the author.



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Received on 17.12.2019           Modified on 12.02.2020

Accepted on 09.04.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(1):143-145.

DOI: 10.5958/0974-360X.2021.00025.1