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Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 37–46

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journa l homepage: www.e lsev ier .com/ locate / jpba

apid, sensitive and selective HPLC–MS/MS method for theuantification of topically applied besifloxacin in rabbit plasma andcular tissues: Application to a pharmacokinetic study

iao-Fei Gu a,1, Bai-Yang Mao b,1, Min Xia c, Yang Yang c, Jia-Li Zhang c, Da-Song Yang c,ei-Xin Wu c, Ying-Xiang Du a,d, Bin Di c,d,∗, Meng-Xiang Su c,d,∗

Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, PR ChinaChangzhou Yabang Pharmacy Research Institute Company Ltd., Changzhou 213163, PR ChinaDepartment of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR ChinaKey Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, PR China

r t i c l e i n f o

rticle history:eceived 4 June 2015eceived in revised form 14 August 2015ccepted 19 August 2015vailable online 22 August 2015

eywords:esifloxacinluoroquinoloneC–MS/MS

a b s t r a c t

Besifloxacin is a fourth-generation broad-spectrum fluoroquinolone registered for the topical treatmentof bacterial conjunctivitis. In this study, a rapid, sensitive and selective liquid chromatography tan-dem mass spectrometry (LC–MS/MS) method was developed for quantification of besifloxacin in rabbitplasma and ocular tissues using nateglinide as the internal standard (IS). The analyte and IS were sepa-rated on a Sepax GP-Phenyl column by isocratic elution with methanol–acetonitrile-5 mM ammoniumformate–formic acid (29:55:16:0.1, v/v/v/v) as the mobile phase at a flow rate of 1.2 mL/min, and the totalrun time was 3.0 min. An electrospray ionization (ESI) source was applied and operated in the positiveion mode; multiple reaction monitoring (MRM) mode was used for quantification, and the monitoredtransitions were 394.2 → 377.1 for besifloxacin and m/z 318.3 → 166.1 for the IS. The calibration curve

abbitopicalcular pharmacokinetics

was linear over the range of 0.103–206 ng/mL for plasma and 2.06–2060 ng/mL for tears, aqueous humor,conjunctiva and cornea with correlation coefficient (r) greater than 0.99. The lower limit of quantification(LLOQ) for besifloxacin was 0.103 ng/mL for plasma and 2.06 ng/mL for other ocular tissues with goodaccuracy and precision. Intra- and inter-batch precision were both lower than 15% and accuracy rangedfrom 85% to 115% at all QC levels. The method was successfully applied to the pharmacokinetic study ofbesifloxacin in rabbit plasma and ocular tissues after single and multiple topical administrations.

. Introduction

Besifloxacin, a fourth-generation chiral fluoroquinolone, devel-ped by Bausch & Lomb has been widely used for the topicalreatment of bacterial conjunctivitis [1,2]. Chemically, besifloxacins 7-[(3R)-3-aminohexahydro-1H-azepin-1-y1]-8-chloro-1-

yclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxyliccid [1]. The N-1 cyclopropyl group can provide broad-spectrumctivity against aerobic bacteria. The mechanism of action ofesifloxacin involves inhibition of both bacterial DNA gyrase and

∗ Corresponding authors at: Department of Pharmaceutical Analysis, Chinaharmaceutical University, No. 24 Tongjia lane, Nanjing 210009, PR China.ax: +86 25 8327 1269.

E-mail addresses: ddw888@vip.sina.com (B. Di), sumengxiang@cpu.edu.cnM.-X. Su).

1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.jpba.2015.08.023731-7085/© 2015 Elsevier B.V. All rights reserved.

© 2015 Elsevier B.V. All rights reserved.

topoisomerase IV enzymes which are essential for the synthesisand replication of bacterial DNA, and this activity is enhancedby a C-8 chloride substituent in besifloxacin. Therefore, it dis-plays potent efficacy against a wide range of Gram-positive andGram-negative ocular pathogens, including multidrug-resistantstrains [3–6]. Besifloxacin is the only fluoroquinolone not used forsystemic infections but specifically developed for ophthalmic use,which makes it unique in its class and theoretically reduces therisk for the development of resistance due to decreased systemicexposure [7].

