gradient hplc of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and...

Ashwini Kumar1

Ashok Kumar Malik1

Dhananjay Kumar Tewary2

Baldev Singh1

1Department of Chemistry,Punjabi University, Patiala, India

2Institute of HimalayanBioresource Technology,Palampur, India

Original Paper

Gradient HPLC of antibiotics in urine, ground water,chicken muscle, hospital wastewater, andpharmaceutical samples using C-18 and RP-amidecolumns

A simple and highly sensitive high pressure liquid chromatographic (HPLC-UV)method has been developed for the determination of ofloxacin, lomefloxacin, cinox-acin, and nalidixic acid, in mobile phase citrate buffer (0.001 M) of pH 4.5 preparedin water (X), methanol (Y), and ACN (Z) using gradient at a flow rate of 1.0 mL/minby direct UV absorbance detection at k = 280 nm. Separation of analytes was studiedon the C-18 and RP-amide columns and best results were observed on the RP-amidecolumn with LODs (3.36S/m) 0.89, 0.55, 0.67, and 1.41 ng/mL for ofloxacin, lome-floxacin, cinoxacin, and nalidixic acid, respectively, and better RSD than the C-18column. The recovery of Fluoroquinolones (FQs) in urine, ground water, hospitalwastewater, and chicken muscle using this method is more than 90%. The methodwas successfully applied to the analysis of ofloxacin, lomefloxacin, cinoxacin, andnalidixic acid in urine, ground water, pharmaceutical dosage forms, hospital waste-water, and chicken muscle.

Keywords: Cinoxacin / HPLC-UV / Lomefloxacin / Nalidixic acid / Ofloxacin /

Received: August 6, 2007; revised: October 9, 2007; accepted: October 7, 2007

DOI 10.1002/jssc.200700373

1 Introduction

Fluoroquinolones (FQs) are a class of important syntheticantibacterials, which are active against both Gram (+)and Gram (– ) bacteria through the inhibition of theirDNA gyrase [1], also they have some activity against myo-bacteria, mycoplasmas, and rickettsias. They can entercells easily and therefore are often used to treat intracel-lular pathogens such as Legionella pneumophila and Myco-plasma pneumoniae. Pseudomonas aeruginosa the broad-hostopportunistic pathogen is the major cause for bacterialinfections in immunocompromised and cystic fibrosispatients and individuals with severe burns [2, 3].

Due to their use in medicine [4], antibiotics have beenreaching the environment by excretion for decades,albeit in trace amounts. The remainders of the antibiot-ics, with effluents, are disposed off to surface waters likecreeks, rivers, or streams. The excretions are usually col-lected in manure storage tanks, where sometimes onlylittle degradation takes place [5]. After top soil dressingwith the contaminated manure, there are several path-ways possible leading into the aquatic cycle: input via

drift during manuring, even an accidental direct input,surface runoff from treated fields, and eventually leach-ing of residues to deeper soil layers and finally into thegroundwater. Antibiotics have been found in diverseenvironmental compartments around the world, includ-ing: (i) sewage water [6–8], (ii) hospital wastewater [9, 10],(iii) ground and river water [11, 12], (iv) sludge [13–16],(v) soil and manure [17–19], and (vi) chicken muscle [20,21]. The presence of FQs in environment in excessamounts may lead to the development of resistance toFQs and of human pathogens to these molecules [22, 23].Therefore, it is very important to develop a method forthe determination of FQs in different samples to protectthe health of consumers.

Several chromatographic methods have been reportedfor the determination of ofloxacin [24–26], lomefloxacin[24, 25, 27–29], cinoxacin [25, 26], and nalidixic acid [24,30, 31] by HPLC using the C-18 column. In the presentstudy, separation of these FQs was achieved on the C-18as well as the RP-amide column. The method shows bestresults on the RP-amide column. The developed methodis highly sensitive as compared to reported methods. TheLODs reported in the literature for ofloxacin [31, 32],lomefloxacin [27], and nalidixic acid [31] are 0.2, 0.05,and 0.1 lg/mL, respectively. Some of them are reportedin the hospital wastewater with concentrations of 0.7–

