Journal of Clinical Pharmacy and Therapeutics (2004) 29, 381–387
Determination of ciprofloxacin concentrations in human
serum and urine by HPLC with ultraviolet and
fluorescence detection
K. M. Sowinski and M. B. Kays
Department of Pharmacy Practice, School of Pharmacy and Pharmacal Sciences, Purdue University,
Indianapolis, IN, USA
SUMMARY
Objective: To develop a simple and sensitive high
performance liquid chromatography method for
the determination of ciprofloxacin concentrations
in human serum and urine.
Method: Serum proteins were removed by ultra-
filtration through a filtering device after the
addition of a displacing reagent. Urine samples
were diluted with mobile phase prior to injection.
Separation was achieved with a C18 reverse-
phase column and using ultraviolet (UVD) and
fluorescence detection (FD) for serum samples
and UVD for urine samples.
Results: The quantitation limits of the assay were
20 ng/ml (FD) and 100 ng/ml (UVD) in serum and
1 lg/ml in urine. The assay was successfully
applied to a pharmacokinetic study of ciprofl-
oxacin in healthy volunteers.
Conclusion: The method presented for ciprofl-
oxacin assay in human serum and urine requires
less sample clean up and is more sensitive than
those reported in the literature.
Keywords: ciprofloxacin, fluorescence detection,
ultraviolet detection
INTRODUCTION
Fluoroquinolone antibiotics, including ciprofloxa-
cin, have evolved from antibacterial agents with a
limited gram-negative spectrum to a class of anti-
biotics with a broad spectrum of activity and
extensive indications for the treatment of bacterial
Received 12 January 2004, Accepted 28 May 2004
Correspondence: Dr K. M. Sowinski, W7555 Myers Building,
1001 West 10th Street Indianapolis, IN 46202-2879, USA. Tel.:
+1 317 613 2315x310; fax: +1 317 613 2316;
e-mail: ksowinsk@iupui.edu

2004 Blackwell Publishing Ltd
infections. High performance liquid chromatogra-
phy (HPLC) with either ultraviolet or fluorescence
detection is the most common analytical method
utilized to determine ciprofloxacin concentrations
in human biological fluids during pharmacokinetic
studies. An exhaustive review article describes
these methods (1), in addition to numerous other
published methods for determining ciprofloxacin
in human biologic fluids (2–18). Two reviews (1, 19)
are available that describe the current status of
analytical techniques for ciprofloxacin and other
fluoroquinolone antibiotics. We recently published
a method for the separation of six different
fluoroquinolone antibiotics and application and
validation for determination of levofloxacin con-
centrations in human serum (20). In the current
study, we developed a method for the determin-
ation of ciprofloxacin in human serum and urine
and applied the method to a ciprofloxacin phar-
macokinetic study in normal healthy volunteers.
This method has several advantages over other
ciprofloxacin assays. First, the method avoids
multiple time-consuming extraction steps used in
previous studies by utilizing ultrafiltration of
serum samples and dilution of urine samples.
Second, the method was developed to allow for
quantitation of very low ciprofloxacin concentra-
tions from human serum that was necessary for the
study in which it was applied. The oral bioavaila-
bility of ciprofloxacin is adversely affected by the
concomitant administration of medications con-
taining divalent and trivalent cations (21, 22).
Namely, when administered concomitantly with an
aluminum-, magnesium- or calcium-containing
product, the bioavailability of ciprofloxacin is sig-
nificantly reduced. Given that divalent cations will
reduce ciprofloxacin absorption, we required an
analytical method that would allow quantitation of
ciprofloxacin at very low serum concentrations.
Two recent (16, 23) papers describing ciprofloxacin
381
382 K. M. Sowinski and M. B. Kays
assays reported lower limits of detection of 41 and
50 ng/mL, respectively. But, we required an assay
procedure with an even lower quantitation limit.
Thus, the aim of the present study was to develop
a sensitive, simple, efficient, reliable, accurate and
economical method for the determination of
ciprofloxacin in human serum and urine.
EXPERIMENTAL
Drug standards, dosage forms and chemicals
Ciprofloxacin and levofloxacin (internal standard)
were kindly provided by Bayer Corporation (West
Haven, CT, USA) and RW Johnson Pharmaceutical
Research Institute (Raritan, NJ, USA). Ciprofloxacin
tablets were manufactured by Bayer Corporation.
All chemicals were analytical-reagent grade:
sodium phosphate monobasic dihydrate (NaH2-
PO4Æ2H2O), sodium dodecyl sulfate (SDS) from J. T.
Baker (Phillipsburg, NJ, USA); citric acid, aceto-
nitrile, and methanol from EM Science (Gibbstown,
NJ, USA); sodium phosphate dibasic heptahydrate
(Na2HPO4Æ7H2O) from VWR Scientific Products
(West Chester, PA, USA); tetrabutylammonium
acetate (TBAA) from Sigma Chemical Co. (St Louis,
MO, USA). The stock solutions of 0Æ3 M NaH2-
PO4Æ2H2O and Na2HPO4Æ7H2O were first prepared
and then diluted to 75 mM with water and adjus-
ted to pH 7Æ5 and 0Æ5% (mg/ml) SDS was added
into the buffer. The displacing reagent consisted of
the buffer containing 0Æ5% SDS with acetonitrile
(4 : 1). All of the above solutions were filtered
through a 0Æ45 lm membrane filter and degassed in
an ultrasonic bath for 10 min before use. Water
used for preparation of mobile phase solutions,
stock solutions, etc. was produced from a Barn-
stead, Nanopure Infinity water purification system
(Barnstead/Thermolyne, Dubuque, IA, USA). The
47 mm 0Æ45 lm membrane filter was from Alltech
(Alltech Associates, Inc., Deerfield, IL, USA). Stock
solution of ciprofloxacin was prepared by dissol-
ving ciprofloxacin hydrocholoride in water. The
standard and control ciprofloxacin samples were
prepared by spiking blank serum and urine with
freshly prepared ciprofloxacin stock solution. The
blank serum and urine was obtained from the
drug-free healthy volunteers enrolled in the study.
All samples were stored at )70 C and protected
from light until assayed.