Several in vitro assay methods for besifloxacin have beenreported. A precolumn derivatization chiral HPLC method has beendeveloped for the determination of the enantiomeric impurity in

besifloxacin [8], and a HPLC method with ultraviolet detectionhas been proposed for quantitative determination of besifloxacinin ophthalmic suspension [9]. However, these analytical methodsmay not well meet the requirement of desired sensitivity, speed

38 X.-F. Gu et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 37–46

A) bes

achPa

Fig. 1. Product ion scan spectra of [M + H]+ for (

nd throughput in bio-sample analysis. In addition, the pharma-

okinetic (PK) properties of besifloxacin in rabbits, monkeys andumans have been reported previously [1,10], and then the ocularK/PD of besifloxacin and other two fluoroquinolones, moxifloxacinnd gatifloxacin following topical administration to pigmented

ifloxacin and (B) internal standard nateglinide.

rabbits was investigated [11]. In these in vivo studies, LC/MS or

LC–MS/MS was chosen for the quantitation of besifloxacin in bio-logical samples. These investigations provide useful references forour experimental design and analysis, and have demonstrated thatbesifloxacin can be rapidly absorbed and distributes extensively

X.-F. Gu et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 37–46 39

F interb ) aqueo

iitmamas

LtwltaoaeseWowesmusfp

ig. 2. Representative LC–MS/MS chromatograms of besifloxacin (up panel) and thelank aqueous humor spiked with besifloxacin and the IS at LLOQ concentration, (Cphthalmic suspension in rabbits.

nto ocular tissues with very low systemic exposure, thus suggest-ng that besifloxacin is a promising drug for the safe and effectivereatment of ocular infections [10–14]. Nevertheless, these phar-

acokinetic studies did not provide detailed information about thenalytical process, neither did they report the chromatograms andethodology data of the analytical method [1,10,11]. Besides, they

lmost refer to the ocular pharmacokinetics of besifloxacin after aingle topical ocular administration [10–14].

In this paper, we present a rapid, selective and highly sensitiveC–MS/MS method with simple pretreatment procedures to quan-itate besifloxacin in rabbit plasma and ocular tissues in only 3 minith LLOQ of 0.103 ng/mL for plasma and 2.06 ng/mL for other ocu-

ar tissues. This assay method has a lower quantification limit forear, cornea and conjunctiva compared with the previous report [1],nd has been successfully applied to the quantitative determinationf besifloxacin in rabbit plasma and ocular tissues following singlend multiple topical administrations. To the best of our knowl-dge, this is the first fully validated method for pharmacokinetictudy of besifloxacin in rabbits and the first report of PK param-ters of besifloxacin after multiple-dose administration in rabbits.

hat is more, the standard deviations of ocular PK parameters werebtained according to the principle of propagation of error dealingith destructive sampling. Propagation of error is defined as the

ffects on a function exerted by variable uncertainty. It is a derivedtatistical calculation designed to combine the uncertainties from

ultiple variables with a view to provide a reasonable estimate of

ncertainty [15,16]. Based on the principle of error propagation, thetandard deviations of main ocular PK parameters were obtainedrom the standard deviations of drug concentration at each timeoint.

nal standard (down panel) in rabbit aqueous humor: (A) blank aqueous humor, (B)ous humor obtained at 4 h after a single topical administration of 0.6% besifloxacin

2. Experimental

2.1. Chemicals and reagents

Besifloxacin hydrochloride reference substance (lot 100830,99.7% HPLC pure) and 0.6% besifloxacin ophthalmic suspension (lot140508) were supplied by Jiangsu Yabang Aipusen Pharmaceuti-cal Company Ltd. (Yancheng, China), and the internal standard (IS)nateglinide was purchased from the National Institute for the Con-trol of Pharmaceutical and Biological Products (Beijing, China, lot100619-200501). Methanol and acetonitrile of HPLC grade werepurchased from Tedia Company Inc. (Fairfield, OH, USA), whereasammonium formate and formic acid of analytical grade wereobtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,China). Deionized water was prepared through a PL5242 PurelabClassic UV (PALL Co., Ltd., USA).

