Temocillin (6-�-methoxy ticarcillin) is well known for its resistance to degradation by �-lactamases including most ESBL. 1,2 It is therefor

A. Bastos

A. Spinewine

F. Van Bambeke

P. M. Tulkens

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Temocillin (6--methoxy ticarcillin) is well known for its resistance to 
degradation by -lactamases including most ESBL.1,2 It is therefore 
considered as a sparing drug for carbapenems.2
Its pharmacokinetic is unknown in haemodialysis patients, which often 
present a wide pharmacokinetic variability that can lead to subtherapeutic 
concentrations. 
Determining temocillin in the serum of these patients is challenged by the 
fact they receive several other medications and accumulate metabolites 
that can interfere in the assay.  
This poster will be made available for download after the meeting: http://www.facm.ucl.ac.be/posters.htm
INTRODUCTION METHODS
RESULTS
CONCLUSIONS REFERENCES
This fully validated method enables detection and 
quantification of temocillin in serum of haemodialysis 
patients in a simple, robust and reproducible manner.  It 
can prove beneficial for optimal management of 
haemodialysis patients.
AIM
To develop and validate a new method to analyse temocillin in serum from 
haemodialysis patients, and to quantify it by an HPLC system.
Chromatogram of a representative human serum sample from 
haemodialysis patient.  The elution profiles of temocillin (R [TMO 
R] and S [TMO S]) isomers are well separated from that of other
substances absorbing at the detection wavelength (235 nm) and 
from ticarcillin (internal standard, with also the corresponding R 
and S isomers). 
Nominal 
concentration (µg/mL)
Freeze-Thaw Stability* 
(%)
Benchtop Stability* 
(%)
Autosampler Stability*
(%)
6months
Stability* (%)
5 89.81 ± 3.20 95.67 ± 2.34 93.50 ± 2.46 93.73 ± 3.07
100 94.14 ± 1.05 94.67 ± 1.40 96.91 ± 7.26 90.97 ± 6.64
400 95.78 ± 0.12 95.40 ± 2.5 98.10 ± 1.98 88.96 ± 2.26
* Mean ± SD
0 100 200 300 400
-25
-20
-15
-10
-5
0
5
10
15
20
25
Temocillin concentration (µg/mL)
R
e
l
a
t
i
v
e
 
E
r
r
o
r
 
(
%
)
0 100 200 300 400
0
100
200
300
400
Introduced concentration (µg/mL)
M
e
a
s
u
r
e
d
 