2004 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 29, 381–387
Apparatus
Chromatography was performed on HPLC equip-
ment consisting of a 118 solvent module, a 166 UV
detector, and a 507e autosampler (Beckman
Instrument, Inc., Fullerton, CA, USA), and FP-920
fluorescence detector (Jasco Corporation, Tokyo,
Japan). Data and chromatograms were collected
using Gold NOUVEAU software (Beckman Coulter,
Inc., Fullerton, CA, USA). The pH of the solutions
was adjusted with Corning pH/ion meter 450
(Corning Incorporated, Corning, NY, USA).
Human serum samples were prepared using
Amicon Centrifree micropartition device (Millipore
Corporation, Bedford, MA, USA).
Chromatographic conditions
The mobile phase consisted of 10 mM SDS, 10 mM
TBAA, 25 mM citric acid with 43% acetonitrile.
Prior to use, the mobile phase was filtered through
a 0Æ45 lm filter and degassed by sonication. The
mobile phase was pumped through the system at a
rate of 1 ml/min. Analytical separation and guard
columns were Adsorbosphere HS C18 5 l (Alltech
Associates, Inc., Deerfield, IL, USA). The dimension
of the separation column was 250 mm L · 4Æ6 mm
ID with 5 lm particle size. The guard column
cartridge was 7Æ5 mm L · 4Æ6 mm ID with 5 lm
particles of identical chemistry to the separation
column. Prefilter elements were 4Æ0 mm diameter
with 2 lm particles. Between the sample injections,
the injection needle was washed with 70% meth-
anol. All experiments were carried out at ambient
temperature at approximately 23 C. The UV
detector wavelength was set at 280 nm and the
fluorescence detector wavelength was set at exci-
tation 293 nm, emission 450 nm.
Sample preparation – serum assay
The blank human serum, calibrator, quality control
and unknown samples were thawed and vortexed
for 30 s. Four hundred and fifty microliters of dis-
placing reagent (80 % [0Æ5 mg/ml SDS in 75 mM
phosphate at pH 7Æ5]: 20 % acetonitrile) and 50 ll
of internal standard (30 lg/ml levofloxacin) or
water (blank samples) were added to 500 ll of
sample and vortexed for 30 s. Each sample was
transferred to an Amicon Centrifree micropartition