2.2. LC–MS/MS conditions

Liquid chromatography was performed on a Shimadzu LC-2010CHT series chromatographic system (Shimadzu, Nakagyo-ku,Kyoto, Japan) consisting of an on-line solvent degasser, a qua-ternary gradient pump, an auto-sampler and a column oven.Quantification was achieved with a Thermo-Finnigan TSQ Quan-tum Ultra AM LC–MS/MS system equipped with an electrospray

ionization (ESI) source (Thermo-Finnigan, San Jose, CA, USA). Dataacquisition was performed with Xcalibur 1.4 software (Thermo-Finnigan, San Jose, CA, USA). Chromatographic separation wasperformed using a Sepax GP-Phenyl column (150 mm × 4.6 mm,5 �m, Sepax Co., Ltd., Suzhou, China) maintained at 35 ◦C. The

40 X.-F. Gu et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 37–46

F intero suspe

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ig. 3. Representative LC–MS/MS chromatograms of besifloxacin (up panel) and thebtained at 4 h after a single topical administration of 0.6% besifloxacin ophthalmic

socratic mobile phase was methanol–acetonitrile–5 mM ammo-ium formate–formic acid (29:55:16:0.1, v/v/v/v) at a flow rate of.2 mL/min, and 30 percent of the effluent was split into the MS inletor the determination. The auto-sampler temperature was adjustedt 4 ◦C to achieve optimal stability.

The mass spectrometer was operated in the positive ion detec-ion mode with a spray voltage of 5 kV. The heated capillaryemperature was 350 ◦C. The nitrogen sheath gas and the auxil-ary gas were set at 350 kPa and 15 kPa, respectively. Quantification

as performed in multiple reaction monitoring (MRM) mode withrgon at a pressure of 0.17 Pa for collision-induced dissociationsCIDs) of the monitoring transitions of m/z 394.2 → 377.1 for besi-oxacin and m/z 318.3 → 166.1 for nateglinide, with a collisionnergy of 16 eV. The positive parent ion mass spectra and prod-ct ion mass spectra of besifloxacin and the internal standard (IS)re shown in Fig. 1.

.3. Preparation of standard solutions, calibration and qualityontrol samples

A stock solution of besifloxacin hydrochloride in a mixture ofethanol and water (50:50, v/v) at a concentration of 1030 �g/mLas prepared. The stock solutions were prepared separately for

alibration standard samples and quality control (QC) samplesy serially diluting with methanol–water (50:50, v/v) to provide

orking solutions of desired concentrations. The stock solution

f IS (nateglinide) was prepared in methanol at a concentrationf 114.2 �g/mL, and the IS working solution of 571 ng/mL wasrepared by diluting the IS stock solution with methanol. The besi-oxacin hydrochloride and nateglinide stock solutions were found

nal standard (down panel) in rabbit (A) plasma, (B) tear, (C) cornea, (D) conjunctivansion.

to be stable in a refrigerator (4 ◦C) for 10 days. All stock solutionswere stored at 4 ◦C and brought to room temperature before use.

Plasma calibration standard samples of besifloxacin (0.103,0.206, 0.515, 2.06, 10.3, 51.5, 164.8 and 206 ng/mL) wereobtained by spiking 20 �L of the appropriate working solu-tions to 250 �L blank rabbit plasma and short vortex mixing.For validation, plasma quality control (QC) samples were pre-pared in the same way as the calibration standard samplesat three concentrations (low quality control/LQC = 0.206 ng/mL,medium quality control/MQC = 10.3 ng/mL, and high quality con-trol/HQC = 164.8 ng/mL). The calibration standards for oculartissues were prepared by adding 20 �L of the appropriate work-ing solutions to 50 �L homogenized blank ocular tissue matrix toobtain the concentrations as follows: 2.06, 5.15, 20.6, 103, 515, 1648and 2060 ng/mL. Quality controls (QCs) samples for ocular tissueswere processed in a similar manner at concentrations of 5.15, 103,1648 ng/mL. The calibration standards and QCs samples were thenprocessed according to the Sample preparation section describedbelow.

2.4. Sample preparation

A simple and rapid protein precipitation method was used fortears, aqueous humor, conjunctiva and cornea samples, while theplasma samples treatment included the protein precipitation, cen-trifugation, solvent evaporation, and preconcentration.

An aliquot of 250 �L plasma sample was placed in a 2 mL Eppen-dorf tube followed by addition of 20 �L IS working solution andvortex-mixing for 30 s. The mixture was extracted with 750 �L ofmethanol by vortex-mixing at a high speed for 3 min, and then thesamples were centrifuged at 25,000 × g for 3 min. The supernatant

X.-F. Gu et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 37–46 41

F umora n + SD

wn2waw

aa2

ig. 4. Concentration-time curves of besifloxacin in rabbit (A) plasma, (B) aqueous hdministration of 0.6% besifloxacin ophthalmic suspension. Data are shown as mea

as transferred into a new Eppendorf tube and evaporated to dry-ess under vacuum at 50 ◦C. The dry residue was reconstituted with00 �L of the mobile phase, after vortexing for 3 min, the mixtureas transferred into 0.5 mL Eppendorf tube and then centrifuged

t 16,000 × g for 3 min; a 10 �L aliquot of the supernatant obtainedas then injected into the LC–MS/MS system for analysis.

