ISSN 0377-9556 (PRINT) ISSN 2383-9457 (ONLINE)

약학회지 제 62 권 제 1 호 1~6 (2018)

Yakhak Hoeji Vol. 62, No. 1
DOI 10.17480/psk.2018.62.1.1

종설
Short Report

Quantification of Ceftriaxone in Rat Plasma Using Hydrophilic Interaction

Chromatography-Tandem Mass Spectrometry

Ju-Hyun Kim and Hye Suk Lee#

BK21 PLUS Team for Creative Leader Program for Pharmacomics-based Future Pharmacy and Integrated Research Institute of
Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon 14662, Republic of Korea
(Received December 29, 2017; Revised January 9, 2018; Accepted January 10, 2018)

Abstract — Ceftriaxone is a parenteral cephalosporin with a broad spectrum of activity against gram-positive and gram-
negative pathogens. For the quantification of ceftriaxone in rat plasma, a selective and sensitive hydrophilic interaction chro-
matography-tandem mass spectrometric method was developed and validated using protein precipitation with acetonitrile
as a sample clean-up procedure. The analytes were separated in a Luna HILIC column with a gradient elution of 5% ace-
tonitrile in 100 mM ammonium formate and 100% acetonitrile in 0.1% formic acid, and mass-to-charge ratios (m/z) were
determined in selective reaction monitoring mode using tandem mass spectrometry with m/z 554.9>395.7 for ceftriaxone
and m/z 172.2>154.2 for gabapentin (internal standard). The standard curve was linear over the concentration ranges of 5−
5000 ng/mL using 30 µL rat plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four
QC levels were 5.2 to 11.6% and −7.3 to 14.0%, respectively. The overall recovery of ceftriaxone from rat plasma using pro-
tein precipitation was 90.9±3.8%. This method was successfully applied to the pharmacokinetics of ceftriaxone after its
intravenous administration at a dose of 10 mg/kg in male Sprague-Dawley rats.

Keywords ceftriaxone, hydrophilic interaction chromatography-tandem mass spectrometry, rat plasma, pharmacokinetics

Introduction detector4-11), tandem mass spectrometry (MS/MS)12), high-res-

Ceftriaxone is a parenteral cephalosporin with a broad spec-
trum of activity against gram-positive and gram-negative patho-
gens and prevalently used in the treatment of severe community-
acquired infections such as pneumonia, meningitis, intra-
abdominal infections orsepsis.1) A pharmacokinetic evaluation
revealed that ceftriaxone is mainly eliminated by the renal
route, in which glomerular filtration and secretion via organic
anion transporter 1 (OAT1) were involved.2,3)
Bioanalytical methods for the quantification of ceftriaxone in
various biological samples such as plasma, urine, tissues,
bones or cerebrospinal fluid were reported: reverse-phase high
performance liquid chromatography (RPLC) systems with UV
#Corresponding Author
Hye Suk Lee
Drug Metabolism & Bioanalysis Lab., College of Pharmacy, The
Catholic University of Korea, Jibongro 43, Wonmi-gu, Bucheon,
Gyeonggi-do14662, Korea
Tel.: 02-2164-4061 Fax.: 032-342-2013
E-mail: sianalee@catholic.ac.kr
olution mass spectrometry13), or spectrofluorometric meth-
od14,15) were used. Hydrophilic interaction chromatography
(HILIC) has been increasingly used as an attractive comple-
ment to RPLC in the analysis of polar or hydrophilic drugs and
metabolites for bioanalysis and metabonomic studies.16-28)
HILIC uses polar stationary phases, such as bare silica, amino,
cyano, and sulfobetaine-type zwitter ionic columns, and low
aqueous/high organic mobile phase. The high organic mobile
phase in HILIC increases the sensitivity due to more favor-
able desolvation and electrospray (ESI) ionization conditions,
and can provide direct injection of the organic extracts
obtained from protein precipitation, liquid-liquid extraction, or
solid-phase extraction, resulting in the removal of time-con-
suming evaporation and reconstitution steps during sample
preparation.
HILIC has been used for the analysis of the antibiotics such
as aminoglycosides, β-lactam, tetracyclines, and others in dif-
ferent matrices.23) However, there was no report on HILIC-
MS/MS method for determination of ceftriaxone in rat plasma.
A simple, rapid, selective, and sensitive HILIC-ESI-MS/MS

