Sensitive inexpensive chromatographic determination of an antimicrobial combination in human plasma and its pharmacokinetic application

Sensitive inexpensive chromatographic determination of an antimicrobial combination in human plasma and its pharmacokinetic application

Journal of Chromatography B 1097–1098 (2018) 94–100 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.else...

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Journal of Chromatography B 1097–1098 (2018) 94–100

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb

Sensitive inexpensive chromatographic determination of an antimicrobial combination in human plasma and its pharmacokinetic application

T



Eman I. El-Kimarya, , Essam F. Khamisa, Saeid F. Belala, Mona M. Abdel Moneimb a b

Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, University of Alexandria, Khartoum Square, El-Messalah, Alexandria 21521, Egypt Faculty of Pharmacy and Drug Manufacturing, Department of Pharmaceutical Chemistry, Pharos University in Alexandria, Alexandria, Egypt

A R T I C LE I N FO

A B S T R A C T

Keywords: Antimicrobial agents HPLC-DAD Human plasma Pharmacokinetics Ciprofloxacin Tinidazole

This study represents simple inexpensive chromatographic determination of ciprofloxacin (CIP) and tinidazole (TIN) simultaneously in human plasma using HPLC-DAD followed by a pharmacokinetic application. C18 column was used as stationary phase with isocratic elution of a mobile phase composed of acetic acid solution (2%) and acetonitrile (85: 15, v/v) and ornidazole as internal standard (IS) with UV detection at 318 nm. The two drugs and the IS were separated at 6.55, 7.91 and 11.07 min for CIP, TIN and IS, respectively, with good selectivity and sensitivity for their analysis in presence of plasma matrix components and the drugs' metabolites. Sample preparation involved only protein precipitation without any complicated extraction procedures decreasing analysis time. For method validation, FDA regulations for analysis in biological fluids were followed. Pharmacokinetic (PK) study on six healthy volunteers was conducted after single oral dose administration of 500 and 600 mg of CIP and TIN, respectively. Dugs' plasma levels were followed for 12 or 72 h post dosing for CIP and TIN, respectively, and different PK data for the two drugs were calculated and they were comparable to the reported values demonstrating successful future application of the presented method in PK, bioequivalence and bioavailability studies.

1. Introduction

bacteria. The literature survey revealed that spectrophotometry [13], HPLC [14,15] and UPLC [16] methods have been reported for their analysis in combined dosage forms. However, the literature survey shows that there are no reported methods for simultaneous analysis of CIP and TIN in real human plasma. Monitoring plasma concentration of antibacterial drugs avoids either sub-therapeutic or excessive antibiotic levels thus avoiding toxicity and warranting efficacy. Therefore, the target of the present work is the development of an HPLC method for the in-vivo analysis of CIP and TIN binary mixture in human plasma and checking the applicability of this method in conducting a pharmacokinetic study for the studied drugs after one oral dose administration of their combined pharmaceutical formulation to healthy human volunteers. For high throughput analysis which is an important requirement for a successful pharmacokinetic study, the proposed method involved very simple sample treatment (protein precipitation) and optimized chromatographic separation using only 15 min analysis time. The proposed method analyzed simultaneously the two drugs with the internal standard ornidazole (ORI), in presence of plasma interferences and the drugs metabolites at a single wavelength (318 nm). The method was also validated in accordance to the FDA [17].

Ciprofloxacin (CIP), 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1yl)-1,4-dihydroquinoline-3-carboxylic acid, (Fig. 1a) is a fluoroquinolone-type antibiotic agent acting against many bacteria [1,2]. There are many works published regarding HPLC determination of CIP in biological fluids. Some of the reported methods about CIP quantitation in human plasma used switching devices [3], tedious extraction methods [4] and derivatization methods [5]. Meanwhile, many articles show the determination of CIP in human plasma after simple protein precipitation treatment [6–9]. Tinidazole (TIN), 1-[2-(Ethylsulfonyl) ethyl]-2-methyl-5-nitro-1Himidazole (Fig. 1b) is a second-generation member of the 5-nitroimidazole group. It is used in Europe widely and in developing countries as well acting against bacteria and protozoa [1,2]. The literature survey revealed that several analytical methods were reported for TIN assay in human plasma by HPLC using protein precipitation treatment with 70% perchloric acid [10], liquid/solid extraction method [11] and liquid/liquid extraction method [12]. Meanwhile, CIP and TIN combination is marketed in several countries with the benefit of effectiveness against both protozoa and



Corresponding author. E-mail address: [email protected] (E.I. El-Kimary).

https://doi.org/10.1016/j.jchromb.2018.09.008 Received 16 July 2018; Received in revised form 27 August 2018; Accepted 7 September 2018 Available online 07 September 2018 1570-0232/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. Chemical structures of (a) ciprofloxacin and (b) tinidazole.

