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MEASUREMENT OF FLUOROQUINOLONEANTIBIOTICS FROM HUMAN PLASMAUSING HOLLOW FIBER LIQUID-PHASEMICROEXTRACTION BASED ON CARRIERMEDIATED TRANSPORTAli Esrafili a , Yadollah Yamini a , Mahnaz Ghambarian a , ShahabShariati b & Morteza Moradi aa Department of Chemistry, Tarbiat Modares University, Tehran, Iranb Department of Chemistry, Faculty of Sciences, Islamic AzadUniversity, Rasht Branch, Rasht, Iran

Available online: 07 Feb 2012

To cite this article: Ali Esrafili, Yadollah Yamini, Mahnaz Ghambarian, Shahab Shariati & MortezaMoradi (2012): MEASUREMENT OF FLUOROQUINOLONE ANTIBIOTICS FROM HUMAN PLASMA USINGHOLLOW FIBER LIQUID-PHASE MICROEXTRACTION BASED ON CARRIER MEDIATED TRANSPORT, Journal ofLiquid Chromatography & Related Technologies, 35:3, 343-354

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MEASUREMENT OF FLUOROQUINOLONE ANTIBIOTICS FROMHUMAN PLASMA USING HOLLOW FIBER LIQUID-PHASEMICROEXTRACTION BASED ON CARRIER MEDIATED TRANSPORT

Ali Esrafili,1 Yadollah Yamini,1 Mahnaz Ghambarian,1 Shahab Shariati,2

and Morteza Moradi1

1Department of Chemistry, Tarbiat Modares University, Tehran, Iran2Department of Chemistry, Faculty of Sciences, Islamic Azad University,Rasht Branch, Rasht, Iran

& Due to the existence of both amine and carboxylic acid groups on ofloxacin and ciprofloxacin,the direct passive transport from aqueous sample to organic solvent is difficult. Therefore, the trans-port of the drugs was carried out in the presence of methyltrioctylammonium chloride (Aliquat 336),as a carrier. The drugs were extracted from 10.0mL of the aqueous solution which had a pH of 8.5,into the organic extracting solvent (1-octanolþ 10% (w=v) carrier) which had been impregnatedinto the pores of hollow fiber; this was followed by a back extraction into 24 lL aqueous solutionlocated inside the lumen of a hollow fiber (pH 1.0, 1mmol=L NaCl). Under the optimized con-ditions, the preconcentration factors (PFs) were achieved up to 137, and the relative standard devi-ation (RSD) of the method was less than 4.9%. The calibration curves were obtained in the ranges of1–500lg=L with a reasonable linearity (R2> 0.997), and the limit of detection (LOD) was0.5lg=L for both of the drugs. Finally, the applicability of the proposed method was evaluated bythe extraction and determination of the drugs from plasma and tap water samples.

Keywords carrier mediated transport, ciprofloxacin, high performance liquid chroma-tography, liquid phase microextraction, ofloxacin

INTRODUCTION

Fluoroquinolones (FQs) are a class of relatively new and entirelyman-made, non-steroidal antibiotics=antibacterials. They are used in thetreatment of infection throughout many parts of the body. They do thisby killing the harmful bacteria or by preventing their growth. Their activityis based on the inhibiting of the DNA enzyme gyrase or topoisomerase II,which is responsible for the preservation of the DNA’s biological activity in

Address correspondence to Yadollah Yamini, Department of Chemistry, Tarbiat ModaresUniversity, P.O. Box 14115-175, Tehran, Iran. E-mail: yyamini@modares.ac.ir

Journal of Liquid Chromatography & Related Technologies, 35:343–354, 2012Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.601488

Journal of Liquid Chromatography & Related Technologies, 35:343–354, 2012Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.601488

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bacteria.[1] The first FQ was synthesized by converting nalidixic acid into aquinolone structure; by adding a fluorine atom and a piperazine ring, thesegroups increase the antibacterial activity of the molecules.[2] Ofloxacin(Oflo) and ciprofloxacin (Cipro) (Figure 1) are two extensively used drugsfrom this group and these drugs have a narrow therapeutics index andshow less protein binding and higher drug tolerance, with a lower toxicity,and longer half-life.[2]

