QUANTIFICATION OF LEVOFLOXACIN IN HUMAN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND THE IMPACT OF THE ANTICOAGULANTS TYPE

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1 Journal of International Research in Medical and Pharmaceutical Sciences 7(2): 54-62, 2016 ISSN: (P), ISSN: (O) International Knowledge Press QUANTIFICATION OF LEVOFLOXACIN IN HUMAN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND THE IMPACT OF THE ANTICOAGULANTS TYPE YAHDIANA HARAHAP 1*, AGUS AL IMAM BAHAUDIN 1, HARMITA 1 AND SANTI PURNA SARI 1 1 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: 26 th August 2015 Accepted: 15 th September 2015 Original Research Article Published: 11 th 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 Sunfire TM (250 x 4.6 mm), 5 µm; temperature of 45 C, mobile phase consisting of 0.5% triethylamine ph.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 ng/ml with r> 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), between citrate-edta plasma and heparin-edta plasma for low concentration and between citrate-heparin plasma and citrate-edta plasma for mid and high concentration. There was interference on retention time of less than 8 minutes in blank EDTA plasma, while on citrate and heparin plasma there was no interference. The method can be applied for pilot bioequivalence study using heparin as anticoagulant with six healthy subjects which were administered two 500 mg levofloxacin formulations. Keywords: Citrate; ciprofloxacin; EDTA; heparin; levofloxacin; HPLC-photodiode array. *Corresponding author: yahdiana0@yahoo.com, yahdiana14@gmail.com;

2 1. INTRODUCTION Levofloxacin is a broad spectrum antibiotic and is one of the compulsory drugs for bioequivalence testing, so that determination of its level in plasma is needed to be done for monitoring and ensuring its quality [1,2]. Plasma is the most used biology matrix in bio-analysis and when it is used in an analysis, one of important factors to be noticed is the types of anticoagulant used []. In some researches, the types of anticoagulant used are often different [4]. Anticoagulant is mostly used to avoid instant coagulation in handling the blood sample. Etilendiamintetraacetate (EDTA), heparin, and citrate are anticoagulants often used in drug analysis in human blood [,5]. Common type of anticoagulant that The Indonesia Red Cross used is citric phosphate dextrose adenine or CPD-A while in bioequivalence study usually uses EDTA and heparin as anticoagulant to isolate plasma from whole blood. Different anticoagulants in drug analysis can affect small molecule, metabolism profile, and clinical parameter of analyte and metabolite [5]. Some anticoagulants (generally EDTA and citrate) can cause suppression or increase formation of ion from drug and metabolite that can affect the biological matrix in its analysis [4]. There has been many developed analysis method to determine levofloxacin level in blood plasma [6-10]. During method development, different anticoagulants are often used, however there has been no research explaining about the effect of different anticoagulants to analysis and stability testing. It is mentioned in EMEA Guideline for Bio-analytical Method Validation [11], that if there is different anticoagulant used in method validation then partial validation is needed to be done. This study was performed to develop levofloxacin method analysis in human plasma and also observing the effect of anticoagulant types such as EDTA, citrate and heparin to parameters stability, recovery and the chromatogram profile to observe if there is any interferences. Full validation was performed using plasma with citrate anticoagulant while for heparin and EDTA, only partial validation was performed. The validated method will be applied to pilot bioequivalence study with six healthy subjects which were administered two 500 mg levofloxacin formulations. This research is aimed to evaluate the effect of different types of anticoagulant in levofloxacin analysis in citrate, heparin, and EDTA plasma, among other is stability in plasma, shape of chromatogram and peak response with its recovery from plasma. 2. EXPERIMENTAL 2.1 Chemicals and Reagents Levofloxacin and Ciprofloxacin HCl were obtained from USP, methanol and acetonitrile (HPLC grade), 85% phosphate acid (analytical grade), methanol (analytical grade), triethylamine (analytical grade) were manufactured by Merck (Darmstadt, Germany). Aquabidestilata from Ikapharmindo was used. Drug free control human plasma was obtained from Indonesia Red Cross (Jakarta, Indonesia). 2.2 HPLC Instrument and Condition High Performance Liquid Chromatography (Waters Alliance 2695) that consists of pump, autosampler, C18 column Sunfire TM (250 x 4.6 mm) 5 µm at temperature of 45 C, photodiode array detector (Waters 2996), degasser, and data processor. The mobile phase was a mixture of 0.5% triethylamine ph.0 - acetonitrile (88:12 v/v); flow rate of 1.25 ml/minute; and detected using photodiode array at 29 nm. Ciprofloxacin HCl was used as internal standard. 2. Calibration Standards and Quality Control Samples A stock solution of levofloxacin was prepared in methanol at 1.0 mg/ml and further diluted with methanol to obtain the following concentration 10 µg/ml, 5 µg/ml, 1 µg/ml, 0.5 µg/ml, 0.2 µg/ml, 0.1 µg/ml, and 0.05 µg/ml and Quality Control (QC) solutions at 7.5 µg/ml, 5 µg/ml and 0.15 µg/ml, respectively. All of the standard solutions were stored in refrigerator ( 8 C) until being used. The calibration curve standards and quality control samples were prepared by spiking them with levofloxacin and is working solutions. It is used for method validation. 2.4 Blood Sample Separation The anticoagulants being used was citrate as Citrate Phosphate Dextrose Adenine (CPD-A), heparin, and EDTA. Citrate plasma was obtained from Indonesia Red Cross, while heparin and EDTA plasma were obtained from blood sample separation that were taken from six healthy subjects. The blood sample were divided into two vacuum tubes in whereas in each tube there was already EDTA and Li-Heparin anticoagulant. The blood samples were then centrifuged at 000 rpm for 10 minutes at room temperature, afterward they were stored in a freezer at -20 C until the analysis was performed. 55