TIN standard solution into two separate 25-mL volumetric flasks and then diluted to volume using mobile phase to get working solutions of 10 and 100 μg·mL−1, respectively. Also, ORI, the internal standard, solution was prepared by transferring accurate weight of 12.5 mg of ORI drug substance into a 25-mL volumetric flask, then dissolved in and diluted to volume using double distilled water to get standard solution of 500 μg·mL−1. The stock solutions of all drugs were stored in light protected vessels at 4 °C for at least 14 days with no changes.

2. Experimental 2.1. Instrumentation and chromatographic conditions An Agilent 1200 series (Santa Clara, USA), HPLC-DAD system was used with diode array detector which is connected to a computer with Agilent Chem Station Software. The HPLC system is supplied with automatic injector, quaternary pump and vacuum degasser. The separations were done on a reversed phase Agilent Zorbax SB-C18 (250 × 4.6 mm) column at 25 °C. Isocratic elution using acetic acid (2%) and acetonitrile (85: 15, v/v) was used. The mobile phase filtration was done using a 0.45 μm membrane filter and 1 mL.min−1 flow rate and 20 μL injection volumes were applied all over the runs. The chromatograms were extracted at 318 nm.

2.4. Calibration and quality control (QC) standards A 500 μL control human plasma in 5 mL centrifuge tubes was spiked with accurate volumes of 5–1000 μL of CIP working solution and 10 μL and 5–200 μL of 10 and 100 μg·mL−1 TIN working solutions, respectively, to achieve concentrations in the range of 0.1–20.0 and 0.2–40.0 μg·mL−1 plasma for CIP and TIN, respectively. Five microliters of ORI (IS) standard solution was added to each centrifuge tube followed by 750 μL of acetonitrile using a micropipette. The tubes were vortex mixed for 3 min. The samples were centrifuged for 15 min at 40,000 rpm. The supernatant was poured into another 5 mL centrifuge tubes and evaporated to dryness using a Christ rotational vacuum concentrator. The dried residue was reconstituted with 1000 μL mobile phase, vortex mixed for 3 min and re-centrifuged at 40,000 rpm for 5 min. Initially, the supernatant layer was filtered by a 0.45 μm syringe adapter before injection into the HPLC but this caused great loss of CIP peak which is in compliance to CIP assay previous report [6]. Thus, a second centrifugation step was used before injection into the HPLC system to obtain a clearer supernatant layer and 20 μL volume of it was injected in triplicate and chromatographed with the previously stated conditions in Section 2.1. The ratios of analyte to IS peak areas versus the corresponding drug concentrations were used for calibration graphs construction. Calibration standards were prepared at 0.1, 0.3, 0.4, 1, 4 and 20 μg·mL−1 plasma for CIP and 0.2, 1, 2, 5, 30 and 40 μg·mL−1 plasma for TIN. As per FDA guidelines [17] for selecting QCs, for accuracy and precision studies, QCs were prepared as six replicates at 4 concentration levels, including lower limit of quantitation (LLOQ), low (L: defined as three times the LLOQ), mid (M: defined as mid-range), and high (H: defined as high-range). While for other runs (during analysis of volunteers' samples), duplicates at only 3 concentration levels (LQC, MQC and HQC) were used. For CIP, QCs were prepared at 0.1, 0.3, 4 and 20 μg·mL−1 for LLOQ, LQC, MQC and HQC, respectively, while for TIN, 0.2 (LLOQ), 0.6 (LQC), 5 (MQC) and 40 μg·mL−1 (HQC) were prepared.