In Europe and other parts of the world, these FQs are still widely usedin many human and animal applications. The way in which antibiotics con-taminate the environment depends on the specific use of them. In the caseof antibiotics used against human diseases, they are mainly spread throughurban wastewaters. In fact, it has been reported that the actual proceduresin the treatment of wastewaters are unable to completely remove thesecompounds.[3] Concerning the antibiotics used in veterinarian medicine,the use of animal excretions for manure has been the main factor in thecontamination. In this case, antibiotics are directly spread to the soil, andafter rainfall, ground surface waters may therefore also become polluted.[4]

Therefore, it is necessary to identify the antibiotics through variousenvironmental and biological samples. Previously, numerous techniqueshave been developed for their analysis in biological fluids[5–7] and in wastewater samples.[8–11]

Recently, in order to enhance the extraction of hydrophilic drugs, car-rier mediated liquid phase microextraction (LPME) has been reported asan active transport mode of LPME. In this method a carrier is added tothe sample solution[12–15] or is dissolved in the impregnated solvent insidethe pores of the hollow fiber.[16,17]

In the present study, hollow fiber liquid membrane containing Aliquat336 as an anionic carrier in the three-phase LPME mode was used for theextraction and determination of Oflo and Cipro in plasma, urine, and tap

FIGURE 1 Structures of Ofloxacin and Ciprofloxacin and their pKa values that were calculated usingthe ACD=pKa (ver. 1.20) software program (Advanced Chemistry Development, Toronto, Canada).

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water samples. The extraction recovery was studied as a function of the nat-ure of the immobilized organic phase, the pHs of source phase (SP) andreceiving phase (RP), salt addition in the SP and RP, extraction time,and stirring rate. These parameters were optimized and the system finallywas applied to extract Oflo and Cipro drugs from various real samples.

EXPERIMENTAL

Chemicals and Reagents

Ofloxacin and ciprofloxacin were obtained from the Jaber Ibne HayyanDrug Company (Tehran, Iran). HPLC grade acetonitrile was purchasedfrom the Caledon Company (Georgetown, ONT, Canada). Tetrabutylammonium chlorate, sodium n-hexyl sulfate, and citric acid were allobtained from the Merck Company (Darmstadt, Germany). Methyltrio-ctylammonium chloride (Aliquat 336) was purchased from Fluka (Buchs,Switzerland). Sodium hydroxide, nitric acid (65% w=w) and 1-octanol andother reagents were all of analytical grade and obtained from Merck. Thewater was purified on a Milli-Q ultra-pure water purification system fromMillipore Company (Milford, MA, USA). Plasma and urine samples wereobtained from Iranian Blood Transfusion Organization (Tehran, Iran).

Standard Solutions and Real Samples

Stock standard solutions of Oflo and Cipro (1000 mg=L) were preparedseparately by dissolving the proper amounts of each drug in methanol.Then, they were diluted with deionized water to reach a secondary mixedstock solution with a concentration of 5 mg=L. All of the working standardsolutions were freshly prepared by diluting the mixed standard solutionwith deionized water to the required concentration.

All of the standard solutions were stored at 4�C and were prepared againevery three weeks. Tap water sample was collected from the laboratory inTarbiat Modares University, (Tehran, Iran). The pH of the samples wasadjusted at 8.5 by using a dropwise addition of NaOH solution (0.01 mol=L).Before extraction, the plasma samples were diluted to a 1:3 ratio using water.

HPLC Instrument

Chromatographic separations were carried out by the use of a Varian9012 HPLC pump (Mulgrave Victoria, Australia) equipped with a Varian9050 UV=Vis detector. All injections were performed manually with aRheodyne 7725 manual injector (Cotati, CA, USA) using a 20 mL sampleloop. Chromatographic data was recorded and analyzed using a home

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designed computerized software. Separations were carried out on a ZorbaxExtend C18 Column (100 mm� 2.1 mm, with 3.5mm particle size) from theAgilent Company (Wilmington, DE, USA). Based on the literature[18] andafter some try and error a mixture of 88% of buffer solution with pH 3.4(containing 10 mmol=L tetrabutyl ammonium chlorate and 10 mmol=Lsodium n-hexyl sulfate and 25 mmol=L citric acid) and 12% acetonitrilewas used as a mobile phase in isocratic elution mode at the flow rate of0.2 mL=min. The detection wavelength was set at 280 nm, and the injectionvolume was 20 mL for all of the samples and standards.