device and centrifuged at 1500 g for 30 min. Two
hundred microlitres of each ultrafiltrate was
transferred to a vial for HPLC analysis and the
autosampler was programmed to inject 20 ll.
Calibration standards – serum assay
Calibration curves were produced by injecting
processed samples prepared from serum stocks
that were spiked to ciprofloxacin concentrations of
10 000, 5000, 2000, 1000, 500, 200 and 100 ng/ml for
UV detection and 1000, 500, 200, 100, 50, and
20 ng/ml for fluorescence detection. The linear
equation describing the relationship between
ciprofloxacin concentration and the peak area ratio
was determined using weighted linear regression
analysis, with the reciprocal of the square of the
concentration for each standard as the weight. The
coefficient of determination (regression sum of
squared residuals/total sum of squared residuals)
was used as an estimate of goodness-of-fit. The
final decision on the suitability of the daily calib-
ration curves was made based on the control
sample results.
Within-day variability and precision were deter-
mined using quality control samples made by spi-
king blank serum to ciprofloxacin concentrations of
6000, 1500, 375 and 150 ng/ml. Five processed
samples were injected at each control concentration
on 1 day. Between-day variability and precision
were evaluated by injecting one processed sample at
each control concentration each day for 7 days.
Control sample concentrations were determined
from the standard curve run on each day of analysis.
Statistics for the inter-assay study were calculated by
using the mean of the replicates for each day. Pre-
cision was expressed as the percent coefficient of
variation. Accuracy was expressed as the ratio of the
mean observed to theoretical concentration as:
Accuracy (%) = 100 · (predicted concentration/
nominal concentration).
Sample preparation – urine assay
The blank human urine, calibrator, quality control
and unknown samples were thawed and vortexed
for 30 s. Fifty microliters of urine sample was
diluted with 1000 ll of mobile phase, to which
50 ll of internal standard (30 lg/ml levofloxacin)
or water (blank samples) was added. The samples

2004 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 29, 381–387
Ciprofloxacin determination by HPLC 383
were vortexed for 30 s, transferred to a vial for
HPLC analysis and the autosampler was pro-
grammed to inject 20 ll.
Calibration standards – urine assay
Calibration curves were produced by injecting
diluted samples prepared from blank urine stocks
that were spiked to ciprofloxacin concentrations of
500, 100, 50, 10, 5, 2 and 1 lg/ml. Analyses were
performed as described in the serum section above.
Within-day variability and precision were
determined using quality control samples made by
spiking blank urine to concentrations of 375, 37Æ5
and 3Æ75 lg/ml. Five samples were injected at each
control concentration on one day. Between-day
variability and precision were evaluated by inject-
ing one processed sample at each control concen-
tration each day for 6 days. Control sample
concentrations were determined from the standard
curve run on each day of analysis. Statistics for the
inter-assay study were calculated by using the
mean of the replicates for each run (i.e. six runs for
each quality control sample). Precision was
expressed as the percent coefficient of variation.
Accuracy was expressed as the ratio of the mean
observed to theroretical concentration as: Accuracy
(%) = 100 · (predicted concentration/nominal con-
centration).
Application of method to a ciprofloxacin
pharmacokinetic study
Subjects were caffeine and alcohol free for at least
12 h before and during the study day, and avoided
all medications for at least 24 hours before and
during the study day. Each subject received 750 mg
ciprofloxacin orally. Blood samples were obtained
at 0 (baseline), and 0Æ5, 1, 1Æ5, 2, 3, 4, 6, 8, 12 and
24 h after the oral dose. All samples were obtained
into non-heparinized evacuated blood collection
tubes, allowed to clot at room temperature, and
centrifuged at 3000 rpm for 15 min. After serum
separation, all samples were stored at )70 C until
analysis. Urine was collected from each volunteer
at the following times after the ciprofloxacin dose:
0–2, 2–4, 4–8, 8–12 and 12–24 h. The volume was
recorded and a 5 ml sample was collected and
stored at )70 C until analysis. The concentra-
tions of ciprofloxacin in serum and urine were
384 K. M. Sowinski and M. B. Kays

Fig. 1. Chromatograms of blank serum sample spiked with ciprofloxacin and internal standard (levofloxacin) (Panel A),
subject serum sample during ciprofloxacin pharmacokinetic study spiked with internal standard (levofloxacin) (Panel B),
blank subject serum sample during ciprofloxacin pharmacokinetic study (Panel C), Peak 1: levofloxacin (internal
standard); Peak 2: ciprofloxacin. Detection was by ultraviolet detection as described in the methods.

2004 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 29, 381–387
 Ciprofloxacin determination by HPLC 385

determined based on the daily calibration curves. Table 1. Calibration curve statistics in serum and urine
During the analysis of unknown samples from the by ultraviolet and fluorescence detection
pharmacokinetic study, the method validation was
continued. Acceptance of assay results was deter- Assay Slope Intercept
mined by monitoring the predicted calibrators and
Serum 0Æ00088 ± 0Æ00005 0Æ00541
quality control results.
Urine 0Æ112 ± 0Æ0059 )0Æ0093