2.2. Materials and reagents Pharmaceutical grades of CIP, TIN and ORI were kindly supplied by Pharco Pharmaceuticals (El Amriya, Alexandria, Egypt), Medical Union Pharmaceuticals-MUP (Abou Sultan – Ismailia, Egypt) and Pharaonia Pharmaceuticals (New Borg El-Arab City, Alexandria, Egypt), respectively, and certified to contain 99.99%. 99.95% and 99.98%, respectively. HPLC-grade acetonitrile and acetic acid (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) were used. High purity double distilled water was also used in the whole work. Pooled drug-free human plasma was obtained from the Alexandria blood bank. Tinifloxacin® capsules (labeled to contain 500 and 600 mg of CIP and TIN, respectively, per capsule) was purchased from the commercial market and is manufactured by Organo Pharma MS, El-Obour Industrial Zone, Egypt. 2.3. Stock solutions A 1000 μg·mL−1 CIP stock solution was initially prepared in methanol with a minimum amount of HCl that allows CIP base to dissolve. During analysis, 2 peaks were noted for CIP. In response to this, double distilled water again with the least amount of HCl was used to prepare CIP stock solution. When the solution was analyzed, peak shouldering was still observed in the chromatogram. For this reason, the mobile phase was chosen to dissolve CIP which gave a very good symmetrical peak for CIP. Standard solutions of CIP and TIN were prepared by transferring accurate weights of 25 mg of each drug substance separately into two 25-mL volumetric flasks, then dissolved in and diluted to volume using mobile phase and methanol for standard solutions of CIP and TIN, respectively, to obtain standard solutions of 1000 μg·mL−1. CIP standard solution of 1000 μg·mL−1 was further diluted by transferring an accurate volume of 0.25 mL of CIP standard solution into a 25-mL volumetric flask and diluted to volume using mobile phase to get a working solution of 10 μg·mL−1. Meanwhile, TIN standard solution of 1000 μg·mL−1 was further diluted to get two working solutions by transferring separately two accurate volumes of 0.25 and 2.5 mL of

2.5. Pharmacokinetic study 2.5.1. Selection of subjects Before conducting the study, ethical approval has been granted from the “Clinical studies ethics committee”, Faculty of pharmacy, 95

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For optimization of the stationary phase, the separation was tried on various columns such as C8 (250 × 4.6 mm), C18 (150 × 4.6 mm), and C18 (250 × 4.6 mm) to achieve the highest sensitivity and best peak shape with short run time. The Agilent C18 (250 × 4.6 mm) column became the column of choice giving good symmetrical peaks shape and response for both the analytes and IS within reasonable retention times. Several mobile phases were tried to reach the optimum separation between the drugs. Both acetonitrile and methanol were attempted as organic modifiers. Acetonitrile was considered better as it eluted CIP much earlier than methanol giving a sharp peak. Then, different ratios of acetonitrile were tried. It was noticed that increasing the acetonitrile ratio caused CIP peak to appear much earlier interfering with plasma peaks and loss of resolution between TIN and ORI (IS) occurred. However, at lower concentrations of acetonitrile, separation was better till the best separation within reasonable analysis time was achieved using 15% acetonitrile. Regarding the aqueous part of the mobile phase, phosphate buffer with different pH and acetic acid aqueous solution (2%) were tried but the latter gave better peak shape and symmetry for CIP. The mobile phase composed of acetic acid (2%) and acetonitrile was the most suitable for faster elution, better reproducibility and peak shape. The amphoteric character of CIP as a fluoroquinolone also has an impact on its separation on a reversed phase column. Thus, careful pH adjustment or ion-pairing reagents offered solutions in previous reports [7]. However, in this work, a simple mobile phase of only two solvents (acetic acid solution (2%) and acetonitrile) without using any buffered solution or ion-pairing reagent was used emphasizing the simplicity of the developed method. In our study, 318 nm was found satisfactory for detection of both analytes with good sensitivity and excellent reproducible results without interference of plasma matrix (Fig. 2). Different internal standards were tried including ofloxacin, metronidazole and ornidazole but good UV absorption at 318 nm and similar chemical behavior were only achieved using ornidazole (ORI) as an internal standard. Using the optimized chromatographic conditions, CIP, TIN and ORI (IS) were resolved at retention times of 6.55 ± 0.15, 7.91 ± 0.35 and 11.07 ± 0.20 min for CIP, TIN and ORI (IS), respectively, which allowed their determination in human plasma. Fig. 2 shows HPLC chromatogram for the separation of CIP and TIN, with the ORI (IS) spiked in human plasma. System suitability parameters of the optimized HPLC system were checked and were found to be within the acceptable ranges as repoted by the FDA guidance on validation of chromatographic methods [21,22] which ascertain the suitability and effectiveness of the operating system. The satisfactory method performance data obtained are shown in Table 1 and they indicate good specificity of the proposed HPLC method for the analysis of CIP and TIN in plasma samples.