Extraction Procedure

All of the LPME experiments were performed using an Accurel Q3=2polypropylene hollow fiber membrane (600mm i.d., 200 mm wall thickness,0.2 mm pore size) from the Membrana Company (Wuppertal, Germany).The whole fiber was cut into small pieces with length of 8.8 cm. In orderto avoid a memory effect, each piece of hollow fiber was used just once.Before the extraction, fibers were sonicated in acetone for 5 min to removeany possible contaminants. Then, a 25-mL Hamilton syringe (Bondaduz,Switzerland) was employed to introduce the receiving phase, RP, into thelumen of the hollow fiber. Extraction and injection processes were per-formed by the following steps: (1) 10.0 mL of a sample solution was trans-ferred into a 12 mL glass vial already containing a 14 mm� 4 mm magneticstirring bar; (2) the sample vial was placed on the IKA multi-station mag-netic stirrer (Staufen, Germany); (3) the fiber was submerged into theorganic solution (1-octanolþ 10% (w=v) carrier) for 10 s and then intowater for 10 s to wash off the extra organic solution from the surface ofthe fiber; (4) a carefully measured portion of 24 mL of the receiving phasewas injected into the hollow fiber and then its end sealed by a piece ofaluminum foil; (5) the fiber was bent to a U-shape and together with asmall part of the supporting syringe needle was submerged in the samplesolution; (6) the vials were covered with Para Film and stirred for a pre-scribed period of time; (7) at the end of the extraction time, the hollowfiber was removed from the sample solution, and its closed end was openedand the receiving phase was withdrawn into the syringe; and (8) finally,20 mL of the receiving phase was injected into the HPLC.

RESULTS AND DISCUSSION

Method Development

Two main processes are followed when employing a successful threephases LPME method: (1) to separate the analytes from the matrices in

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order to clean them up and (2) to preconcentrate the analytes to increasethe sensitivity and lower the limits of quantification. Cipro and Oflo(Figure 1) are particularly challenging analytes due to the existence of boththe amine and carboxylic acid groups; quantitative extraction of the drugsfrom aqueous samples into the organic phase is not possible. Therefore,by utilizing a cationic carrier in the membrane solvent, it is possible toactively transport Cipro and Oflo from the aqueous samples (SP) into theRP. According to our previous experiments[16,19] methyltrioctylammoniumchloride (Aliquat-336, R3NCHþ

3 Cl�) was chosen as the cationic carrier in themembrane. In this process, the driving forces for transporting the drugs arethe gradient of the pH and the gradient of the Cl� concentration betweenthe SP and RP. In order to achieve maximum sensitivity, all of the parametersinfluencing the extraction recovery were studied and optimized. Accordingto the preliminary trials, an organic solvent containing 10% w=v Aliquat 336was utilized as the membrane solvent and the concentration of the targetdrugs in optimization experiments were 100 mg=L. Each extraction wasrepeated at least three times and the means were used in the studies.

Membrane Solvent Selection

Selection of a suitable organic solvent in HF-LPME is of great impor-tance for an efficient analyte preconcentration. There are several require-ments for an organic solvent selection. The solvent has to be easilyimmobilized in the hollow fiber’s pores; it should have a high capacityfor dissolving carrier and also allow ion-pair formation between analytesand carrier. It should have a low tendency to dissolve or take up interfer-ences existing in the source phase; its water solubility and also volatilityshould be low in order to separate it from the aqueous phases of SP andRP, also to prevent solvent loss during extraction. Also, it should have alow viscosity because this promotes a high analyte fluxes through the mem-brane and, finally, it is better that the solvents have a low toxicity. There-fore, five different organic solvents including n-dodecane, 1-octanol, aswell as a mixture of them, methyl isobutyl ketone, and ethyl acetate wereevaluated for usage as membrane solvents. Each experiment was carriedout by adding 10% (w=v) of the carrier into the organic solvent. The resultsshowed that 1-octanol has the highest extraction recovery. Therefore,1-octanol was used as the membrane solvent in all subsequent experiments.

Effect of Compositions of the Source and Receiving Phases

Because of the use of a charged carrier in the membrane phase (Aliquat336), transportation of the drugs was achieved by ion-pair formation. Inthis case, the optimization of the SP pH is crucial to change the drugs to

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their anionic forms. Therefore, the pH of the SP was adjusted between theranges of 1.0 to 12.0 by the dropwise addition of 0.01 and 1 mol=L of HNO3

or NaOH.Based on the obtained results (Figure 2), the pH of 8.5 was selected as

the optimum value for SP. Subsequently, the effect of the pH of the RP onextraction efficiency was studied in the ranges of 1–13. The obtainedresults, indicated that the highest extraction recovery was obtained atpH¼ 1. The low pH of the RP plays two roles: first, the amine functionalgroups can be protonated, accelerating the transport of the drugs intoRP; and second, binding of the protons to the drugs at the carboxylic sitecan dissociate carrier-drug bonding.