3 2.5 Sample Preparation A plasma sample (00 µl) was transferred to polypropylene tube, the 50 µl of internal standard working solution 100 µg/ml ciprofloxacin in methanol was added. The mixture was vortex mixed for minute and centrifuged at 10,000 rpm for 10 minutes. Afterward 20 µl of aliquot was injected and analyzed into HPLC. 2.6 Method Validation Method validation using different types of anticoagulant such as citrate, EDTA and heparin. Full validation was done using citrate plasma with parameters such as selectivity, carry-over, lower limit of quantitation (LLOQ), linearity of calibration curve, accuracy, precision, recovery, dilution integrity, and stability. Partial validation would be done to EDTA and heparin plasma thus stability test toward EDTA and heparin plasma was also conducted to identify the effect of anticoagulant type being used. Plasma samples were quantified using the ratio of the peak area of levofloxacin to that of IS as the assay parameter. For the calibration standards, peak area ratios were plotted against analyte plasma concentrations. A linear regression was used with a 1/x 2 weighting factor applied. The acceptance criterion for calibration curve was a correlation coefficient (r) of or better. Limit of quantification (LOQ) was defined as the lowest concentration at which the precision was expressed by relative standard deviation that is lower than 20% and inaccuracy (bias) was expressed by relative difference of the measurement and true value is within 20%. The method specificity was evaluated by screening six lots of blank plasma. Accuracy and precision were assessed by determination of QC samples with five replicates for four concentration levels on the same batch (intrabatch) and on different batch (inter-batch). The acceptance criteria for intra-and inter-batch precision was 15% or better, and the inaccuracy was within 15% or better while for LOQ was 20%. The absolute recovery of the levofloxacin and IS were determined by comparing the peak areas of extracted plasma standards to the peak areas of post extraction plasma blanks spiked at corresponding concentration. The stability of the analytes and IS in plasma were assessed by analyzing QC samples at two concentrations (low and high), respectively, in triplicate (n = ), under different temperature and timing conditions. The results were compared with those for freshly prepared QC samples, and the percentage concentration deviation was calculated. 2.7 Comparison of Citrate, Heparin, and EDTA as Anticoagulant to Analysis Levofloxacin The effect of anticoagulant type on levofloxacin analysis was evaluated by observing the shape of chromatogram from the plasma, recovery, area of levofloxacin in each plasma, and stability of levofloxacin in plasma was analyzed statistically. 2.8 Application the Method to Pilot Bioequivalence Study The method was applied for pilot bioequivalence study with six Indonesian healthy subjects. The blood was collected until 24 hours after administration two formulations of levofloxacin 500 mg. The next step was blood sampling on the following time sequence 0, 0.25, 0.5, 1, 1.5, 2, 2.5,, 4, 6, 8, 12, and 24 hours after drug administration using heparin as anticoagulant. Afterward the acquired plasma were separated by centrifugation and was stored at -20 C before analysis using High Performance Liquid Chromatography.. RESULTS.1 Specificity The chromatograms of blank plasma and blank plasma spiked with levofloxacin at LLOQ and IS which used citrate as anticoagulant were shown in Fig. 1. The retention times of levofloxacin and IS were 8.1 and 9.4 min, respectively and no significant interfering peak was observed around the levofloxacin and IS. Comparation blank plasma of three anticoagulants (citrate, heparin, and EDTA) were shown in Fig Carry-over The test did not show carry-over effect in blank samples after injecting high concentration standard. The results of carry-over test were shown in Table 1. Table 1. Carry-over of levofloxacin in blank plasma after injecting highest concentration standard Blank sample number Carry-over of analyte / levofloxacin Blank Blank Blank Blank Blank Mean Carry-over of internal standard 56