Homogenized rabbit ocular matrices (conjunctiva and cornea)nd fluid samples (tear solution and aqueous humor) were thawedt room temperature. Aliquots of 50 �L samples were placed into.0 mL Eppendorf tubes followed by addition of 20 �L IS solution

, (C) tear, (D) cornea and (E) conjunctiva following single and multiple topical ocular (n = 4 for plasma; n = 8 for aqueous humor, tear, cornea and conjunctiva).

and 20 �L methanol–water solution (50:50, v/v). These solutionswere vortexed for 30 s, to which 50 �L of methanol was added,and again vortexed for 3 min to precipitate proteins. After centrifu-gation at 25,000 × g at 4 ◦C for 3 min, a 10 �L of the supernatantobtained was injected into the LC–MS/MS system.

2.5. Assay validation

The method for plasma, tear and conjunctiva was fully validatedfor specificity and selectivity, linearity, LLOQ, precision and accu-

42 X.-F. Gu et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 37–46

Table 1Precision, accuracy, extraction recovery and matrix effect of besifloxacin in rabbit plasma and ocular tissues.

Sample Nominal concentration (ng/mL) Precision (%) Accuracy (%) Recovery Matrix effect

Intra-batch Inter-batch (%, mean ± SD) (%, mean ± SD)

Plasma 0.206 11.5 10.3 80.9 92.8 ± 1.2 92.3 ± 6.910.3 4.6 4.8 81.4 107.2 ± 6.3 102.5 ± 3.0

164.8 11.6 7.3 106.3 100.4 ± 7.7 91.5 ± 10.35.15 6.4 9.4 101.2 95.9 ± 6.9 100.7 ± 11.4

Tear 103 3.7 5.4 95.1 104.2 ± 3.5 98.3 ± 4.65.8

4.2

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acy, extraction recovery and matrix effect, stability and dilutionest according to guidelines set by the United States Food and Drugdministration (FDA) for bioanalytical method validation [17]. Due

o the small amount of aqueous humor and its similar propertyith tear sample extraction, the analytical method of aqueous

umor is partly validated. Similar to this, the method validationas partly performed for cornea, because the homogenized rab-

it ocular matrices including conjunctiva and cornea have similaromponents and properties.

.5.1. SelectivityThe selectivity was investigated by preparing and analyzing six

amples of rabbit blank plasma and ocular matrices samples atandom. Each blank sample was tested using the above-describedample preparation procedure and LC–MS/MS chromatographiconditions to ensure no interferences of IS and besifloxacin fromlank samples.

.5.2. Linearity of calibration curves and lower limit ofuantification

The calibration curve consisted of eight calibration samples cov-ring the range from 0.103 ng/mL to 206 ng/mL for plasma and.06–2060 ng/mL for other ocular tissues, respectively. To evalu-te the linearity of the method, calibration curves were preparednd assayed on five different days. The calibration curves werebtained by plotting the peak-area ratios of besifloxacin to the IS (y)ersus the concentrations of besifloxacin (x), using a weighted least-quares linear regression (the weighting factor used was 1/x2). Theower limit of quantification (LLOQ) was defined as the lowestoncentration on the calibration curves that could be quantifiedeliably with acceptable accuracy (80–120%) and precision (<20%).he LLOQ was established using six samples that were independentf the standards.

.5.3. Accuracy and precisionAccuracy, precision, intra- and inter-batch reproducibility were

valuated using QC samples covering the calibration range. Intra-atch accuracy and precision were evaluated from replicate

able 2tability data of short-term, freeze-thaw and long-term stability.

Sample Nominal concentration (ng/mL) Calculated concentration (n

Room temperature for 5 h

Plasma 0.206 0.234

10.3 9.9

164.8 162.7

5.15 4.98

Tear 103 101.36

1648 1650.8

5.15 5.33

Conjunctiva 103 106.4

1648 1774.6

96.6 109.1 ± 5.2 101.9 ± 1.5111.7 101.0 ± 6.2 90.9 ± 11.4103.1 105.5 ± 4.0 97.7 ± 3.2106.7 95.9 ± 5.8 94.6 ± 4.4

analysis (n = 5) of QC samples at different concentrations on thesame day. Inter-batch accuracy and precision were also assessedfrom the analysis of the same QC samples on 3 consecutive daysin replicate (n = 5). QC samples were analyzed against calibrationcurves. Mean, standard deviation, and relative standard deviation(RSD) were calculated from QC samples and used to estimate theintra-batch and inter-batch precision. Accuracy was assessed bycomparison of the calculated mean concentrations with the knownconcentrations.

