1. Methods
Dog Plasma Sample Preparation and Extraction Procedure
• Dog plasma with dipotassium ethylenediaminetetraacetic acid (K2EDTA)
was treated with 100 µg/mL of tazobactam.
• Samples were extracted from 50 µL of dog plasma using protein
precipitation with 300 µL of acetonitrile.
• The supernatant was transferred, evaporated, and reconstituted
in 100 µL of 20:80 acetonitrile/0.1% formic acid in water.
• Calibration range was 10.0 to 5000 ng/mL.
Human Plasma Sample Preparation and Extraction Procedure
• Human plasma with K2EDTA was treated with 100 µg/mL of tazobactam.
• Samples were extracted from 20 µL of human plasma using protein precipitation with 300 µL of
acetonitrile.
• The supernatant was transferred, evaporated, and reconstituted in 500 µL of 20:80
acetonitrile/0.1% formic acid in water.
• Calibration range was 100 to 50000 ng/mL.
Human Intestinal Chyme Sample Preparation and Dilution
• Human intestinal chyme was diluted 1:1 with 8 M guanidine hydrochloride in
100 mM ammonium bicarbonate.
• The standard curve was prepared in fasted-state small intestinal fluid conditions (FaSSIF) buffer
proxy matrix and then diluted 1:1 with 8 M guanidine hydrochloride in 100 mM ammonium
bicarbonate.
• Dilution was performed in a wet ice bath for stability.
• 50 µL of human intestinal chyme was diluted using 50 µL of 1:1 blank FaSSIF buffer/
8 M guanidine hydrochloride in 100 mM ammonium bicarbonate.
• All samples were diluted using 400 µL of 0.1% formic acid in water and 50 µL of the diluted
sample was transferred to a second collection plate.
• 500 µL of internal standard working solution was added, and, after vortex mixing, 50 µL of sample
was transferred to a collection plate for further dilution in 150 µL of 0.1% formic acid in water.
• Calibration range was 1.00 to 2000 µg/mL.
References
1. Kokai-Kun JF, Bristol JA, Setser J, et al. Nonclinical safety assessment of SYN-004: An oral
beta-lactamase for the protection of the gut microbiome from disruption by biliary-excreted,
intravenously administered antibiotics. Int. J. Toxicol. 2015 (In Press).
2. Hyo-Eon J, Su-Eon J and Han-Joo M. Recent bioanalytical methods for quantification
of third‑generation cephalosporins using HPLC and LC-MS/MS and their applications in
pharmacokinetic studies. Biomed. Chem. 2014;28:1565–1587.
3. Crowther GS and Wilcox MH. Antibiotic therapy and Clostridium difficile infection-primum non
nocere‑first do no harm. Infect. Drug Resist. 2015;8:333–337.
Overview
• This method was developed for quantification of ceftriaxone in various matrices to support
clinical and non-clinical studies of a novel oral β-lactamase enzyme.
• Ceftriaxone is a class III cephalosporin antibiotic used to treat a variety of infections.
• Digestive issues, including Clostridium difficile infections, related to the administration of
ceftriaxone are common and can be quite severe.
• SYN-004 is a first-in-class, recombinant β-lactamase drug that is administered orally in
conjunction with intravenous ceftriaxone (and other β-lactam antibiotics). SYN-004 is intended
to degrade antibiotics excreted into the intestines and thus has the potential to protect the gut
microbiome from disruption by these antibiotics [1].
• The co-administration of the oral β-lactamase enzyme mitigates the harmful gastrointestinal
effects of ceftriaxone while allowing the beneficial targeted action of the drug to combat
bacterial infections in the respiratory, circulatory, and central nervous systems.
• Existing analytical methods have quantitation levels in the microgram per milliliter range;
however, because ceftriaxone exhibits high permeability through biological membranes,
lower limits of quantitation resulted in a more complete and accurate analysis of the
compound in various biological fluids across time to fully understand the scope of action of
the drug in vivo.
Introduction
• Ceftriaxone has been analyzed using high-performance liquid chromatography with ultraviolet
detection (HPLC-UV); however, there is a notable lack of analytical methods using HPLC with
tandem mass spectrometry detection (HPLC/MS/MS) for quantification in biological matrices [2].
• Method development challenges included the development of a novel LC/MS/MS assay and the
extraction of the drug from plasma and human intestinal chyme.
• Selection of an enzymatic inhibitor to control for the action of β-lactamase enzyme following
collection in human intestinal chyme was required to inactivate the co-administered enzyme to
accurately quantify ceftriaxone and establish stability of ceftriaxone in matrix.
• Determination of a suitable surrogate matrix control matrix for analysis of the intestinal chyme
samples was achieved through comparative analyses.
• This method has sensitivity to reach nanogram per milliliter analytical concentrations of
ceftriaxone for quantification in plasma and was successfully applied to analysis of human
plasma and intestinal chyme, as well as dog plasma samples from pharmacokinetic studies.
• Due to the highly polar nature and the complex atomic heterogeneity of ceftriaxone, extensive
LC method development was undertaken to determine conditions for analysis that provided
acceptable peak shape and retention using reagents that are favorable for electrospray
ionization.