Correspondence: Dr. Ashok Kumar Malik, Department of Chem-istry, Punjabi University, Patiala-147002, IndiaE-mail: [email protected]: +91-175-2283073

i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

294 A. Kumar et al. J. Sep. Sci. 2008, 31, 294 – 300

J. Sep. Sci. 2008, 31, 294 – 300 Liquid Chromatography 295

124.5 lg/L [33]. The present method provides improvedLODs as compared to the already reported methods. Onthe RP-amide column, the elution of FQs is rapid andachieved in 13 min by using C-18 that is 24 min. Thisimprovement in the procedure of the present work isalso an additional advantage of this method. Structuresof ofloxacin (A), lomefloxacin (B), cinoxacin(C), and nali-dixic acid (D) are shown here.

2 Experimental

2.1 Apparatus

The HPLC system consisted of a Dionex P680 HPLC pump,a Dionex 4.66250 mm id C-18 5 lm RP analytical col-umn (Acclaim 120), a Supelco 4.66150 mm RP-amide5 lm column (AscentisTM), and a Dionex UVD 170U detec-tor and operated at a wavelength of 280 nm.

2.2 Materials and reagents

Ofloxacin, lomefloxacin, cinoxacin, and nalidixic acidwere purchased from Sigma–Aldrich (Germany). HPLC-grade ACN and methanol used were purchased from J.T.Baker (Mexico, USA) and S.D. Fine-chem (India), respec-tively. Sodium citrate (Merck, India), citric acid (Rankem,India), nylon 6,6-membrane filters (Rankem), filtrationassembly (from Perfit, India) were used for the study.

2.3 Standard preparations

Ofloxacin and cinoxacin (100 mg/L) solutions were pre-pared in ACN, lomefloxacin in 50 mM acetic acid, andnalidixic acid in water. All the FQ solutions were storedat 48C.

2.4 Sample preparation

2.4.1 Urine sample

Urine of a healthy person was taken for a sample prepara-tion. Urine was diluted 100 times with triply distilled

water, filtered with nylon 6,6-membrane filters, and wasthen degassed with an ultrasonic bath. Prior to use forHPLC-UV studies, this urine sample was spiked withofloxacin, lomefloxacin, cinoxacin, and nalidixic acidsolutions at a concentration of 5 ng/mL.

2.4.2 Ground water sample

Tube-well water was obtained from Punjabi UniversityPatiala (Punjab, India) filtered, and then degassed withan ultrasonic bath. Prior to HPLC-UV studies, groundwater sample was spiked with ofloxacin, lomefloxacin,cinoxacin, and nalidixic acid solutions at a concentra-tion of 5 ng/mL.

2.4.3 Pharmaceutical dosage forms or tablets

The following available commercial preparations wereanalyzed:

(i) Festive-200 (Laborate Pharmaceuticals India, India)labeled to contain 200 mg of ofloxacin per tablet.

(ii) Lomitas (Intas Pharmaceutical, Ahmedabad, India)labeled to contain 400 mg of lomefloxacin per tablet.

(iii) Cinobac (Ranbaxy, India) labeled to contain200 mg of cinoxacin per tablet.

(iv) GramoNeg (Ranbaxy) labeled to contain 500 mg ofnalidixic acid per tablet.

Formulations of ofloxacin, cinoxacin, lomefloxacin,and nalidixic acid were obtained from the market. Oflox-acin and cinoxacin were dissolved in ACN while lome-floxacin and nalidixic acid were dissolved in 50 mM ace-tic acid and water, respectively. Solutions of 5 ng/mL ofeach formulation were prepared, filtered, and thendegassed with an ultrasonic bath prior to use for HPLC-UV studies.

2.4.4 Hospital wastewater

Hospital wastewater was obtained from Rajendra Hospi-tal Patiala (Punjab, India), filtered, diluted 100 times,and then degassed with an ultrasonic bath. Prior toHPLC-UV studies, wastewater sample was spiked withofloxacin, lomefloxacin, cinoxacin, and nalidixic acidsolutions at a concentration of 5 ng/mL.