2.5.4. Extraction recoveryThe recovery values of besifloxacin and the IS from rabbit plasma

and ocular tissues were expressed as the mean of area ratios ofextracted QC samples divided by those of samples at the sameconcentrations obtained by spiking extracted blank samples withanalytes of the working standard solutions.

2.5.5. Matrix effectsTo evaluate the matrix effect, 5 different blank rabbit plasma

and ocular matrice samples were extracted and spiked with the QCsamples. The corresponding peak areas were then compared withthose of the analytes resolved in the mobile phase at equivalentconcentrations, and this peak area ratio was defined as the matrixeffect (ME). The ME of IS was evaluated at the working concentra-tion (571 ng/mL) in the same manner. An ME value that is not in therange between 85 and 115% indicates an exogenous matrix effect.

2.5.6. StabilityThe stability of besifloxacin in rabbit plasma and ocular tissues

was evaluated by analyzing six replicates of samples containingbesifloxacin at QC sample concentrations. The short-term stabilitywas determined after exposure of the spiked samples to room tem-

perature conditions for 5 h. The freeze/thaw stability was evaluatedafter three successive freeze/thaw cycles (−20 ◦C). The long-termstability was assessed after storage of the standard spiked samplesat −20 ◦C for 40 days. The samples were analyzed against calibra-tion curves obtained from newly prepared standards.

g/mL)

Three freeze-thaw cycles at –20 ◦C Frozen (–20 ◦C) for 40 days

0.201 0.2129.4 10.9

147.3 146.45.74 4.94

103.33 98.841449.8 1565.6

5.62 5.79107.2 96.77

1799.5 1703.5

al and Biomedical Analysis 117 (2016) 37–46 43

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X.-F. Gu et al. / Journal of Pharmaceutic

.5.7. Dilution testIn order to determine the feasibility of the method to analyze

amples whose concentrations are higher than the upper limitf the calibration range, a dilution test was performed. For thisurpose, the tear sample was spiked with the appropriate besi-oxacin solution to obtain final concentrations of 515, 10300 and64800 ng/ml. The solutions of each concentration were furtheriluted by 100-fold to give six replicates. Concentrations of theeplicates were calculated using a valid calibration curve. Further-

ore, the resulting mean concentration, precision and accuracyere calculated.

.6. Pharmacokinetic study

All animal experimental procedures and protocols werepproved by the Animal Ethical Committee of China Pharmaceuti-al University. New Zealand white male rabbits weighing 2–2.5 kg,ree of any signs of ocular inflammation or gross abnormalities,

ere obtained from Anlimo Technology Development Co., Ltd.Nanjing, China), and were acclimated to standard housing andnvironmental conditions (25 ◦C, 50% relative humidity, and 12 hight/dark cycle). Food and water were supplied and libitum. Prioro study, eighty-four rabbits were divided into two groups of 36nd 48 animals each.

Rabbits in the single-dose administration group (n = 36) wereandomized into 9 groups corresponding to a total number of 9ampling points at 0.25, 0.5, 1, 2, 4, 8, 12, 24 and 36 h after dos-ng. 48 rabbits were randomly divided into 12 groups (3 groups forhe steady-state test and 9 groups for multiple administrations).or the multiple-dose study, each rabbit received a 50 �L of 0.6%esifloxacin three times a day with a dosing interval of 8 h for 7onsecutive days, then plasma and ocular tissues were collectedrior to dosing on days 5, 6 and 7 (0 h prior to dosing) and at 0.25,.5, 1, 2, 4, 8, 12, 24 and 36 h post-dose on day 7. At the zero-hourime point, each rabbit received a 50 �L instillation of 0.6% besi-oxacin ophthalmic suspension into the conjunctival sac of eachye as a single bolus dose via a micropipette (n = 4 rabbits per col-ection time). To facilitate even distribution of the suspension overhe surface of the eye and minimize runoff, the eyelids of each rabbitere gently held closed for several seconds after dosing.