• Using an antibiotic inactivation approach can reduce the risk of negative side effects that arise
from the elimination of the normal microbiota in the gut and allow the continued use of broad-
spectrum cephalosporins, which are widely prescribed for treatment of a variety of serious
bacterial infections [3].
Results
Conclusions
Acknowledgments
• Suzanne Spencer for poster preparation
QC sample statistics indicate good accuracy and precision from three validation runs for dog and
human plasma (n = 18) and three method development runs for human intestinal chyme (n = 18).
Rapid Analysis of Ceftriaxone in Human Intestinal Chyme, Human Plasma, and Dog Plasma by HPLC/MS/MS
Todd Lusk1, John F. Kokai-Kun2, Michael Schlosser3, Stacey Zeman1, Sara Brady1, Thad Yousey1, Dan Mulvana1
1Q2 Solutions, Ithaca, NY USA; 2Synthetic Biologics, Inc., Rockville, MD USA; 3MSR Pharma Services, Inc., Lincolnshire, IL USA
Copyright ©2016 Q2 Solutions. All rights reserved.
• Three novel LC/MS/MS analytical methods for the quantitation of ceftriaxone in various
matrices that were successfully applied in support of clinical and non-clinical studies.
• The LC/MS/MS methods are capable of achieving the LLOQ requirements associated with
analysis of ceftriaxone in various biological fluids.
• These methodologies may be applied to other drugs in the cephalosporin family.
• Inhibition of ex vivo enzymatic activity is an important aspect of analyzing highly reactive
drugs in order to accurately assess in vivo interactions of therapeutic enzymes with their
target drugs.
• Rapid LC/MS/MS methods were developed to increase throughput of the assay, thereby
increasing sample capacity and maximizing instrument throughput.
Challenges In Method Development
Extraction
• The matrix selection was based on the pKa of ceftriaxone in solution.
• Selection of FaSSIF buffer for control matrix was determined to be the best option for human
intestinal chyme samples.
• Tazobactam (a β-lactamase inhibitor) was added to the plasma samples during sample
collection to inhibit any effect of SYN-004, a β-lactamase, which theoretically could be present
in trace amounts in plasma. This allowed the plasma samples to be accurately analyzed for
ceftriaxone without ex vivo degradation.
• The action of the SYN-004 was not completely inhibited by using the tazobactam added to the
human intestinal chyme matrix, therefore a chaotrope reagent, 8 M guanidine hydrochloride, was
selected to inhibit SYN-004’s β-lactamase activity in the chyme during processing.
• An LC/MS/MS method sensitive enough to quantify the lower limit of quantitation (LLOQ) in
the nanogram per milliliter range required for the plasma methods was achieved using protein
precipitation. The microgram per milliliter analytical range of the human intestinal chyme
method allowed for a high dilution of the chyme samples that was sufficient for injection
directly to LC/MS/MS.
• The nature of the intestinal chyme matrix made dilution of samples above the analytical range
problematic; therefore, a wider dynamic range was validated to account for larger variance in
concentration in the clinical samples.
• The complex heteroatomic structure of ceftriaxone results in the presence of multiple ionic
species across a wide range of solution pH (shown below).
+Q1: Ceftriaxone
Max. 2.8 x 106
cps
m/z
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
RelativeIonAbundancee
555.1
[M+H]+
200 500300 400 600
S
N
N
O O
N
H
N
S
O
OH
S
N
N
N
OH
O
O
NH2
H
S
N
N
O
13
C
D
D
D
O
N
H
N
S
O
OH
S
N
N
N
OH
O
O
NH2
H
Chromatography
• Multiple LC column and mobile phase combinations were examined during method
development but the majority yielded poor peak shape and retention due to the highly polar
nature of ceftriaxone and the structural complexity of the molecule.
• Hydrophilic interaction chromatography (HILIC) was investigated in order to retain
ceftriaxone in highly aqueous conditions but peak shape was highly variable.
• The polar reverse phase (RP) column using an acidified methanol gradient yielded the best
peak shape and retention.
Mobile Phase Selection
• Methods for analysis of ceftriaxone in the literature commonly employ UV detection and,
as such, LC conditions use phosphate buffers; therefore, the methods are not transferrable
directly to LC/MS/MS.
• 1% formic acid was found to yield the best peak shape and sensitivity.
• A Synergi Polar-RP 80 column was selected based on the polar nature of the molecule and
the need for greater retention in gradient elution.
