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
Date
Authors
Kokwaro, G.
Muchohi, Simon N.
Thuo, Nahashon
Karisa, Japhet
Muturi, Alex
Maitland, Kathryn
Journal Title
Journal ISSN
Volume Title
Publisher
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