At the predetermined sample collection time point after top-cal ocular dosing, tears were collected using tear film, which

as removed after 30 s, immediately weighed, and then stored at20 ◦C. Blood samples (3 mL) were collected by a syringe aroundeart into heparinized tubes, and then plasma was isolated fromhe blood by centrifugation at 600 × g for 10 min, and stored at20 ◦C until analysis. Then animals were euthanized under anes-

hesia. Immediately after death, a 100 �L sample of aqueous humoras obtained with an intraocular cannula from the treated eye via

aracentesis. Then, conjunctiva and cornea were dissected, afterinsing with physiological saline, they were put on filter paper toemove excessive water, weighed afterwards, then homogenizedn physiologic saline and stored at −20 ◦C.

.7. Pharmacokinetic calculation and statistical analysis

The pharmacokinetic parameters including Cmax, Tmax, AUC,nd t1/2 etc., were calculated by noncompartmental analysis,sing pharmacokinetic analysis package DAS (Drug and Statistics)oftware (version 2.0, Mathematical Pharmacology Professionalommittee of China, Shanghai, China). All data were expressed as

he mean ± standard deviation (SD). SD values were calculated byhe derived formula in statistical moments based on the principlef propagation of error proposed by the authors’ previous research16]. The accumulation index (R) was estimated using the follow-ng equation: R = AUCss/AUC0 − � = AUC0 − ∞/AUC0 − � , where AUCss Ta

ble

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s area under the concentration–time curve from zero to 36 h atteady state after multiple dosing, and AUC0 − � exhibits AUC fromero to 36 h during the first dose interval.

. Results and discussion

.1. Method development

.1.1. Optimization of mass and chromatographic conditionsThe MS spectra of besifloxacin and IS were analyzed in the pos-

tive ion mode. Because of the presence of a carboxyl group in thehemical structure of besifloxacin, the negative ion mode was alsoested, but the response intensity obtained was very low. Operationarameters, such as the sheath gas, auxiliary gas, CID and collisionnergy, were adjusted to achieve the detection sensitivity of besi-oxacin and IS. The optimum MS conditions are listed in Section.2.

To obtain chromatograms with appropriate retention time andatisfactory resolution, different types of columns and mobilehases were evaluated to optimize the analytical performance.he retention times of besifloxacin and IS were extremely dif-erent on C18 and C8 columns, while when a phenyl column150 mm × 4.6 mm, 5 �m) with a flow rate of 1.2 mL/min was used,he retention times were reasonable and the peak shapes wereymmetric, which could be due to interactions between the benzylroups of the column and those of besifloxacin and IS. Then severalobile phases were compared and the percentage of acetonitrileas optimized to obtain a proper retention time of besifloxacin

nd IS. At the previously described chromatographic conditions,oth besifloxacin and IS were rapidly eluted with retention times of.9 min for besifloxacin and 2.6 min for the IS. The total chromatog-aphy analysis run time per sample using the current method washorter (3.0 min for all matrices) when compared with the reportedun time of 4.0 min for human tear samples in an earlier study [14].

.1.2. Selection of internal standardAnalogs of the analytes are usually used as the IS because similar

roperties can be obtained during the sample preparation, chro-atographic elution and mass spectrometric detection. In previous

tudies, deuterium-labeled analog [10,13], sparfloxacin [1,14] andlinafloxacin [10,11] have been used as internal standards. The usef deuterium-labeled analog can minimize analytical variation dueo the same extraction recovery, ionization response in ESI masspectrometry and the same chromatographic retention time withhe analyte, but it is difficult to obtain, thus increasing the cost.n the earlier stage of our experiment, several fluoroquinolonesuch as lomefloxacin and ciprofloxacin were tested as IS. However,erious instability was observed in the ionization efficiency, lead-ng to considerable variation in peak areas of the IS, thus couldot satisfy the requirement of precision. Finally, nateglinide waselected as the IS since it also contains a carboxyl group as withesifloxacin, and shows stable ionization efficiency as well as ade-uate and reproducible extraction recovery employing the sampleretreatment protocol described above.

.1.3. Sample preparationMoreover, a simple and efficient method for bio-sample cleanup

o remove protein and potential interference from endogenous sub-tances prior to LC–MS/MS analysis is an important step for theevelopment of a robust bioanalytical method. Besifloxacin is aind of hydrochloride with polar carboxyl group, thereby almost

nsoluble in nonpolar solvents. Based on this, two kinds of polarolvents, methanol and acetonitrile, were tested as the protein pre-ipitation solvent in order to obtain higher extraction recovery ofesifloxacin. By comparison, methanol exhibited a behavior supe-ior to acetonitrile, thus was finally selected to extract besifloxacin.