Alexandria University. Based on acceptable medical histories, six healthy (confirmed by physical and medical exanimation) male subjects aged 25–35 years and their bodyweights in the range of 67–82 kg were selected. Other acceptance criteria include absence of hepatic, renal or other disorders that require long term use of medications. 2.5.2. Dosage scheduling and samples The volunteers' meals on the evenings before drug administration and on days of administration were standardized. The volunteers were forbidden to administrate other drugs throughout the study. Caffeine or smoking or alcohol is prohibited 48 h prior to, and throughout the study. Each volunteer fasted for 12 h before administration of the Tinifloxacin® capsule and also for 4 h after administration of Tinifloxacin® capsule to prevent any interference from any food or drinks on the pharmacokinetic profile of each drug. In the morning, all volunteers administrated orally a single dose of Tinifloxacin® capsule containing 500 and 600 mg of CIP and TIN, respectively, with 250 mL water. Five mL blood samples were taken from the volunteers, into ethylene diamine tetra-acetic acid (EDTA) anticoagulant blood-drawing tubes at the scheduled times. After collection, the blood was centrifuged to plasma and conserved at −20 °C. All collected plasma samples were thawed at ambient temperature prior to analysis. CIP and TIN plasma concentrations were monitored over a 72 h period. Thus, the collecting times were 0, 0.5, 1, 1.5, 2, 3, 4, 8, 12, 24, 48 and 72 h after a single capsule containing 500 and 600 mg of CIP and TIN, respectively was orally administrated to the volunteers. 2.5.3. Analysis and in-study validation A 500 μL aliquot of each plasma sample was dispensed into 5 mL centrifuge tubes, then a 5 μL volume of IS standard solution and a 750 μL of acetonitrile were added to each tube using a micropipette. Then the samples were processed as described earlier in Section 2.4. As per FDA guidelines [17], calibration standards and duplicates of QC samples at three concentration levels (LQC, MQC and HQC) should be included and analyzed each run during the analysis of volunteers' samples to ensure good method performance in each run. The run acceptance criteria include that at least 2/3 of the QCs show recoveries from 85 to 115% of their nominal values with at least one sample at each concentration level meeting these criteria. 2.5.4. Statistical analysis and pharmacokinetic data Time of maximum plasma concentration (Cmax) is (Tmax). The elimination rate constant (kel) was calculated by Ln-linear regression of terminal segment of plasma concentration versus time curve. Optimal regression fit was calculated with the three last concentrations as the period with highest possible coefficient of correlation. The -ve value of the slope of the fitted linear regression line represents kel and Ln 2/kel denoting the terminal elimination half-life (t1/2). AUC0–∞ was calculated using the linear trapezoidal rule starting 0 h after dose to time of the last measured concentration, plus the quotient of last measured concentration divided by kel. The apparent oral clearance (CL/F) was measured as quotient of dose to AUC0–∞ while the apparent volume of distribution (VL/F) was assessed as quotient of apparent oral clearance divided by kel [18–20].

3.2. Sample pretreatment and extraction procedure The extraction procedure described was chosen after extensive investigation of suitable methods for the extraction of CIP and TIN from human plasma. As shown in previous reports, analysis of fluoroquinolones such as CIP is troublesome due to their amphoteric character. Thus, protein precipitation is described in most of the reported methods for CIP analysis as no need for pH adjustment [7]. In our study, both methanol and acetonitrile were tested as precipitating agent; acetonitrile was superior giving clearer supernatant. Evaporation of the supernatant to dryness and reconstitution of the residue with mobile phase was necessary to significantly improve the chromatography of CIP peak, and correct any distortions in it if injected directly without the evaporation step and allowed the sensitivity to be increased significantly.