2.4.5 Broiler chicken muscle

Chicken was purchased from a local market and was ana-lyzed for FQs absence before work. Fortified muscle sam-ples were prepared by spiking 1 g of minced blankmuscle tissue with the adequate volume (1 mL) of mix-ture standard solutions of ofloxacin, cinoxacin, lome-floxacin, and nalidixic acid at a concentration of 5 ppbeach. Before the analysis, samples were allowed to standfor 20 min in the dark at room temperature.

2.4.5.1 Extraction procedure

The muscle (1 g) was homogenized with phosphate buf-fer (10 mL). Dichloromethane (40 mL) was added to the



homogenate, vortexed for 5 min, and centrifuged at4000 rpm for 20 min. The upper aqueous layer was dis-carded, the organic phase was transferred to a cleantube, and the tissue was again extracted with 30 mL ofdichloromethane. Organic layers were combined andevaporated at 308C under nitrogen stream. The extractwas redissolved with 1 mL of mobile phase and used forHPLC analysis. The quantification in nanogram of anti-microbial/g of muscle was performed in relation to thecorrespondent peak area of the standard curve.

2.5 HPLC determination of FQs

The solvents used were citrate buffer (0.001 M) of pH 4.5,prepared in triply distilled water (X), methanol (Y), andACN (Z), using gradient. For the C-18 column, the compo-sition of mobile phase was X:Y (60:40) for 14 min, thenthe composition was set to change from 14 to 16 min toX:Z (60:40), and was constant later on. For the RP-amidecolumn, the mobile phase X:Y (60:40) was used for first7 min, then set to change to X:Z (60:40) in next 2 min,and remains constant throughout. All the analyses wereconducted at ambient/laboratory temperature, viz.approximately 258C.

3 Concluding remarks

3.1 HPLC-UV studies

The separation of these FQs was studied using directinjection of samples and different parameters like selec-tion of suitable wavelength, effect of flow rate, and com-position of mobile phase were optimized. The composi-tion of mobile phase was X:Y (60:40) for the separation ofofloxacin and lomefloxacin, then the composition wasset to change for cinoxacin and nalidixic acid to X:Z(60:40) and was constant later on. At higher flow ratesthan 1.0 mL/min, the separations were not up to the base-line and with lower flow rates peak tailing was observed,so the flow rate was optimized to 1.0 mL/min. Wave-length for the detection is selected from the UV absorp-tion spectra of FQs and it was selected as 280 nm.

3.2 Method performances

3.2.1 Preparation of calibration curve

The calibration curves were constructed for the detec-tion of ofloxacin, lomefloxacin, cinoxacin, and nalidixicacid under optimized conditions using the HPLC with UVdetection in the range of 5–1000 ppb of these FQs withthe C-18 and RP-amide columns. The calibration curveswere linear over this range. Characteristics of the calibra-tion curves are summarized in Table 1. The LODs forthese FQs were calculated using 3.36S/m (S = SD,

m = slope of calibration curve). Characteristic chromato-grams with HPLC-UV detection at 280 nm are shown inFig. 1a (C-18) and Fig. 1b (RP-amide) for the separation ofthese FQs.

3.2.2 Accuracy, repeatability, and LODs

The method LODs were calculated for these FQs accord-ing to ICH Harmonised Tripartite Guidelines (www.i-ch.org/LOB/media/MEDIA417.pdf). Similarly, the methodLOQs can be estimated as 106S/m. The accuracy (% recov-ery) and precision (% RSD) of SPME-HPLC-UV method wasevaluated for each analyte by analyzing a standard ofknown concentration (5 ng/mL) five times, and quantify-ing it using calibration curves established above. Theresults for LOD and RSD are given in Table 1. The LODand RSD values for the RP-amide column are better than


Figure 1. HPLC-UV chromatogram for the separation ofofloxacin (A), lomefloxacin (B), cinoxacin (C), and nalidixicacid (D), 5 ppb standard each, at a flow rate of 1.0 mL/min.The solvents used were citrate buffer (0.001 M) of pH 4.5prepared in triply distilled water (X), methanol (Y), and ACN(Z) and detection at 280 nm. (a) On the C-18 column, thecomposition of mobile phase was X:Y (60:40) for 14 min thenthe composition was set to change from 14 to 16 min to X:Z(60:40) and was constant later on. (b) On the RP-amide col-umn, the composition of mobile phase was X:Y (60:40) for7 min then the composition was set to change from 7 to9 min to X:Z (60:40) and was constant later on.


the C-18 column. The method gave satisfactory resultswhen used to quantify these FQs in pharmaceutical dos-age form, urine, ground water, chicken muscle, and hos-pital wastewater.