Biomedical Analysis 117 (2016) 37–46

When dealing with plasma samples, the supernatant treated withmethanol was further concentrated by evaporating to drynessunder vacuum, which improved the concentration and could becompatible with the desired sensitivity.

Under the conditions established for the sample preparation,chromatographic separation and mass spectrometric detection,besifloxacin and IS displayed appropriate retention times with agood chromatographic resolution and relatively high extractionrecoveries, which suggested that this method could well meet therequirement for speed, throughput and sensitivity in bioanalysis.

3.2. Assay validation

3.2.1. SelectivityThe typical chromatograms of blank aqueous humor, blank

aqueous humor spiked with besifloxacin (LLOQ level) and IS(572 ng/mL) and aqueous humor obtained at 4 h after a single top-ical administration of 0.6% besifloxacin ophthalmic suspension areshown in Fig. 2. Typical chromatograms of plasma, tear, corneaand conjunctiva obtained at 4 h are shown in Fig. 3. No interfer-ence peak was detected for besifloxacin and IS from blank plasmaand ocular tissues. The retention times of besifloxacin and IS wereapproximately 1.9 min and 2.6 min, respectively.

3.2.2. Calibration curves and LLOQThe calibration curve was linear in the range from 0.103 to

206 ng/mL in plasma, and 2.06–2060 ng/mL in other ocular tis-sues including aqueous humor, tear, conjunctiva and cornea. Thelinear regression equation was y = 22.892x − 0.01207 (r = 0.9983)for plasma, y = 68.73x + 0.46638 (r = 0.9983) for tear and aque-ous humor, y = 66.758x + 0.41892 (r = 0.9989) for conjunctiva andcornea. Based on the range of the calibration curve, the LLOQ was0.103 ng/mL for plasma and 2.06 ng/mL for other ocular matrices.

3.2.3. Precision, accuracy, extraction recovery and matrix effectThe results of intra- and inter-batch precision, accuracy, extrac-

tion recovery and matrix effect of the QC samples for besifloxacinin rabbit plasma and ocular matrices are presented in Table 1.The intra- and inter-batch relative standard deviation (RSD) for allmatrices were <15%. The accuracy for besifloxacin in each matrixwas within 85–115% of their nominal values at all concentrationsanalyzed. The mean extraction recovery of besifloxacin for eachmatrix at QC levels was within 90–110%. The matrix effects forbesifloxacin and the IS (572 ng/mL) in each matrix were within90–110%. As a result, the matrix effects of the analyte and IS werenegligible under the present LC–MS/MS conditions. These availabledata demonstrate that the proposed method is accurate, reliableand reproducible for the quantitation of besifloxacin in plasma andocular tissues.

3.2.4. StabilityThe stability of besifloxacin in rabbit plasma and other ocular tis-

sues investigated at QC concentrations under different conditionsis shown in Table 2. The data indicated that besifloxacin was stablein plasma and ocular tissues that were either stored at room tem-perature for 5 h, subjected to three freeze/thaw cycles or stored at−20 ◦C for 40 days.

3.2.5. Dilution testAt the first several time points after administration, the tear col-

lection strips may be contaminated by the eye drops, thus leading to

a high drug concentration, while low concentration was observedafter elimination for several days. Therefore it is necessary to dilutethe sample to an appropriate concentration before analysis. Theaccuracy of the measured besifloxacin concentration in tear follow-ing a 100-fold dilution was within 85–115% of the nominal values,

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nd the precision (RSD) was within 15%. Hence, this method coulde used to quantify the tear samples with besifloxacin concentra-ion exceeding the calibration range.