The effect of salt addition on the extraction recovery was examined byadding sodium chloride to both source and receiving phases in concen-tration levels of 0, 0.5, 1, 1.5, 2, and 2.5 mol=L. The extraction recoveryof drugs decreased by increasing the salt concentration in SP, which maybe due to increasing in viscosity, and then decreasing in the mass transferof the analytes. Also, Cl� ion competes with the drugs to form ion pairingwith the carrier. As a result, all of the subsequent experiments were per-formed in the absence of salt in SP. The effect of increasing the NaClconcentration in the RP was evaluated on the extraction recoveries ofthe drugs. Figure 3 illustrates the effect of the NaCl concentration in theextraction recoveries of the drugs. Results showed that by increasing theNaCl concentration in the RP, the extraction recoveries rapidly increased.The maximum extraction recoveries were obtained when the NaCl concen-tration was 1.0 mol=L. As it was described in a previous article,[17] one ofthe driving forces in the transporting of the drugs is the concentration

FIGURE 2 Effect of the pH of the source phase on the transport of the drugs. Conditions:RP¼ 0.01 mol=L of HNO3, VSP¼ 10 mL, CA¼ 100.0mg=L, VRP¼ 24mL, MP¼ 1-octanol, stirringspeed¼ 600 rpm, t¼ 30 min. (Color figure available online.)

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gradient of Cl�, and by increasing Cl� concentration, the driving forceincreases. But, by further increasing of NaCl concentration (over 1 mol=L),the extraction recoveries of the drugs decreases; this may be due to increas-ing the thickness of the inner diffusion layer of the hollow fiber that gives adecrease in the mass transfer of the analytes to the RP. Thus, further experi-ments were performed using 1 mol=L of NaCl in the RP.

Agitation Speed

As in other microextraction techniques, the extraction rate in HF-LPMEcan be enhanced by agitating the sample solution, thereby reducing thetime required to attain thermodynamic equilibrium especially for thehigher molecular mass analytes.[20,21] In HF-LPME the organic solvent isprotected by the hydrophobic hollow fiber membrane, so it is easier to han-dle and can tolerate the higher stirring speeds. In the present work, theeffect of the stirring rate was investigated in the range of 300–1000 rpm.The partitioning of the analytes into the RP was enhanced when the stirringspeed was increased to 1000 rpm. As a result, the chosen maximum stirringspeed of 1000 rpm was utilized for the rest of the experiments.

Extraction Time

Due to the fact that there are two liquid–liquid interfaces in this extrac-tion system (i.e., source phase-organic membrane phase and organicmembrane phase-receiving phase), solute molecules require a long timeto pass through these interfaces. LPME is not an exhaustive extraction

FIGURE 3 Effect of NaCl addition in receiving phase on the transport of the drugs. Conditions: pHof SP¼ 8.5, pH of RP¼ 1, VSP¼ 10 mL, CA¼ 100.0mg=L, VRP¼ 24mL, MP¼ 1-octanol, stirring speed¼600 rpm, t¼ 30 min. (Color figure available online.)

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technique, thus maximum sensitivity is attained at the equilibrium con-ditions. On the other hand, a complete equilibrium needs not to beattained for an accurate and precise analysis.[22,23] Therefore, the extrac-tion time is one of the most important factors influencing the extractionrecovery. In this study, the extraction recoveries of the drugs were investi-gated as a function of time in the range of 15 to 75 min. The extractionrecoveries of the drugs were increased by enhancing the extraction timeuntil 45 min and subsequently were decreased. The decrease of extractionefficiency may be due to loss of organic membrane solvent or receivingphase at long times. As shown in Figure 4 the optimal extraction timewas found to be 45 min. Thus, the extraction time of 45 min was chosenfor the remainder of the experiments.

Method Performance

Calibration curves were plotted against the concentration levels of thedrugs in the ranges of 1–500 mg=L. For each level, three replicate extrac-tions were performed. The corresponding coefficients of determination(R2), linear dynamic ranges (LDRs) and the limits of detections (LODs)were calculated and are summarized in Table 1.