3.3 Applications

At present, in India and other countries these FQs arecommercially available and are used very frequently.Therefore, it was considered to analyze these FQs in


Table 1. Comparison of HPLC-UV characteristics of the ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid on the C-18 andRP-amide columns

C-18 RP-amide

FQ Ofloxacin Lomefloxacin Cinoxacin Nalidixic acid Ofloxacin Lomefloxacin Cinoxacin Nalidixic acid

R2 0.9932 0.9903 0.9994 0.9925 0.9987 0.9995 0.9997 0.9996Regression equation 0.0007x + 0.073 0.0008x + 0.0229 0.002x - 0.0311 0.0007x - 0.0294 0.0013x + 0.0217 0.0021x + 0.0083 0.0022x – 0.0013 0.0019x + 0.0003Slope (m) 0.0007 0.0008 0.002 0.0007 0.0013 0.0021 0.0022 0.0019Retention time (min) 5.5 6.2 8.75 23.5 3.5 3.95 4.8 12.9Average peak area (5 ng/mL, mAu6min)a)

0.0742 0.0356 0.0842 0.0174 0.0924 0.1174 0.1108 0.0453

Working range (ng/mL) 5 – 1000 5 – 1000 5 – 1000 5 – 1000 5 – 1000 5 – 1000 5 – 1000 5 – 1000LOD = 3.36S/m (ng/mL) 1.88 1.47 1.40 2.59 0.89 0.55 0.67 1.41LOQ = 106S/m (ng/mL) 5.69 7.50 4.25 7.84 2.69 1.67 2.04 4.27Intraday RSDa) (%) 0.54 1.68 1.01 3.16 0.38 0.30 0.41 1.81Interday RSDa) (%) 0.94 2.14 1.96 5.14 0.71 0.69 0.97 2.73Retention factor 0.37 0.55 1.18 4.87 0.76 0.98 1.41 5.48Selectivity factor 1.47 2.16 4.11 1.28 1.45 3.88Resolution 1.102 3.42 17.15 0.82 1.54 15.0

a) Each reading is mean of five experiments.

Figure 2. HPLC-UV chromatogram of the urine samplespiked with ofloxacin (A), lomefloxacin (B), cinoxacin (C),and nalidixic acid (D), 5 ppb each, on (a) C-18 column, (b)RP-amide column. Other conditions are the same as inFig. 1.

Figure 3. HPLC-UV chromatogram of the ground water sam-ple spiked with ofloxacin (A), lomefloxacin (B), cinoxacin (C),and nalidixic acid (D), 5 ppb each, on (a) C-18 column, (b)RP-amide column. Other conditions are the same as inFig. 1.



Figure 4. HPLC-UV chromatogram of the hospital waste-water sample spiked with ofloxacin (A), lomefloxacin (B),cinoxacin (C), and nalidixic acid (D), 5 ppb each, on (a) C-18column, (b) RP-amide column. Other conditions are thesame as in Fig. 1.

Figure 5. HPLC-UV chromatogram of the pharmaceuticalsamples of ofloxacin (A), lomefloxacin (B), cinoxacin (C),and nalidixic acid (D), 5 ppb each, on (a) C-18 column, (b)RP-amide column. Other conditions are the same as inFig. 1.

Figure 6. HPLC-UV chromatogram of the pharmaceuticalsamples of ofloxacin (A), lomefloxacin (B), cinoxacin (C),and nalidixic acid (D), 50 ppb each, on (a) C-18 column, (b)RP-amide column. Other conditions are the same as inFig. 1.