4 Fig. 1. The chromatograms of blank plasma and blank plasma spiked with levofloxacin at LLOQ and IS which used citrate as anticoagulant Fig. 2. Chromatogram blank plasma of citrate, heparin, and EDTA Carry-over value after high levofloxacin concentration injection was 2.70% from LLOQ respond, while % carry-over of internal standard was 0.05%. The percentage of carry-over was still suitable with the requirement for analyte of < 20%, while the internal standard was <5%.. Calibration Curve and LLOQ The calibration curves showed a good linearity in the concentration range of 50 10,0000 ng/ml with correlation coefficient (r2> 0.99) and LLOQ for levofloxacin was 50 ng/ml with CV and bias were 10.57% and 15.45%, respectively..4 Dilution Integrity The dilution effect was investigated to ensure that human plasma samples exceeding the highest concentration of the calibration range (10.0 µg/ml) could be diluted with blank human plasma and accurately quantified without losing reproducibility. In this test, standard solution was made with concentration above upper limit of quantification (15 µg/ml). Based on the result, the dilution fulfilled the requirement because the % diff and CV did not exceed 15%. The results of dilution integrity test were shown in Table 2..5 Precision and Accuracy The results of intra-batch and inter-batcand accuracy were shown in Table. Both precision precision values (RSD) were less than 12.11%.. Intra-batch and inter-batch accuracy ranged from -5.52% to 6.18% and -0.14% to 9.17%, respectively..6 Stability The storage stability of levofloxacin was evaluated to determine whether degradation occurred during long term storage. Stability was determined by analyzing QC samples stored at -20 C over a period of 14 and 1 days. The data indicated that levofloxacin was stable at -20 C for at least 1 days (Table 4). The analytes were also tested for freeze/thaw stability. The freeze/thaw samples were assayed and mean concentrations were compared to those values obtained for the intra-day analysis of QC samples. The results indicate that levofloxacin was stable after three freeze/thaw cycles (Table 5). 57