The limit of detections, based on signal to noise ratio of three (LOD¼ 3S=N) were 0.5 mg=L for both of the drugs. The preconcentration factor(PF) was calculated based on the following equation:

PF ¼CRP ;final

CSP ;initialð1Þ

FIGURE 4 Effect of extraction time on the transport of the drugs. Conditions: pH of SP¼ 8.5, pH ofRP¼ 1, VSP¼ 10 mL, CA¼ 100.0mg=L, VRP¼ 24mL, MP¼ 1-octanol, stirring speed¼ 1000 rpm. (Colorfigure available online.)

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where CRP,final and CSP,initial are the final and initial concentrations of thedrugs in the organic and the aqueous phases, respectively. CRP,final of theextracted drugs was calculated from the calibration curve that was obtainedbased on the direct injection of the drugs solutions into the HPLC. The PFsof 115 and 137 were obtained for Oflo and Cipro, respectively.

The main advantages of the proposed method are: low detection limit,good precision, rapidity, low consumption of organic solvent, and simplicityof operation.

Real Samples

In order to study the suitability of the proposed HF-LPME method forthe determination of the target analytes in the real samples, it was appliedfor the extraction and determination of the drugs in the tap water andplasma samples. In order to reduce the matrix effect, the plasma samplewas diluted to 1:3, using water. Then, each real sample was extracted atits optimal conditions by the proposed procedure and no target analytes

TABLE 1 Figures of Merit of the Proposed Method

AnalyteLOD

(mg=L) R2LDR

(mg=L)RSD(%)

PreconcentrationFactor

ExtractionRecovery (%)

Ofloxacin 0.5 0.9984 1–500 4.9 115 27Ciprofloxacin 0.5 0.9967 1–500 3.1 137 33

FIGURE 5 Chromatograms of the target drugs after extraction from a plasma sample (A) without spike(B) after spiking of 30 mg=L of target drugs.

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were found in the real samples. Thus, the samples were spiked with thedrugs in the concentration ranges of 5.0–50.0 mg=L and the procedurewas followed. The typical chromatogram of the plasma sample beforeand after spiking of 30mg=L concentration level of the target drugs isshown in Figure 5A and 5B. Table 2 shows that the results of three replicateanalysis of each real sample obtained by the proposed method are in satis-factory agreement with the spiking amounts. When the proposed methodwas applied to urine sample, some additional components co-extractedwith the drugs resulting in peaks that overlapped the drug peaks in theHPLC chromatogram. Thus, this method could not be applied for theurine samples. On the other hand, the obtained chromatogram reveals agood clean-up of the proposed approach to determine drug concentrationsin plasma sample.

CONCLUSION

The present study exhibited the excellent performance of the carrierbased HF-LPME technique for the analysis and extraction of trace levelof popular antibacterial drugs from water and biological samples. The pro-posed method provided high PFs for the target analytes, along with anexcellent clean-up capability. The whole operation is very convenient tohandle because the receiving phase is protected by the hollow fiber. Theconcentration-time profile for ciprofloxacin and ofloxacin in plasmabetween 2.5 hr and 20 hr after administration of a single 200-mg and300-mg oral dose to a healthy volunteer were changed in the range of2.25 mg=L–50mg=L and 4.5 mg=L–250mg=L, respectively.[24] Thus therecommended procedure is applicable for determining of the drugs inplasma samples. Also, by applying high sensitive instruments such asHPLC-MS-MS, the method is applicable to monitor the drugs in environ-mental waters.

TABLE 2 Determination of the Drugs in Different Spiked Samples

Sample Ofloxacin Ciprofloxacin

Tap water (5.0mg=L added) Initial concentrationa – –Foundb 4.4 4.5Relative recovery (%) 88.0 90.0RSD (%) (n¼ 3) 6.8 8.8

Plasma (50.0mg=L added) Initial concentration – –Found 54.3 53.2Relative recovery (%) 108.6 106.4RSD (%) (n¼ 3) 6.2 8.6

aAll concentrations are in mg=L.bConcentration of the drugs in the spiked sample.

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Compared to traditional methods, this method exhibits low limits ofdetection with a higher enrichment factor. It gives good precision with awide linear dynamic range. Results showed that HF-LPME combined withHPLC was viable, easy to use, and economical for the analysis of Ofloand Cipro in the environmental and biological samples.

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at 0

3:46

16

Febr

uary

201

2