Figure 7. HPLC-UV chromatogram for the determination ofofloxacin (A), lomefloxacin (B), cinoxacin (C), and nalidixicacid (D), 5 ppb, on (a) C-18 column, (b) RP-amide column, inchicken muscle extract. Other conditions are the same as inFig. 1.


urine, ground water, and hospital wastewater samplesand spiking the urine, ground water, and hospital waste-water sample with these FQs validated the method. Nointerfering peaks appeared in the retention time of thecompounds studied. The urine (Fig. 2), ground water (Fig.3), and hospital wastewater (Fig. 4) samples spiked withofloxacin, lomefloxacin, cinoxacin, and nalidixic acidand their pharmaceutical sample (Figs. 5 and 6) givepeaks at the retention time of ofloxacin, lomefloxacin,cinoxacin, and nalidixic acid. We have performed theanalytical validation for the simultaneous quantificationof ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid,with a good resolution of the four antimicrobials inchicken muscle (Fig. 7). The present method lasted for3 h from the reception of the muscle to the analysis ofthe sample with good recovery, linearity, and LOQ and itis feasible for laboratories, avoiding the importation ofcartridges of SPE. The general procedure was applied forthe analysis of these FQs and results of the determinationin urine, ground water, pharmaceutical formulations,hospital wastewater, and chicken muscle sample aregiven in Table 2.

4 Conclusion

The proposed method is simple, rapid, reproducible, pre-cise, specific, economic, and highly sensitive and can beused for the assay of the studied FQs either in bulk or intheir corresponding dosage forms without interferencefrom commonly used excipients as well as their detec-tion in the biological fluids and various environmentalsamples due to high sensitivity of the method. Thepresent method provides improved LODs as compared tothe already reported methods. The use of a shorter RP-amide column in the present method also reduced theretention time for the separation and determination ofthese FQs and the analysis becomes rapid. The RSD valuesfor RP-amide column are better than the C-18 column.The method validation parameters yielded good resultsand included the range, linearity, precision, accuracy,specificity, and recovery.

The authors are thankful to the CSIR, New Delhi for supportingthis research work (no. 80(0063)/07/EMR-I).


Table 2. Comparison for the determination of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, 5 ppb standard, each on theC-18 and RP-amide columns in urine, ground water, pharmaceutical formulations, hospital wastewater, and chicken muscle sam-ples

Sample FQ Added(ng/mL)

Recovery(ng/mL)

Recovery(ng/mL),

RSDa) (%) C-18 RSDa) (%) RP- amide

C-18 column RP-amidecolumn

Intraday Interday Intraday Interday

Urine Ofloxacin 5 4.51 4.52 0.21 0.29 2.79 3.85Lomefloxacin 5 4.42 4.62 3.86 5.45 2.14 2.89Cinoxacin 5 4.63 4.72 3.65 5.02 2.19 2.94Nalidixic acid 5 4.37 4.68 18.9 25.2 2.52 3.47

Ground water Ofloxacin 5 4.73 4.51 0.21 0.29 2.75 3.81Lomefloxacin 5 4.52 4.72 3.80 5.35 2.09 2.87Cinoxacin 5 4.73 4.80 3.55 5.01 2.15 2.93Nalidixic acid 5 4.45 4.79 12.8 18.35 2.46 3.41

Pharmaceutical Ofloxacin 5 4.64 4.59 0.21 0.30 2.76 3.80formulation Lomefloxacin 5 4.47 4.69 3.83 5.39 2.10 2.89

Cinoxacin 5 4.63 4.75 3.66 5.13 2.18 2.98Nalidixic acid 5 4.42 4.76 14.6 23.2 2.47 3.38

Pharmaceutical Ofloxacin 50 45.2 45.6 0.22 0.27 2.78 3.84formulation Lomefloxacin 50 44.3 46.5 3.82 5.44 2.16 2.85


Hospital Ofloxacin 5 4.49 4.53 0.22 0.23 2.72 3.81wastewater Lomefloxacin 5 4.41 4.64 3.85 5.37 2.10 2.83


Chicken muscle Ofloxacin 5 4.47 4.49 0.20 0.32 2.83 3.87Lomefloxacin 5 4.39 4.59 3.87 5.47 2.17 2.92Cinoxacin 5 4.58 4.68 3.68 5.09 2.23 2.97Nalidixic acid 5 4.33 4.65 18.7 25.3 2.54 3.49

a) Each reading is mean of five experiments.


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gradient hplc of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and...

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