5 To evaluate autosampler stability of levofloxacin sample, two concentrations of low and high QCs in triplicate were maintained for 24 hours immediately after preparation at the autosampler temperature used during analysis. The results of autosampler stability were shown in Table 6. The stock solution stability of levofloxacin and IS were examined under room temperature condition for 6 hours, while the stock solution of levofloxacin and ciprofloxacin HCl (internal standard) were stored under temperature 8 C after 1 days storage. Short term stability test of levofloxacin in citrate plasma, the % diff value for QCL and QCH were -1.99% until 1.60% and -11.0% until 1.98%. The % diff in heparin plasma for QCL and QCH were 12.6% until 11.74% and -1.4% until 14.8%. The % diff value of EDTA plasma for QCL and QCH were -1.56% until 11.88% and % until 14.69%..7 Recovery Levofloxacin recovery test was also performed on citrate, heparin, and EDTA plasma. The absolute recovery value was acquired by comparing the analyte peak response in plasma with standard analyte solution with similar concentration (Table 7), while for modified recovery that used plasma matrix was acquired by comparing analyte peak response spiked to plasma after extraction process to extracted plasma analyte peak response (Table 8). In this research, levofloxacin absolute recovery value was 9.26% until 50.% in citrate plasma, 5.98% until 4.7% in heparin plasma, and 5.55% until 49.15% in EDTA plasma. The modified recovery value using plasma matrix was 80.5% until % in citrate plasma, 77.87% until 11.45% in heparin plasma and 69.44% until % in EDTA plasma..8 Comparison of Citrate, Heparin, and EDTA as Anticoagulant to Analysis of Levofloxacin Beside of chromatogram shape for each of anticoagulant, the effect of anticoagulant types were investigated by comparing value of recovery, peak area ratio, and stability of levofloxacin in each plasma which was analyzed statistically. The statistic results of comparison three anticoagulants were shown in Table 9. Table 2. Dilution integrity of the method for determining levofloxacin levofloxacin in human plasma Actual concentration Dilution factor Concentration measured (Mean±SD; ng/ml) Intra-batch RSD Bias Concentration measured (Mean±SD; ng/ml) Inter-batch RSD / ± ± / ± ± Table. Precision and accuracy of the method for determining levofloxacin concentrations in plasma samples using three anticoagulants Actual concentration Type of anticoagulant Intra-batch Concentration measured (Mean±SD; ng/ml) RSD Bias Inter-batch Concentration measured (Mean±SD; ng/ml) Citrate 47.14± ± Heparin 50.9± ± EDTA 54.71± ± Citrate 16.80± ± Heparin 14.5± ± EDTA ± ± Citrate ± ± Heparin 468.0± ± EDTA ± ± Citrate ± ± Heparin ± ± EDTA ± ± RSD Bias Bias 58

6 Table 4. Long term stability of levofloxacin in plasma using three anticoagulants Actual concentration Type of Number of 0 day 14 days 1 days anticoagulant samples Mean concentration Bias Mean concentration Bias Mean concentration Bias measured measured measured Citrate ± ± ± Heparin 14.60± ± ± EDTA ± ± ± Citrate ± ± ± Heparin ± ± ± EDTA ± ± ± Table 5. Freeze/thaw stability of levofloxacin in plasma using three anticoagulants Actual concentration Type of anticoagulant Number of samples rd cycle Mean concentration measured Bias Citrate 162.2± Heparin ± EDTA 17.86± Citrate ± Heparin ± EDTA ± Table 6. Autosampler stability of levofloxacin in plasma using three anticoagulants Actual concentration Type of anticoagulant Number of 0 hour 24 hours samples Mean concentration Bias Mean concentration measured Bias measured Citrate ± ± Heparin 14.60± ± EDTA ± ± Citrate ± ± Heparin ± ± EDTA ± ±

7 .9 Application to Pilot Bioequivalence Study of two Formulations of 500 mg Levofloxacin The method has been successfully applied for pilot bioequivalence study with six Indonesian healthy subjects. 4. DISCUSSION This research uses types of anticoagulant such as citrate, heparin, and EDTA to isolate plasma from whole blood. The full validation was performed upon citrate plasma that was spiked with levofloxacin and ciprofloxacin as internal standardd while partial validation was performed on the other anticoagulant. Extraction of levofloxacin from plasma was done by using protein precipitation method d because it is simple, relatively easy, and extracted quite well. The used protein precipitation was methanol because resulting good separation and extraction. Extraction process of levofloxacin in plasma iss acquired with best condition by using methanol (1:1.5 v/v). Fig.. Pharmacokinetic profile of 6 healthy subjects after administration of two formulation of 500mg levofloxacin Table 7. Absolute recovery of determining levofloxacin in human plasma using three anticoagulants Concentration added Type of anticoagulant Number of samples Absolute recovery (Mean±SD; %) Citrate Heparin EDTA 4.7± ± ± Citrate Heparin EDTA 42.24± ± ±.91 RSD Table 8. Modified recovery of determining levofloxacin in human plasma using three anticoagulants Concentration added Type of anticoagulant Citrate Heparin EDTA Citrate Heparin EDTA Number of Modified recovery samples (Mean±SD; %) 85.02± ± ± ± ± ±8.8 RSD