0
10
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80
90
100
0 147 10.53.5
Inter-Lot Accuracy and Precision in Quality Control SamplesTypical Chromatograms of Extracted Dog Plasma Samples
Intensity(cps)Intensity(cps)
Matrix Blank with IS LLOQ (10.0 ng/mL)Control Blank
Time (minutes)
Time (minutes)
Time (minutes)Time (minutes)
Intensity(cps)Intensity(cps)
Typical Chromatograms of Extracted Human Plasma Samples
Matrix Blank with IS LLOQ (100 ng/mL)Control Blank
Time (minutes) Time (minutes)
Typical Chromatograms of Diluted Human Intestinal Chyme Samples
Intensity(cps)Intensity(cps)
Time (minutes)
Matrix Blank with IS LLOQ (1.00 µg/mL)Control Blank
Time (minutes) Time (minutes)
pH
Percentage
Ceftriaxone Ionic Microspecies Distribution (across pH)
Ceftriaxone
C18H18N8O7S3
Exact Mass: 554.0461
Molecular Weight: 554.5710
[13C,2H3]-Ceftriaxone
Internal Standard (IS)
C17
13CH15
2H3N8O7S3
Exact Mass: 558.0682
Molecular Weight: 558.5817
Ceftriaxone Ceftriaxone Ceftriaxone
Ceftriaxone Ceftriaxone Ceftriaxone
Ceftriaxone Ceftriaxone Ceftriaxone
[13C,2H3]-Ceftriaxone [13C,2H3]-Ceftriaxone [13C,2H3]-Ceftriaxone
[13C,2H3]-Ceftriaxone[13C,2H3]-Ceftriaxone[13C,2H3]-Ceftriaxone
[13C,2H3]-Ceftriaxone [13C,2H3]-Ceftriaxone [13C,2H3]-Ceftriaxone
Ceftriaxone Q1 and Product Ion Spectra
Ceftriaxone Concentration in Dog Plasma
LLOQ QC
10.0 ng/mL
QC1
30.0 ng/mL
QC2
200 ng/mL
QC3
1500 ng/mL
QC4
3750 ng/mL
Dilution QC
(10-fold dilution)
20000 ng/mL
Mean 10.4 31.2 224 1550 3850 21000
SD 0.542 1.24 6.53 40.3 144 997
CV (%) 5.2 4.0 2.9 2.6 3.7 4.7
RE (%) 4.0 4.0 12.0 3.3 2.7 5.0
n 18 18 18 18 18 18
Ceftriaxone Concentration in Human Plasma
LLOQ QC
100 ng/mL
QC1
300 ng/mL
QC2
2000 ng/mL
QC3
15000 ng/mL
QC4
37500 ng/mL
Dilution QC
(10-fold dilution)
200000 ng/mL
Mean 102 295 2010 15600 37900 214000
SD 11.8 16.7 62.8 868 2000 7040
CV (%) 11.6 5.7 3.1 5.6 5.3 3.3
RE (%) 2.0 -1.7 0.5 4.0 1.1 7.0
n 18 18 18 18 18 18
Ceftriaxone Concentration in Human Intestinal Chyme
LLOQ QC
1.00 µg/mL
QC1
3.00 µg/mL
QC2
45.0 µg/mL
QC3
1500 µg/mL
QC4
2000 µg/mL No Dilution
Mean 0.877 2.74 44.9 1480 1980 —
SD 0.0832 0.117 2.71 80.2 160 —
CV (%) 9.1 4.4 6.0 5.4 8.1 —
RE (%) -12.0 -8.7 -0.3 -1.5 -0.8 —
n 18 18 18 18 18 —
Chromatographic Conditions
Autosampler: LEAP CTC PAL at 4 °C
Injection Volume: 10 µL for dog plasma, 5 µL for human plasma,
5 to 20 µL for human intestinal chyme
LC Pump and Controller: Shimadzu LC-10AD and Shimadzu SCL-10A
HPLC Column: Phenomenex Synergi Polar-RP 80 Å (50 x 2.0 mm, 4 µm particle size)
Column Temperature: 50 °C for dog and human plasma, ambient temperature
for human intestinal chyme
Mobile Phase A: 1% formic acid in HPLC-grade water
Mobile Phase B: 1% formic acid in methanol
LC Gradient: 30% to 95% Mobile Phase B with analyte elution at ~1 minute
Flow Rate: 1000 µL/min
Retention Time: 0.90 ± 0.18 min for dog and human plasma,
1.03 ± 0.2 min for human intestinal chyme
Mass Spectrometry Conditions
Mass Spectrometer: AB SCIEX API 4000
Ionization: TurboIonSpray™ in positive ion mode
Ion Spray Voltage: 2500 V for dog and human plasma, 5500 V for human intestinal chyme
Temperature: 650 °C for dog and human plasma, 700 °C for human intestinal chyme
Declustering Potential: 45 V for dog and human plasma, 50 V for human intestinal chyme
Total Cycle Time: 4.5 min for dog plasma, 5.3 min for human plasma,
and 6.0 min for human intestinal chyme
Transitions Monitored (±0.2 min for each m/z) Dwell time
Ceftriaxone m/z 555.1 → m/z 396.0 125 ms
[13C,2H3]-Ceftriaxone m/z 559.1 → m/z 400.0 125 ms
Tazobactam
C10H12N4O5S
Molecular Weight: 300.289
Precision: %CV (coefficient of variation) = (standard deviation/mean) x 100
Accuracy: %RE = (mean/nominal) x 100
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+MS2: Ceftriaxone
Max. 1.2 x 105
cps
m/z
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
RelativeIonAbundance
396.0
555.1
100 500400300200