A conventional HPLC-MS method for the simultaneous determination of Ofloxacin and

Cefixime in plasma: Development and validation Mahesh Attimarad1, Sree Harsha N1, Ahmed O. Alnajjar2, Bandar E. Aldubhaib 1 Anroop B. and Ibrahim A. Alhaider1

1Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, KSA.

2College of Science, King Faisal University, Al-Ahsa, KSA.

Introduction

A combination of ofloxacin and cefixime is available in the market,

which is highly active against many bacterial infections such as

typhoid fever, urinary and respiratory tract infections, noscomial

infections, soft tissue and intra abdominal infections.

High performance liquid chromatographic (HPLC) and high

performance thin layer chromatography (HPTLC) methods were

reported for the simultaneous determination of ofloxacin and

cefixime in formulations, but no method has been reported so far for

simultaneous estimation of ofloxacin and cefixime in human plasma.

Some of the reported methods for determination of ofloxacin and

cefixime were in single or with other drugs in pharmaceutical

preparations and biological fluids were spectrophotometry,

fluorometry, HPLC, LC/MS/MS and capillary electrophoresis.

Most of the reported methods for the determination of ofloxacin and

cefixime in biological fluids involve tedious sample preparation

procedures (liquid/liquid or solid phase extraction), low extraction

yields and low sensitivity.

To bypass these difficulties, we have develop more conventional

procedures for determining ofloxacin and cefixime using liquid

chromatography coupled with mass spectrometry (LC-MS). This

assay is simple and robust, as well as sufficiently sensitive for the

pharmacokinetic studies. Conclusion

Research Methodology

HPLC-MS instrumentation and conditions : Agilent LCMS system

consisted of Quat pump, an auto injector, degasser with Agilent Chem

station data module (Agilent 1200 series, Germany) and Quadrupole

LC/MS 6120 detector.

HPLC column : Zorbax eclipse C18 (150 mm×4.6 mm i.d, 5 μm)

The mobile phase : 23%:10%:67% acetonitrile, methanol : formic acid

(0.5%) in water.

Flow-rate : 0.6 mL/min at ambient temperature (25OC)

MS : Selected ion monitoring (SIM) [M+H]+ for ofloxacin: m/z 362.0,

cefixime : m/z 453.8 and IS : m/z 402.0.

Preparation of plasma samples

Blood samples were collected from healthy drug-free human

volunteers. And spiked with appropriate volumes of standard

solutions of OFL and CEF followed by MOX. Samples were

centrifuged at 6,000 rpm for 5 min. Plasma (200 μL) was deproteinized

using acetonitrile (400 μL). vortexed for 5 min and centrifuged at 9000

rpm for 10 min Then, 20 μL of clear supernatant was injected into the

chromatograph.

Acknowledgement

Table 2. Stability of ofloxacin and cefixime trihydrate in human plasma

Ofloxacin Spiked concentration (ng mL-1) 10 100 400

Measured concentration (ng mL-1 )

Freeze and thaw stability

Mean ± SD 9.88 ± 0.3 98.02 ±2 .9 393.21 ± 15.2

RE (%) -2.2 -1.2 -1.7

Post-preparative stability (24 h at room temperature)

Mean ± SD 10.27 ± 0.2 98.1 ±3.8 391.65 ± 16.6

RE (%) 2.7 -1.9 -2.08

Stability for 15 days at -20OC

Mean ± SD 9.75 ± 0.3 97.3 ± 3.2 395.5 ± 20.8

RE (%) -2.5 -2.7 -1.12

Cefixime Spiked concentration (ng mL-1) 100 2000 4000

Measured concentration (ng mL-1)

Freeze and thaw stability

Mean ± SD 98.6 ± 2.2 1965.7 ± 43.9 3945.9 ± 135.2

RE (%) -1.4 -1.71 -1.35

Post-preparative stability (24 h at room temperature)

Mean ± SD 101.2± 4.6 1958.1 ± 69.8 3853.5 ± 116.6

RE (%) 1.27 -2.1 -3.65

Stability for 15 days at -20OC

Mean ± SD 97.31 ± 2.8 1949.3 ± 57.2 3909.8 ± 110.8

RE (%) -2.69 -2.53 - 2.25

RE: Relative Error, SD: Standard Deviation

Objectives The aim of this study was to develop and validate simple analytical

method for simultaneous determination ofloxacin and cefixime in

human plasma.

Newly develop method could be used for pharmacokinetic and

therapeutic drug monitoring.

Table 1. Intraday and Interday Precision and Accuracy for ofloxacin and cefixime

Table 1: Intraday and Interday Precision and Accuracy for

ofloxacin and cefixime

Interday (n-15) Intraday (n-5)

Quotient*(%)

(Ion.Supp %)

Recovery

(%)

Accurac

y (%)

RSD

(%)

Measured

(ng mL-1)

RSD

(%)

Measured

(ng mL-1)

Expected

(ng mL-1)

Ofloxacin

95.4 (4.6) 90.7±8.1 98.4±7.7 7.09 10.16±0.72 5.40 9.81±0.53 10

93.8 (6.2) 95.2±3.1 99.2±5.5 6.74 98.09±6.61 2.81 99.82±2.8 100

96.4 (3.6) 93.4±5.0 98.6±4.6 4.77 396.05±18.9 3.30 392.69±12.95 400

93.1±5.4 98.6±5.9 Average

Cefixime

95.6 (4.5) 93.1±4.5 99.1±4.8 4.43 99.07±4.39 3.07 98.38±3.02 100

92.9 (7.1) 90.5±5.1 99.4±2.4 5.14 2041.28±104.8 4.09 1962.39±80.23 2000

91.3 (8.7) 94.3±4.8 98.3±5.3 5.08 3922.21±199.4 4.34 3942.73±171.09 4000

92.6±4.8 98.9 ±4. Average

Fig. 3. LCMS chromatograms (SIM

mode) of blank human plasma

Fig. 4. LCMS chromatograms (SIM mode) of

human plasma spiked with OFL (500 ngmL-1 ),

MOX (500 ng mL-1 ) and CEF (4000 ng mL-1 )

Results and Discussion

We have developed a specific, rapid, sensitive, and

inexpensive LCMS method for determination of OFL and CEF

in plasma and validated.

The assay involves a simple sample preparation procedure

followed by separation on a reversed phase column with MS

detection.

The specificity test showed that no additional peaks due to

endogenous substances were observed that would interfere

with the OFL and CEF.

The accuracy, precision, recovery, and quantitation and

detection limits enable use of the procedure in

pharmacokinetic, clinical and residues studies.

Because of the short chromatographic run time (5 min) and

simple sample preparation procedure, the reported method is

suitable for processing of many samples on a daily basis.

Authors are thankful to College of Clinical Pharmacy, King Faisal University,

for providing facilities.