RESULTS AND DISCUSSION

Selectivity and chromatography
Representative chromatograms are illustrated in
Fig. 1. These chromatograms include a processed
blank serum sample, processed subject serum sam-
ple and processed calibrator sample. As is illustrated
in each of these chromatograms, the retention times
of the levofloxacin (internal standard) and ciprofl-
oxacin are approximately 7 and 9 min, respectively.
The chromatograms show that ciprofloxacin and
levofloxacin are completely resolved from one
another. No interferences were seen in any individ-
ual study subject’s baseline drug-free serum or urine
(Representative subject shown in Fig. 1).
Linearity
The serum calibration curve was constructed of
seven calibrators (100–10,000 ng/ml) for the UV
detection assay and five calibrators (20–1000 ng/ml)
for the FD assay. It was necessary to use two calib-
ration curves with two different detection methods
as the UV detection assay did not achieve sufficient
sensitivity and the calibration curve for the FD assay
was not linear above 1000 ng/ml. Both calibration
curves were linear over the specified ranges. The
urine calibration curve was constructed of six cali-
brators (1–500 lg/ml). The summary statistics for
the calibration curves in serum and urine are illus-
trated in Table 1. The coefficient of determination
was >0Æ995 on all calibration curves in serum and
urine.
Precision and accuracy
The minimum quantifiable serum concentration
was 100 ng/ml (UV detection) and 20 ng/ml (FD).
The inter-assay CV at these concentrations were
2Æ26 and 2Æ86 %, respectively. The minimum
quantifiable urine concentration was 1 lg/ml. The
inter-assay CV at this concentration was 1Æ26 %.
The results of the intra-assay (within day) and
inter-assay (between day) study for the serum
assay are listed in Table 2. The results of the intra-
assay (within day) and inter-assay (between day)
study for the urine assay are listed in Table 3. The
results indicate that the method is reliable, repro-
ducible and accurate.

Table 2. Intra- and inter-day

precision and accuracy of Nominal Predicted Precision Accuracy
ciprofloxacin in human serum concentration (ng/ml) concentrations (ng/ml) CV (%) (%)
by UV detection and
Inter-day precision and accuracy, (n = 7 days of replicate samples)
fluorescence detection
150UV 155Æ7 ± 11Æ2 7Æ16 103Æ8
375UV 396Æ1 ± 5Æ2 1Æ31 105Æ6
1500UV 1549Æ1 ± 65Æ6 4Æ23 103Æ3
6000UV 5681Æ3 ± 291Æ9 5Æ14 94Æ7
150FD 157Æ3 ± 7Æ4 4Æ70 104Æ9
375FD 385Æ6 ± 16Æ3 4Æ23 102Æ8
Intra-day precision and accuracy, (n = 7 replicate samples)
150UV 153Æ4 ± 5Æ3 3Æ48 97Æ3
375UV 386Æ1 ± 12Æ6 3Æ26 99Æ5
1500UV 1559Æ8 ± 48Æ0 3Æ08 101Æ3
6000UV 5599Æ9 ± 118Æ4 2Æ11 97Æ8
150FD 157Æ4 ± 3Æ4 2Æ19 101Æ6
375FD 386Æ9 ± 7Æ6 1Æ97 96Æ8

2004 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 29, 381–387
386 K. M. Sowinski and M. B. Kays

Table 3. Intra- and inter-day

Nominal concentration Predicted concentrations Precision Accuracy precision and accuracy of
(lg/ml) (lg/ml) CV (%) (%) ciprofloxacin in human urine by
UV detection.
Inter-day precision and accuracy, (n = 5 days of replicate samples)
3Æ75 3Æ74 ± 0Æ16 4Æ27 99Æ6
37Æ5 37Æ2 ± 2Æ1 5Æ64 99Æ3
375 386 ± 35Æ9 9Æ27 103Æ1
Intra-day precision and accuracy, (n = 5 replicate samples)
3Æ75 3Æ84 ± 0Æ10 2Æ69 102Æ4
37Æ5 39Æ8 ± 0Æ73 1Æ82 106Æ0
375 397 ± 17Æ0 4Æ29 105Æ8

10000
CONCLUSIONS
Ciprofloxacin concentration (ng/mL)

The assay described in this paper is a useful, rel-

atively simple, highly sensitive, precise, accurate
1000
and selective HPLC method for determining
ciprofloxacin concentrations in human serum and
urine. The method was applied successfully to a
100 pharmacokinetic study of ciprofloxacin in human
healthy volunteers.

10 ACKNOWLEDGEMENTS
0 5 10 15 20 25

Time (h) Supported in part by grants from Geltex Pharma-

ceuticals (Waltham, MA, USA) and by grant MO1
Fig. 2. Individual ciprofloxacin concentrations vs. time
RR00750 from the National Institutes of Health
in a representative subject. The closed circles (d) repre-
sent the observed data points following a 750 mg (Bethesda, MD, USA).
ciprofloxacin oral dose alone, with calcium acetate ( )
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