8 Table 9. Comparison of citrate, heparin, and EDTA as anticoagulant to analysis of levofloxacin Parameters Type of anticoagulant Differences* P value Citrate Heparin EDTA Absolute recovery (Mean±SD; %) QCL 4.7± ± ±2.47 No significant differences QCH 42.24± ± ±.91 No significant differences Modified recovery (Mean ± SD; %) QCL 85.02± ± ±7.87 No significant differences QCH ± ± ±8.8 No significant differences 0.65 Peak area ratio (Mean±SD) QCL ± ± ± Citrate to EDTA; Heparin to EDTA QCM ± ± ± Citrate to Heparin; Citrate to EDTA QCH ± ± ± Citrate to Heparin; Citrate to EDTA Long-term stablity (-20 C) QCL At least 1 days At least 1 days At least 1 days No difference - QCH At least 1 days At least 1 days At least 1 days No difference - Freeze-thaw stability ( cycles) QCL At least cycles At least cycles At least cycles No difference - QCH At least cycles At least cycles At least cycles No difference - Autosampler stability QCL At least 24 hours At least 24 hours At least 24 hours No difference - QCH At least 24 hours At least 24 hours At least 24 hours No difference - *Comparison among anticoagulants were obtained by ANOVA (LSD Post Hoc test) and Kruskal Wallis test. P value > 0.05 showed that there was no significant differences, P value < 0.05 showed significant differences among each parameter. 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. 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 freeze-thaw cycles, 24 hours in room temperature, 24 hours on auto sampler, and 1 days at -20 C. 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. 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 % with CI 90%. 5. CONCLUSION The method has been validated and can be used to analyze levofloxacin with citrate, heparin, and EDTA 61

9 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. CONSENT All authors declare that written informed consent was obtained from the patient for publication of this case report and accompanying images. ETHICAL APPROVAL All authors hereby declare that all experiments have been examined and approved by the Ethics Committee of the Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia, and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. DISCLAIMER This manuscript was presented in the conference OMICS international 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques available link is /2015/yahdiana-harahap-universities-indonesiaindonesia September 01-0, 2015 Valencia, Spain COMPETING INTERESTS Authors have declared that no competing interests exist. REFERENCES 1. Badan Pengawas Obat dan Makanan Republik Indonesia. Kepala Badan Pengawas Obat dan Makanan Republik Indonesia Nomor HK Tentang Obat Wajib Uji Ekivalensi. Jakarta; Shargel L, Wu-Pong S, Yu ABC. Applied biopharmaceutics and pharmacokinetics, 5 th Edition, United States of America: Appeton & Lange; Bowen RAR, Hortin GL, Csako G, Otañez OH, Remaley AT. Impact of blood collection devices on clinical chemistry assays. Clin. Biochem. 2010;4(1-2): Barri T, Dragsted LO. UPLC-ESI-QTOF/MS and multivariate data analysis for blood plasma and serum metabolomics: Effect of experimental artefacts and anticoagulant. Anal. Chim. Acta. 201;768: Gonzalez-Covarrubias V, Dane A, Hankemeier T, Vreeken RJ. The influence of citrate, EDTA, and heparin anticoagulants to human plasma LC MS lipidomic profiling. Metabolomics. 2012;9(2): Kumar TM, Srikanth G, Rao JV, Rao S. Development and validation of HPLC-UV method for the estimation of levofloxacin in human plasma. Int. J. Pharm. and Pharmaceut. Sci. 2011;(2): Liang H, Kays MB, Sowinski KM. Trovafloxacin and cinoxacin by highperformance liquid chromatography: Application to levofloxacin determination in human plasma. J. Chromatogr. B. 2002;772: Sousa J, Alves G, Fortuna A, Falcão A. First liquid chromatography method for the simultaneous determination of levofloxacin, pazufloxacin, gatifloxacin, moxifloxacin and trovafloxacin in human plasma. J. Chromatogr. B. 201;90: Watabe S, Yokoyama Y, Nakazawa K, Shinozaki K, Hiraoka R, Takeshita K, Suzuki Y. Simultaneous measurement of pazufloxacin, ciprofloxacin, and levofloxacin in human serum by high-performance liquid chromatography with fluorescence detection. J. Chromatogr. B. 2010;878(19): Zhang C, Nightingale CH, Nicolau DP. Development and optimization of a reversed phase high performance liquid chromatographic method for the determination of levofloxacin in growth media, human bal fluid, alveolar macrophages and plasma. J. Infect. Dis. Pharmacother. 2004;6(4): European Medicines Agency. Guideline on Bioanalytical Method Validation: An agency of the European Union. London; Copyright International Knowledge Press. All rights reserved. 62