Quantitative Determination of Ciprofloxacin in Human Plasma by LC-MS-MS
Authors
Christopher J.L. Buggé, Michael P. Sullivan, Melvin S. Tan, William S. Edgemond
Introduction
Ciprofloxacin is a member of the quinolone class of synthetic antimicrobial agents. Older members of this class have been available for many years but have limited therapeutic utility and develop rapid bacterial resistance. The more recent introduction of fluorinated 4-quinolones such as ciprofloxacin represents an important therapeutic advance since these compounds have broad antimicrobial activity and are effective for the treatment of a wide variety of infectious diseases. Relatively few side effects appear to accompany the use of ciprofloxacin and microbial resistance develops more slowly. Additionally, ciprofloxacin is approved for use after inhalation exposure of anthrax to reduce the progression of the disease.
A rugged and sensitive method for the analysis of ciprofloxacin suitable for the pharmacokinetic investigations of a 750 mg tablet was developed and validated. This presentation provides details of the method and validation.
Experimental
Chemicals
Ciprofloxacin hydrochloride was obtained from USP and lomefloxacin hydrochloride from Sigma Chemical Co. All the chemicals used were AR grade and all solvents were HPLC grade or better.
Standards/Solutions
Stock solutions of ciprofloxacin and lomefloxacin and the working internal standard solution of lomefloxacin were prepared in acetonitrile/water, 1:1. The intermediate solutions were prepared from the stock solutions in human plasma and were corrected for their salt content. A series of standard samples were prepared in plasma to cover the required quantification range. The standards were stored in 0.100 mL aliquots at -20 ° C.
Quality control (QC) samples were prepared in human plasma at three different concentrations (75.0, 4000, and 8000 ng/mL). These QCs were stored in 0.100 mL aliquots at -20 ° C and were prepared from different stock solutions than from the standard samples.
Extraction Procedure
To all samples except blank-blank samples, 10 m L of internal standard solution (lomefloxacin) is added. After adding 100 m L of water to all samples, 400 m L of acetonitrile is added to precipitate the proteins. The supernatant is removed and dried under a stream of nitrogen. The residue is reconstituted in 200 m L of mobile phase.
LC-MS-MS
The LC-MS-MS system consisted of a Shimadzu LC pump, Perkin-Elmer autosampler and SCIEX API 3000 mass spectrometer with an APCI interface. The ion transitions monitored were m/z 332 --> 231 for ciprofloxacin and m/z 352 --> 249 for lomefloxacin (internal standard). The reconstituted samples were injected onto the LC system flowing at a rate of 0.800 mL/min. The analytes were separated on a C-18, 4.6 X 50 mm column using acetonitrile/water/formic acid (125:400:1) as mobile phase. Ciprofloxacin and lomefloxacin were retained at approximately 1.2 minutes.
Results
The assay was linear over the range 25.0 to 10,000 ng/mL using a plasma volume of only 100 uL. Three validation runs were performed each on separate days. Precision (CV) and accuracy (% bias) across all levels of the calibration range, including the LLOQ, were within + 10% of the theoretical concentrations. No chromatographic interferences or matrix effects from six different lots of plasma were observed indicating the specificity of the method. Stability of ciprofloxacin in plasma was established for 24 hours at room temperature, 4 cycles of freezing and thawing, and over 46 hours in the final extract.
Conclusions
This accurate method requires few extraction steps leading to high throughput and little error. With one LC-MS-MS instrument, more than 600 samples can be routinely analyzed per day.