Determination of ciprofloxacin in human plasma using high-performance liquid chromatography coupled with fluorescence detection: Application to a population pharmacokinetics study in children with severe malnutrition
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Journal of Chromatography B
Abstract
Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma
drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence
detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin)
was added to plasma aliquots (200uL) prior to protein precipitation with acetonitrile. Ciprofloxacin
and IS were eluted on a Synergi Max-RP analytical column (150mm×4.6mm i.d., 5um particle
size) maintained at 40 ◦C. The mobile phase comprised a mixture of aqueous orthophosphoric acid
(0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A
fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for
ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin
were linear over the concentration range of 0.02–4ug/mL, with correlation coefficients (r2)≥0.998. Intraand
inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105%
for quality control samples (0.2, 1.8 and 3.6ug/mL). The mean (SD) extraction recoveries for ciprofloxacin
from spiked plasma at 0.08, 1.8 and 3.6ug/mL were 72.8±12.5% (n = 5), 83.5±5.2% and 77.7±2.0%,
respectively (n = 8 in both cases). The recovery for IS was 94.5±7.9% (n = 15). The limits of detection and
quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least
one month when stored at −15 ◦C to −25 ◦C and −70 ◦C to −90 ◦C. This method was successfully applied
to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin
in malnourished children.
Description
Article published in Journal of Chromatography B
Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin) was added to plasma aliquots (200uL) prior to protein precipitation with acetonitrile. Ciprofloxacin and IS were eluted on a Synergi Max-RP analytical column (150mm×4.6mm i.d., 5um particle size) maintained at 40 ◦C. The mobile phase comprised a mixture of aqueous orthophosphoric acid (0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin were linear over the concentration range of 0.02–4ug/mL, with correlation coefficients (r2)≥0.998. Intraand inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105% for quality control samples (0.2, 1.8 and 3.6ug/mL). The mean (SD) extraction recoveries for ciprofloxacin from spiked plasma at 0.08, 1.8 and 3.6ug/mL were 72.8±12.5% (n = 5), 83.5±5.2% and 77.7±2.0%, respectively (n = 8 in both cases). The recovery for IS was 94.5±7.9% (n = 15). The limits of detection and quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least one month when stored at −15 ◦C to −25 ◦C and −70 ◦C to −90 ◦C. This method was successfully applied to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin in malnourished children.
Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin) was added to plasma aliquots (200uL) prior to protein precipitation with acetonitrile. Ciprofloxacin and IS were eluted on a Synergi Max-RP analytical column (150mm×4.6mm i.d., 5um particle size) maintained at 40 ◦C. The mobile phase comprised a mixture of aqueous orthophosphoric acid (0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin were linear over the concentration range of 0.02–4ug/mL, with correlation coefficients (r2)≥0.998. Intraand inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105% for quality control samples (0.2, 1.8 and 3.6ug/mL). The mean (SD) extraction recoveries for ciprofloxacin from spiked plasma at 0.08, 1.8 and 3.6ug/mL were 72.8±12.5% (n = 5), 83.5±5.2% and 77.7±2.0%, respectively (n = 8 in both cases). The recovery for IS was 94.5±7.9% (n = 15). The limits of detection and quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least one month when stored at −15 ◦C to −25 ◦C and −70 ◦C to −90 ◦C. This method was successfully applied to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin in malnourished children.
Keywords
Ciprofloxacin, HPLC fluorescence detection, Plasma, Protein precipitation, Validation