c
o
n
c
e
n
t
r
a
t
i
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n
(
µ
g
/
m
L
)
(a) Accuracy profile of the concentration (µg/mL) of temocillin using a linear regression model. The 
continuous line is the relative bias, the dashed line is the upper and lower 80% β-expectation tolerance 
limits, and the dotted lines are the upper and lower FDA acceptance limits. The dots represent the 
relative error of the measured concentrations for each QC sample and are plotted with respect to their 
target concentration.  (b) Linear profile of temocillin in human serum.  The dotted lines are the upper and 
lower FDA acceptance limits, and the dashed lines are the upper and lower 80% β-expectation tolerance 
limits, both expressed in concentration units. The dots represent the data points for the different quality 
control samples across the concentration range 5 – 400 µg/mL.
Sample
Nominal 
concentratio
n (µg/mL)
Trueness Precision Accuracy
Absolute 
bias 
(µg/mL)
Relative 
bias
(%)
Repeatabilit
y
(RSD %)
Intermediate 
precision
(RSD %)
β-expectation 
tolerance 
intervals
(µg/mL)
Relative β-
expectation 
tolerance limits
(%)
LOQ 5 -0.256 -5.129 2.062 5.120 [4.223 ; 5.264] [-16.095 ; 5.836]
QC1 15 -0.553 -3.687 2.336 4.006 [13.230 ; 15.664] [-12.101 ; 4.734]
QC2 100 -0.497 0.497 2.205 2.410 [92.642 ; 108.353] [-7.319 ; 8.314]
QC3 400 -5.889 -1.472 0.957 3.611 [363.344 ; 424.877] [-9.279 ; 6.334]
RSD: relative standard deviation; QC: quality control
Solid phase extraction 
of temocillin from 
human serum
with Waters Oasis® 
MAX cartridges 
(sorbent mass 
1cc/30mg)
HPLC analysis coupled 
to a photodiode array detector 
(HPLC-UV)
RP-18 LiChrospher® column 
(250 x 4 mm, 100Å, 5µm) with 
isocratic elution of 100mM sodium 
acetate buffer pH 7 / acetonitrile 
(95:5, v/v); flow rate 1 mL/min;
ticarcillin was internal standard. 
A
B
Clinical evaluation in 4 haemodialysis patients -
degree of dialyzability of temocillin (extraction 
ratio – ER, haemodialysis clearance – CLHD)
Assay Validation Results for Quantification of Total Temocillin in Human Serum.
Stability Results for Temocillin in Human Serum
1.Jules K, Neu HC. Antibacterial activity and beta-lactamase stability 
of temocillin. Antimicrob Agents Chemother 1982; 22, 453-60. 
PM:6982681 
2.Livermore DM, Tulkens PM. Temocillin revived. J Antimicrob
Chemother 2009; 63, 243-5. PM:19095679.
3.Hubert P, Nguyen-Huu JJ, Boulanger B et al. Harmonization of
strategies for the validation of quantitative analytical procedures. A 
SFSTP proposal--Part I. J Pharm Biomed Anal. 2004;36:579-586. 
ACKOWLEDGMENTS
(a) (b)
We thank Laurence Dinaux from Waters 
Corporation, Zellik, Belgium, and and Dr. 
Arnaud Capron from the Cliniques 
Universitaires Saint Luc, Brussels, Belgium for 
useful suggestions and guidance.  Temocillin 
was the generous gift from Eumedica s.a., 
Manage, Belgium. This work was supported by 
the Region Wallonne.
Development and validation of a HPLC Assay for the 
Determination of Temocillin in Serum of Haemodialysis Patients
A.Bastos1,2, S.Vandecasteele3, P.M.Tulkens1,2, A. Spinewine2, F. Van Bambeke1,2,*
1 Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium;
2 Centre for Clinical Pharmacy, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium;
3 Department Nephrology and Infectious Diseases, AZ Sint-Jan Brugge-Oostende a.v., Bruges, Belgium.
Method validation
The method was validated 
according to the accuracy 
profile methodology, using 
total error to verify the 
trueness, precision and overall 
accuracy.3 The results were 
evaluated according to the 
FDA acceptance criteria. 
Concentration-time profile for 
TMO in serum in a 
haemodialysis patient. Initial 
(t=0) and post dialysis (t=48) 
dose was 2g. The beginning 
(HD on) and end (HD off) of the 
haemodialysis are marked with 
horizontal arrows, and 
haemodialysis periods (4hours) 
are shown by horizontal bars 
(HD).
Temocillin dialysability was investigated in samples from 4 patients, 
showing a mean extraction ratio of 56.9% (range 44.8 - 71.1%), and 
a mean CLHD of 10.87 L/h (range 8.03 – 13.0 L/h). ER – extraction 
ratio, Cs,IN, Cs,OUT – serum concentrations of TMO flowing into and 
out of the dialyser (μg/mL), CLHD – haemodialysis clearance (L/h),  
Qdial – blood flow into and out of the dialyser (L/h)
ER 
Cs,IN Cs,OUT
Cs,IN
CLHD  Qdial ER
Clinical Evaluation in Haemodialysis Patients
(b)
(a)

METHODS

http://www.facm.ucl.ac.be/posters/2014/DrugAnalysis-2014/Bastos-temocillin-HPLC-haemodialysis-2014.pdf

METHODS

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