3. Results and discussion 3.1. HPLC method optimization The work aimed to develop a sensitive and high-throughput HPLCDAD method for simple, rapid and reliable simultaneous determination of CIP binary mixture with TIN in human plasma. Several method parameters were evaluated to optimize the chromatographic separation.

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Fig. 2. HPLC chromatogram of 20 μL injection volume of (a) blank human plasma, (b) human plasma spiked with LLOQ of CIP and TIN with ORI (IS) (c) human plasma spiked with 10 and 20 μg·mL−1 plasma of CIP and TIN, respectively, with ORI (IS) and (d) human plasma 3 h after oral administration of one capsule of Tinifloxacin® containing 500 and 600 mg CIP and TIN, respectively.

high F-values and small significance F-values indicating strong correlation between the peak area ratios and each drug concentrations and confirming good linearity of the calibration graphs. Table 2 presents all the statistical parameters and performance data. An important parameter that indicates random error of estimated “y” value is the standard deviation about regression, Sy/x. The small value of “Sy/x ” indicates the closeness of the points to the regression line [23,24].

3.3. Method validation Validation was performed per the FDA regulations [17]. 3.3.1. Linearity The six-point calibration curve was linear over the concentration ranges of 0.1–20 and 0.2–40 μg·mL−1 plasma for CIP and TIN, respectively, with correlation coefficient values higher than 0.999, 97

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Table 1 HPLC system suitability parameters. Analyte

Retention time (Rt), min

Capacity factor (k′)

Selectivity (α)

Resolution (Rs)

Asymmetry (Af)

Efficiency (plates/m)

CIP TIN ORI

6.55 7.91 11.07

2.02 2.65 4.10

1.31 1.55 –

4.99 10.96 –

1.11 1.01 1.12

8185 15,299 19,052

System suitability recommendations: k′ (2−10), α > 1, Rs > 2, Af (0.8–1.2) and plates·m−1 (> 2000).

components of plasma. The human plasma was also spiked with the LLOQ concentrations of the drugs with IS to confirm the selectivity of the method (Fig. 2). Moreover, matrix effect was investigated by spiking blank samples (from six different sources) with the two drugs at their low and high QC levels along with the IS. The peak area ratio of the spiked drugs in presence of matrix was compared with those acquired from the same drug concentrations in standard solutions. The obtained RSD% was lower than 9% for both CIP and TIN showing lack of matrix interference. Spectra of CIP, TIN and IS of both spiked plasma and plasma obtained from volunteers after their co-administration recorded at different time intervals across their peaks were overlapped confirming peak purity (Fig. 3). Also, the purity angle was within the purity threshold limit in all samples confirming the absence of any interference.

Table 2 Characteristic parameters for the regression equations of the proposed HPLCDAD method for the determination of CIP and TIN in human plasma. Parameter

HPLC (measured at 318 nm)

Linearity range, μg·mL LOQ, μg·mL−1 LOD, μg·mL−1 Intercept Slope Correlation coefficient Sa Sb Sy/x F Significance F

−1

CIP

TIN

0.1–20.0 0.10 0.03 −3.20 × 10−2 0.25 0.9993 3.27 × 10−2 4.24 × 10−3 7.61 × 10−2 3612.73 2.41 × 10−8

0.20–40.00 0.03 0.01 −0.13 0.27 0.9991 9.49 × 10−2 4.99 × 10−3 0.20 2883.69 4.23 × 10−8

LOQ is limit of quantitation, LOD is limit of detection, Sa is standard deviation of intercept, Sb is standard deviation of slope and Sy/x is standard deviation of residuals.

3.3.4. Stability studies Stability tests were performed to measure drugs' stability in plasma samples using LQC and HQC (n = 6) under different sample analysis conditions. Table 4 summarizes the conditions for long-term, shortterm, post-preparative and freeze-thaw stability studies (Table 4). The results of the stability studies yield RSD% not exceeding 4% and Er% not exceeding 10% indicating good stability of the two drugs under all the studies conditions.