1
2 Ju-Hyun Kim and Hye Suk Lee
method was developed and validated for determination of cef-
triaxone in rat plasma.
Experimental
Materials and reagents
Ceftriaxone was a gift from Kyung Dong Pharm Co. Ltd.
(Seoul, Korea). Gabapentin (used as an internal standard, I.S.)
was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).
Acetonitrile and water (LC-MS grade) were obtained from
Fisher Scientific (Pittsburgh, PA, USA). Other chemicals used
were of the highest quality available. Drug-free rat plasma con-
taining sodium heparin as the anticoagulant was obtained from
male Sprague-Dawley rats.
Preparation of calibration standards and quality con-
trol samples
The stock solution of ceftriaxone (1 mg/mL) was prepared in
deionized water and was serially diluted with water to work-
ing standard solutions of 0.1, 0.2, 2, 16, 40, 80, and 100 μg/mL.
Gabapentin stock solution (1 mg/mL) was prepared in deion-
ized water and was diluted to 50 ng/mL with acetonitrile. All
o
standard solutions were stored at 4 C in amber glass vials
when not in use.
Rat plasma calibration standards of ceftriaxone at concentra-
tions of 5, 10, 100, 800, 2000, 4000, and 5000 ng/mL were pre-
pared by adding 1.5 μL of the working standard solutions to
28.5 μL of drug-free rat plasma. To prepare quality control
(QC) samples at 5, 15, 500, 3500, and 50000 ng/mL, 200 μL of
the appropriate working standard solutions at 0.1, 0.3, 10, 70,
and 1000 μg/mL were added to 3800 μL of drug-free rat plasma.
Aliquots (30 μL) of QC samples were transferred into polypro-
o
pylene tubes and stored at −80 C until analysis.
Sample preparation
Thirty μL of rat blank plasma, calibration standards, and QC
samples were vortex-mixed with 100 μL of gabapentin in ace-
tonitrile (50 ng/mL, I.S.) for 3 min at high speed. After centrif-
ugation at 13,000 rpm and 4oC for 8 min, the aliquot (2 μL)
was injected into the LC-MS/MS system.
HILIC-MS/MS Analysis
The LC-MS/MS system consisted of an Agilent Infinity 1290
coupled with a 6490 triple quadrupole mass spectrometer (Agi-
Vol. 62, No. 1, 2018
lent Technologies, Wilmington, DE, USA). Separation was per-
formed on a Luna HILIC column (3 μm, 2.0 mm i.d. × 100 mm,
Phenomenex, Torrance, CA, USA) with a gradient elution of
5% acetonitrile in 100 mM ammonium formate (mobile phase
A) and 100% acetonitrile with 0.1% formic acid (mobile phase
B) at a flow rate of 0.4 mL/min: 85% mobile phase B for 0.5
min, 85% to 65% mobile phase B for 0.7 min, 65% mobile
phase B for 2.8 min, 65% to 85% mobile phase B for 0.01 min,
85% mobile phase B for 6 min. The column and autosampler
were maintained at 50oC and 4oC, respectively. ESI source set-
tings for ionization of ceftriaxone and gabapentin in the posi-
tive mode were as follows: gas temperature, 150oC; gas flow,
11 L/min; nebulizer, 20 psi; sheath gas temperature, 300oC;
sheath gas flow, 10 L/min; capillary voltage, 4500 V. Fragmenta-
tion of molecular ions for ceftriaxone and gabapentin was per-
formed at a collision energy of 10 eV. Selected reaction monitoring
(SRM) mode was used for quantification: m/z 554.9→395.7 for
ceftriaxone and m/z 172.2→154.2 for gabapentin. Mass Hunter
software (Agilent Technologies) was used for LC-MS/MS sys-
tem control and data processing.
Method Validation
In order to complete the method validation, the batches con-
sisting of calibration standards covering the range of 5-5000 ng/
mL and five replicates of QC samples at 5, 15, 500, and
3500 ng/mL were analyzed on three consecutive days. The rel-
ative error (RE), the percentage of deviation of the mean from
the true values, served as a measure of the accuracy, and the
coefficient of variation (CV) indicated the precision.
Recovery of ceftriaxone and gabapentin was determined by
comparing the mean peak areas of the analytes spiked before
extraction into the blank plasma sources with those of the ana-
lytes spiked post-extraction into the blank plasma extracts at
three concentrations, i.e., 15, 500, and 3500 ng/mL.
Post-extraction batch integrity was determined by batch
reinjection after 24 hours of storage in the auto-sampler.
Pharmacokinetic study of ceftriaxone in rats
The developed HILIC-MS/MS method was applied to the
pharmacokinetic study of ceftriaxone after intravenous admin-
istration in male Sprague-Dawley rats (body weight 240−263 g)
(Samtako Co., Osan, Korea). Animals were kept in plastic
cages with free access to standard rat diet (Samtako Co.) and
water. The room was maintained at a temperature of 22−24oC
 HILIC-MS/MS of ceftriaxone in rat plasma 3

with a 12 h light/dark cycle and relative humidity of 50±10%.