.3. Pharmacokinetic study

The mean concentration–time curves of besifloxacin in rab-it plasma and ocular tissues after single-dose and multiple-doseopical administration of 0.6% besifloxacin ophthalmic suspensionre presented in Fig. 4. The main pharmacokinetic parametersre shown in Table 3. The PK parameters were calculated usingoncompartmental analysis by computing the average value ofhe concentration at each time point. However, this method wasot able to provide the estimation for error of these parameters.ecause of the destructive or invasive nature of sampling associ-ted with ocular PK study design, it was not feasible to repeatedlyample from each individual animal. Therefore, the variance of theK parameters after single-dose and multiple-dose administrationsor the destructive sampling was calculated by the formula pro-osed earlier based on the principle of propagation of error [16].he principle can be briefly described as follows: the error is firstlyransferred from the measured values (drug concentration value)o the primary PK parameters and then to the secondary PK param-ters. The standard deviations of the PK parameters are calculatedasing on the basic formula as follows [16]:

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here xi (i = 1, 2, . . . n) are the measured variables from an exper-ment, the desired result y is a function of the measured variables,nd �y and �xi are the standard deviations of y and xi respectively.alculating formulas for the standard deviations of the PK param-ters are summarized in our previously published paper [16].

It can be found from Table 3 that following topical ocular admin-stration of besifloxacin to rabbits, ocular exposure to besifloxacin,ased on Cmax or AUC0 − 36 h, was highest in tears, followed in ordery conjunctiva, cornea and aqueous humor. Maximal concentra-ions were observed within 1 h in these anterior tissues, indicatinghat the absorption of besifloxacin into the eye was rapid.

The concentrations of besifloxacin in ocular tissues observed inhe present study were basically consistent with that of the pre-iously reported results [1,10,11]. Besifloxacin levels in aqueousumor were relatively low with maximal mean concentrations lesshan MIC 0.5 �g/mL, which indicated that the penetration of besi-oxacin through the cornea into the aqueous humor was difficult,nd also suggested that besifloxacin can better concentrate in thearget tissue (i.e., conjunctiva) than other ocular tissues, thereforehe incidence of side effects is extremely low. The t1/2 of besifloxacinn tear and cornea after single administration in our study were sim-lar with those reported, while the t1/2 of besifloxacin in aqueousumor in the present study was 5–6 h, differing greatly with theeported 12 h [10]. In addition, the t1/2 of besifloxacin in conjunc-iva in the present study was 12–13 h, suggesting a longer retentionime in conjunctiva than in other tissues.

After single administration, besifloxacin levels in tear and con-unctiva were higher than the MIC breakpoint of 0.5 �g/g for 24 h,

hile besifloxacin concentrations remained well above the MIC

.5 �g/g for up to 36 h following multiple instillations (three timesaily). There was some accumulation of besifloxacin after multi-le doses with the accumulation index (R) in rabbit plasma andther ocular tissues ranged from 1.36 to 4.38 (see Table 3), and theffective acting time was also prolonged.

[

Biomedical Analysis 117 (2016) 37–46 45

Cmax and AUC0 − 36 h values of besifloxacin in rabbit plasmaafter a single topical instillation were 2.05 ± 0.12 ng/mL and4.70 ± 0.69 ng h/mL, respectively. Following repeated topicaladministrations, systemic exposure to besifloxacin increased onlyslightly with Cmax and AUC0 − 36 h values of 2.55 ± 1.01 ng/mLand 20.60 ± 6.86 ng h/mL, respectively. The low systemic levelsobserved suggested that the incidence of systemic adverse reac-tions of besifloxacin is extremely low after single or multipletopical ocular dosing. The LLOQ for besifloxacin in this presentassay method is as low as 0.103 ng/mL for plasma, which is suf-ficient to meet the requirement for trace-amount determinationof besifloxacin in the systemic circulation after topical ocularadministration.

4. Conclusion

A rapid and reproducible LC–MS/MS method with high selectiv-ity and sensitivity was developed and validated for the quantitativeanalysis of besifloxacin in rabbit plasma and ocular tissues. Thecurrent method has a low quantification limit of 0.103 ng/mL forplasma and 2.06 ng/mL for other ocular tissues with a short chro-matographic run time of 3.0 min. The method has been successfullyapplied to the pharmacokinetic study of besifloxacin in plasma andocular tissues after single-dose and multiple-dose topical adminis-tration to rabbits.

Acknowledgements

This work was financially supported by the National Natural Sci-ence Foundation of PR China (No. 81402900) and the FundamentalResearch Funds for the Central Universities (No. JKQZ2013028).

References

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4 al and

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(FDA), Center for Drug Evaluation and Research (CDER), Guidance forIndustry, Bioanalytical Method Validation. Available at: http://www.fda.gov/

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concentrations of besifloxacin, moxifloxacin, and gatifloxacin after topicalocular application, J. Cataract Refract. Surg. 37 (2011) 1082–1089.

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