3.3.2. Accuracy, precision and recovery Six replicates of standards at four concentrations were used to assess the accuracy and inter-day precision within three different days and intra-day precision within the same day. Table 3 summarizes the accuracy and intra and inter-day precision results. The recovery of the drugs spiked to plasma with LQC and HQC concentrations was calculated compared to the same concentrations without extraction. Mean extraction recoveries of CIP were found to be 96.90 ± 1.50% and 97.25 ± 1.00% while for TIN were 98.25 ± 0.99% and 99.80 ± 1.90%, respectively, at LQC and HQC concentrations of each drug. The extraction recovery of the IS was 98.89 ± 1.73%. These results confirm successful extraction of the drugs and also the internal standard from human plasma after the simple sample pretreatment step used.

3.4. Pharmacokinetic study The proposed method was applied for the determination of CIP and TIN plasma concentrations after a single oral dose of Tinifloxacin® capsule containing 500 and 600 mg of CIP and TIN, respectively, per capsule up to 12 h for CIP and 72 h for TIN post dosing. Fig. 2d shows the chromatogram of plasma sample obtained from a volunteer 3 h after oral administration of one Tinifloxacin® capsule. Following oral administration, CIP shows oral bioavailability of 70–80%. Its plasma protein binding ranges from 20 to 40%. Plasma half-life of CIP is 3–5 h, with about 50–70% of the dose excreted in the urine as the intact drug and 15% as metabolites following hepatic metabolism [25]. Meanwhile, TIN is rapidly and almost completely

3.3.3. Selectivity Six control human plasma samples of different sources were chromatographed to ensure the lack of interference from endogenous

Table 3 Evaluation of intra-day and inter-day precision and accuracy of the proposed HPLC method for the determination of CIP and TIN in human plasma (n = 6). Intra-day

Inter-day

Level

Theoretical concentration (μg·mL−1)

Mean % recovery ± SD

RSD %a

Er %b

Mean % recovery ± SD

RSD%a

Er %b

CIP LLOQ LQC MQC HQC

0.1 0.3 4.0 20.0

104.00 101.33 100.50 100.20

± ± ± ±

1.85 1.20 1.05 0.99

1.78 1.18 1.04 0.99

4.00 1.33 0.50 0.20

110.00 ± 1.98 102.00 ± 1.50 105.75 ± 1.57 99.84 ± 1.00

1.80 1.47 1.48 1.00

10.00 2.00 5.75 −0.16

TIN LLOQ LQC MQC HQC

0.2 0.6 5.0 40.0

104.00 101.00 100.04 100.94

± ± ± ±

0.98 0.50 1.05 1.06

0.94 0.50 1.05 1.05

4.00 1.00 0.04 0.94

110.00 104.00 100.24 102.21

0.99 0.93 1.55 1.37

10.00 4.00 0.24 2.21

a b

Percentage relative standard deviation. Percentage relative error. 98

± ± ± ±

1.09 0.97 1.55 1.40

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Fig. 3. Spectra illustrating peak purity of (a) CIP, (b) TIN and (c) ORI (IS); each is obtained from spiked plasma (1) or from human plasma of a volunteer after single dose administration of CIP and TIN combined pharmaceutical preparation (2) using the proposed HPLC-DAD method.

Cmax is in the range of 1.09–2.4 μg·mL−1 and 10–13.89 μg·mL−1 for CIP [2,8,9,25] and TIN [2,12,25], respectively. Regarding the terminal elimination half-lives (t1/2), the calculated values were found to be 3.17 ± 0.29 and 15.94 ± 2.61 for CIP and TIN, respectively, which met the reported (t1/2) for CIP and TIN [2,25]. These findings showed that pharmacokinetic parameters of the drugs were in good accordance with the reported values following their individual administration confirming that their co-administration had no effect on their individual pharmacokinetics.