Rats were anesthetized by isoflurane and were cannulated with
polyethylene tubing (PE-50, Natsume Co., Tokyo, Japan) in the
jugular vein for blood sampling and in the femoral vein for
intravenous injection. Each rat was housed individually in a rat
metabolic cage and allowed to recover from anesthesia for 1
day prior to the start of the study. Rats were not restrained at
any time during the study. Heparinized isotonic saline (10 IU/
mL) was used to flush the catheters to prevent blood clotting.
Ceftriaxone was dissolved in saline and was injected into the
femoral vein at a dose of 10 mg/kg (n=4). Blood samples
(approximately 0.1 mL) were collected before (control) and at
1, 5, 15, 30 min, and 1, 2, 4, 6, 8, and 24 h after drug adminis-
tration. Plasma samples were harvested by centrifugation at
13000 rpm for 8 min and stored at −80oC until analysis.
The area under the plasma concentration-time curve (AUC),
the terminal elimination half-life (t1/2), the clearance (Cl), and
the volume of distribution at steady state (Vss) were calculated
using a non-compartment analysis (WinNonlin, Pharsight,
Mountain View, CA, USA).

Results and discussion

HILIC-MS/MS
Electrospray ionization of ceftriaxone and gabapentin pro-
duced the abundant molecular ion ([M+H]+) at m/z 554.9 and
172.2, respectively, without evidence of fragmentation or
adduct formation. The [M+H]+ ions of ceftriaxone and gab-
apentin were selected as the precursor ion and were subse-
quently fragmented in MS/MS mode in order to obtain the
product ion spectra, yielding useful structural information (Fig.
1). Ceftriaxone produced the major product ion at m/z 395.7 by
the cleavage of the thioether C-S bond to C3 substituent chain
of the β–lactam ring (Fig. 1A). Gabapentin showed a promi-
nent product ion at m/z 154.2 by loss of water from the
[M+H]+ ion (Fig. 1B). For the quantification of ceftriaxone in
the plasma, SRM mode was used and the transition of the pre-
cursor ion to a product ion was monitored as followings: m/z
554.9 to m/z 395.7 for ceftriaxone and m/z 172.2 to m/z 154.2
for gabapentin.
Ceftriaxone showed better retention and peak shape, and
less matrix effect on a Luna HILIC column, compared with an
Atlantis HILIC silica column (Waters Co., Milford, MA, USA).
Use of 100 mM ammonium formate itself resulted in better
Fig. 1 − Product ion mass spectra of (A) ceftriaxone and (B) gabapentin
(internal standard).
retention and sensitivity of ceftriaxone, compared with pH 3.0
and 4.5 ammonium formate buffers on a Luna HILIC column.
The high acetonitrile content in the mobile phase increased
electrospray ionization efficiency for ESI-MS/MS detection and
resulted in high sensitivity of ceftriaxone.
In the analysis of the blank plasma samples obtained from
six different rats, no interference peak was observed at the
retention times of ceftriaxone (2.2 min) and gabapentin (1.4
min), indicating the selectivity of the present method (Fig. 2A).
Sample carryover effect was not observed.
Method validation
Calibration curve was obtained over the concentration
ranges of 5 to 5000 ng/mL of ceftriaxone in rat plasma. Linear
regression analysis with a weighting of 1/concentration gave
the optimum accuracy (RE, −5.0 to 5.3%) and precision (CV,
≤11.7%) of the corresponding calculated concentrations at each
level (Table 1). The low CV value (3.0%) for the slope indi-

J. Pharm. Soc. Korea

4 Ju-Hyun Kim and Hye Suk Lee

Fig. 2 − SRM chromatograms of (A) a rat blank plasma, (B) a rat plasma sample spiked with 5 ng/mL of ceftriaxone, and (c) a rat plasma
sample obtained 5 min after intravenous administration of ceftriaxoneat a dose of 10 mg/kg to a male Sprague-Dawley rat.