absorbed following oral administration. Its plasma protein binding is about 12%. The plasma elimination half-life of TIN ranged from 12 to 17 h. It is excreted by liver and kidneys with 20–25% of the dose excreted in urine, unchanged, and about 12% in the feces [25]. The aim of our study was to monitor possible pharmacokinetic interaction between CIP and TIN following their co-administration. Fig. 4 shows the mean plasma concentration–time curves of CIP and TIN after their co-administration. The obtained plasma concentration-time curve of each drug was used for pharmacokinetic parameters assessment and they are shown in Table 5. As presented in Table 5, the time for CIP and TIN to reach maximum concentration was 1.33 ± 0.29 and 1.67 ± 0.58 h, respectively, which agree with the previously published observations reporting Tmax to be in the range of 0.5–2 h and 1.5–3 h for CIP [2,8,9,18] and TIN [2,12,18], respectively. Also, the maximum plasma levels of CIP and TIN (Cmax) were determined to be 2.25 ± 0.87 and 13.13 ± 3.05 μg·mL−1, respectively. These values were also in good accordance with previous publications reporting that

4. Conclusion A simple high throughput HPLC-DAD method was successfully developed for the simultaneous analysis of CIP/TIN binary mixture in human plasma. The method involves simple mobile phase without buffer solutions or ion-pairing reagents consisting of only two solvents, aqueous acetic acid and acetonitrile. It also requires a very simple

Table 4 Stability summary of CIP and TIN in human plasma (n = 6). Stability

Short-term room temperature (6 h) Freeze-thaw (3 cycles, at −70 °C) Post preparative 5 °C for 24 h Long-term 45 days at −70 °C

a b c

CIP

TIN

QCa samples

Mean % Recovery ± SD

RSD%b

E r% c

Mean % Recovery ± SD

RSD %b

Er%c

LQC HQC LQC HQC LQC HQC LQC HQC

100.67 ± 1.80 100.92 ± 1.60 98.33 ± 2.33 96.26 ± 1.93 104.67 ± 1.61 108.10 ± 1.95 96.00 ± 1.42 100.14 ± 1.95

1.79 1.59 2.37 2.00 1.54 1.80 1.48 1.95

0.67 0.92 −1.67 −3.74 4.67 8.10 −4.00 0.14

100.40 ± 2.04 102.29 ± 1.24 99.40 ± 2.66 92.70 ± 1.89 101.40 ± 1.23 94.57 ± 1.47 90.00 ± 2.08 91.13 ± 2.79

2.03 1.21 2.68 2.04 1.21 1.55 2.31 3.06

0.40 2.29 −0.60 −7.30 1.40 −5.43 −10.00 −8.87

LQC and HQC samples contain 0.3 and 20 μg·mL−1 for CIP or 0.6 and 40 μg·mL−1 of TIN, respectively. Percentage relative standard deviation. Percentage relative error. 99

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Fig. 4. Plasma concentration–time curves of (a) CIP and (b) TIN following oral administration of a single oral dose of Tinifloxacin® capsules containing 500 and 600 mg of CIP and TIN, respectively, to six healthy male volunteers. Table 5 Pharmacokinetic parameters of CIP and TIN following single oral dose administration of Tinifloxacin® capsule containing 500 and 600 mg of CIP and TIN, respectively, to 6 healthy male volunteers. Pharmacokinetic parameters −1

Cmax (μg·mL ) Tmax (h) AUC0–∞ [mg·h·L−1] AUC0–12h [mg·h·L−1] AUC0–72h [mg·h·L−1] kel (h−1) t½ (h) CL/F (L·h−1) Vd/F (L)

CIP

TIN

2.25 ± 0.87 1.33 ± 0.29 13.06 ± 2.57 12.02 ± 2.67 – 0.22 ± 0.02 3.17 ± 0.29 39.19 ± 6.93 180.89 ± 45.97

13.13 ± 3.05 1.67 ± 0.58 360.28 ± 73.74 – 341.30 ± 62.02 0.044 ± 0.01 15.94 ± 2.61 1.71 ± 0.31 38.54 ± 2.86

sample preparation without the need for pretreatment or solid-phase extraction step and without interference from plasma's endogenous components or drugs' metabolites. The method also demonstrates high throughput capability because of the short time required for analysis. Validation results show that the method can be utilized in pharmacokinetic studies with the desired sensitivity, precision and accuracy. References [1] The British Pharmacopeia, Her Majesty's Stationery Office, London, 2016. [2] S.C. Sweetman, Martindale, The Complete Drug Reference, Thirty-eighth edition, The Pharmaceutical Press, London, UK, 2014.

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