Table I − Calculated concentrations of ceftriaxone in calibration standards prepared with rat plasma (n = 3)

Statistical Theoretical concentration (ng/mL)

slope r2
variable 5 10 100 800 2000 4000 5000
Mean (ng/mL) 5.0 9.5 99.1 842.2 2070.6 3803.2 5085.3 6.8e-5 0.998
CV (%) 4.2 11.7 10.2 7.8 3.3 3.2 2.2 3.0 0.1
RE (%) 0.0 −5.0 −0.9 5.3 3.5 −4.9 1.7

cated the repeatability of the method (Table 1).
Table 2 shows a summary of the intra- and inter-day preci-
sion and accuracy data for QC samples containing ceftriaxone.
Both intra- and inter-day CV values ranged from 5.2% to
11.6% at four QC levels. Intra- and inter-assay RE values were
−7.3% to 14.0% at four QC levels. These results indicated
acceptable accuracy and precision of the present method. The
lower limit of quantification (LLOQ) for ceftriaxone was set at
5 ng/mL using 30 μL of rat plasma with a signal-to-noise ratio
higher than 10 (Fig. 2B). After 50-fold dilution of the dilution
quality control samples (50000 ng/mL), CV and RE for ceftriax-
one were 10.8% and −9.1%, respectively, indicating the accept-
ability of the 50-fold dilution prior to analysis.
The overall recovery of ceftriaxone from rat plasma using
protein precipitation was 90.9±3.8% at 15, 500, and 3500 ng/
mL levels and was consistent over the concentration range of
15-3500 ng/mL. Recovery of gabapentin (I.S.) was 92.2±3.9%.
Re-analysis of the organic extracts stored for 24 h at 4oC
showed that acceptable accuracy (RE: −11.0~−10.7%) and pre-
cision (≤ 11.3%) for the QC samples at 15 and 3500 ng/mL.

Table II − Precision and accuracy of ceftriaxone in quality control samples

Statistical variable Intra-day (n = 5) Inter-day (n = 15)
QC (ng/mL) 5.0 15.0 500.0 3500.0 5.0 15.0 500.0 3500.0
Mean (ng/mL) 5.5 14.5 472.4 3812.9 5.7 13.9 496.9 3748.3
CV (%) 5.2 10.5 7.0 6.0 7.4 10.4 11.6 8.2
RE (%) 10.4 −3.5 −5.5 8.9 14.0 −7.3 0.6 7.1

Vol. 62, No. 1, 2018

 HILIC-MS/MS of ceftriaxone in rat plasma
Korea, 2016 and the Bio & Medical Technology Development
Program of the National Research Foundation (NRF) funded by
the Korean government (MSIT) (NRF-2017M3A9F5028608)
Fig. 3 − Mean plasma concentration-time profiles of ceftriaxone after
its intravenous administration at a dose of 10 mg/kg (n = 4) to
male Sprague-Dawley rats. Each point represents the
mean±S.D.
Pharmacokinetics of ceftriaxone in rats
The method was applied successfully to study pharmacoki-
netics after intravenous injection of ceftriaxone at a dose of
10 mg/kg in male Sprague Dawley rats. Figure 2C shows rep-
resentative SRM chromatograms from the analysis of a plasma
sample obtained 5 min after intravenous administration of cef-
triaxone in a rat.
After intravenous administration of ceftriaxone at 10 mg/kg
in male SD rats, its mean plasma concentration-time curves
declined in a poly-exponential fashion (Fig. 3). The plasma con-
centrations of ceftriaxone at 8 h and 24 h after intravenous
administration were less than the LLOQ (5 ng/mL). After
intravenous administration of ceftriaxone at 10 mg/kg dose, the
AUC, t1/2, Vss, and Cl values of ceftriaxone were 15.5±3.9 μg·h/
mL, 0.78±0.05 h, 608.4±95.6 mL/kg, and 668.4±149.5 mL/h/
kg, respectively.
Conclusion
A rapid, simple, sensitive, reproducible, and selective
HILIC-MS/MS method for the quantification of ceftriaxone in
rat plasma was developed using protein precipitation as the
sample preparation. This study demonstrated the acceptable
sensitivity (LLOQ, 5 ng/mL), selectivity, precision, accuracy,
and stability of the present method. The pharmacokinetic
parameters of ceftriaxone were evaluated after intravenous
administration of ceftriaxone in rats.
5
Acknowledgements
This study was supported by The Catholic University of
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