Rapid Determination of Meropenem

in Biological Fluids by LC: Comparison

of Various Methods for Sample Preparation
and Investigation of Meropenem Stability
2009, 70, 14231427

Karin Kipper1,2,&, Kaili Anier2, Ivo Leito1, Juri Karjagin3, Kersti Oselin2, Koit Herodes1
1
2
3

Institute of Chemistry, University of Tartu, Jakobi 2, 51014 Tartu, Estonia; E-Mail: karin.kipper@gmail.com
Department of Pharmacology, University of Tartu, Ravila 19, 51014 Tartu, Estonia
Anaesthesiology and Intensive Care Clinic, University of Tartu, Puusepa 8, 51014 Tartu, Estonia

Received: 4 May 2009 / Revised: 24 July 2009 / Accepted: 4 August 2009

Online publication: 3 September 2009

Abstract
A rapid and sensitive LC-UV method was developed and validated for the determination of
meropenem, in human plasma and urine. Meropenem retention time was 4.8 min. Method
development was based on comparative analysis of different extraction methods published
as well as careful study of meropenem stability in biological samples under different conditions. Best results in plasma sample preparation were obtained from protein precipitation
with methanol. LOQ was 0.1 lg mL-1 for plasma and 1 lg mL-1 for urine samples. Meropenem in plasma has low stability at room temperature (<20% of original content after
12 h), but had acceptable stability when the whole analysis procedure was designed to
minimize the exposure of meropenem-containing samples and solutions to temperatures
higher than 4 C. The developed method was applied to a human pharmacokinetic study in
patients with acute peritonitis.

Keywords
Column liquid chromatography
UV detection
Pharmacokinetic study in human plasma and urine
Meropenem

Introduction
Meropenem, (4R,5S,6S)-3-[(3S,5S)-5-dimethylcarbamoylpyrrolidin-3-yl-thio]-6[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1azabicyclo[3.2.0]hept-2-ene-2 carboxylic
acid (Fig. 1b), is a broad spectrum

Full Short Communication

DOI: 10.1365/s10337-009-1304-8
0009-5893/09/11

carbapenem antibiotic for intravenous

administration. Meropenem is a white to
pale yellow crystalline powder with
acidic properties (pKa1 = 2.9 and pKa2 =
7.4). It is eective in the treatment of
Gram-negative and -positive infections.
Meropenem penetrates the bacterial cell

wall of susceptible organisms and

inhibits cell wall synthesis [13].
Determination of meropenem by LC
has received wide interest. Several LC
methods have been developed and reported of meropenem pharmacokinetic
studies [136].
Dierent LC eluents contain methanol or acetonitrile (ACN) or both in a
mixture with buer solutions (pH value
varied from 2.8 [35] to 7.4 [36]). Various
sample preparations have been applied to
the plasma: e.g. SPE with dierent sorbents as C18 [35, 17, 30, 32, 34] and C8
[16], protein precipitation methods using
methanol [18, 23] and ACN [1, 34] or both
[8], as well as column switching by
Supelclean LC-NH2 40-lm (50 9 2.1 mm
i.d.) [14] and (20 9 3.9 mm i.d.) precolumn tap-lled with Li-Chroprep RP-8
(2540 lm) [19] as extraction columns
and ltration through syringe lters [7, 31,
36]. To the best of our knowledge, these
sample preparation procedures have not
been comparatively evaluated in the
literature. Table 1 shows dierent sample
preparation methods and chromatographic conditions, published in the
literature and evaluated in the present
study for determination of meropenem
in various biological matrices: plasma,
serum, urine and ultraltrate.
Meropenem samples have low stability at room temperature after sample

Chromatographia 2009, 70, November (No. 9/10)

2009 Vieweg+Teubner | GWV Fachverlage GmbH

1423

preparation. Meropenem aqueous solutions, like all b-lactam antibiotics, are

unstable and subject to hydrolytic degradation [24]. Some papers have been
published about meropenem stability in
solid phase and in aqueous solutions at
dierent conditions [2, 2426] as well as
meropenem stability in infusion step [8].
Meropenem showed degradation of
12.7% after 72 h at 25 C in aqueous
solution [2] and degradation at elevated
temperatures and alkaline conditions
[24]. However, the instability of meropenem in biological samples has received only modest attention in most
published reports on meropenem determination.
The aim of the present work was
threefold: (i) to comparatively evaluate
dierent sample preparation methods
for the meropenem determination in
plasma; (ii) to develop a suitable LC
method to quantify meropenem in human plasma and urine using UV detection; (iii) to investigate the stability of
meropenem in biological samples and to
validate the developed method paying
particular attention to the handling and
storing conditions and stability of meropenem samples and solutions.

Experimental
Chemicals
Meropenem trihydrate was obtained
from AstraZeneca Pharmaceuticals
(AstraZeneca, Tallinn, Estonia). Sodium
phosphate monobasic dihydrate from
Janssen Chimica (Geel, Belgium), ACN
and methanol from Rathburn Chemicals
(Walkerburn, Scotland), 85% orthophosphoric acid from Riedel-deHaen
AG (Seelze, Germany), perchloric acid
from YA Kemira (Helsinki, Finland).
The stock solution of meropenem
(5.0 mg mL-1) was prepared in distilled
water and stored at -80 C.

Conditions for Sample

Preparation
Dierent extraction methods published
previously in literature (Table 1) and our

1424

in-house developed methods were compared: (i) Plasma samples were ltered
through Alltech regenerated cellulose
syringe lters (pore diameter 0.45 lm)
and the ltrate was directly injected into
the LC system [7]. (ii) Plasma sample
(0.5 mL) was extracted with an aliquot
of methanol [23]. After shaking, sample
was centrifuged at 13,000 g for 10 min at
4 C and supernatant was injected into
the LC system. (iiiv) Extraction procedures with ethanol, 12 and 1 M perchloric acid were performed the same
way as in (ii). (vi) Aliquot of plasma was
mixed with ACN, the sample was shaken
and centrifuged. Methylene chloride was
added to supernatant, after shaking and
centrifugation the upper aqueous layer
was injected into the LC column [1, 6].
(vii) In-house developed extraction was
performed by adding an aliquot of ACN
to 1 mL of plasma sample. Sample
was vortexed and kept at -20 C for at
least 30 min. From two separated solution layers the upper organic layer
(*800 lL) was removed and evaporated
to dryness under an air stream [and
compared to mixture of gases, containing no oxygen (85% N2, 10% H2, 5%
CO2)]. The residue was dissolved in
100 lL of mobile phase and 40 lL were
injected into the LC system.
For urine samples two methods were
used. Urine samples were extracted using
SPE Alltech NH2 cartridges [20]. They
were ltered through Alltech syringe lters (pore diameter 0.45 lm) and the
ltrate was injected into the LC system.

LC Apparatus and
Chromatographic Conditions
The LC system consisted of a Waters 717
plus autosampler with sample thermostat (Waters Millipore, USA), Alltech
426 LC pump (Alltech Associates, USA)
and Waters 486 tunable UV absorbance
detector (Waters Millipore, USA).
Chromatography was performed on an
analytical column Inertsil ODS-3 C18
(150 9 4.6 mm I.D.) packed with 5 lm
diameter particles (GL Science, Japan).
The analytical column was protected by
a Platinum C18 (7.5 9 4.6 mm I.D.,
5 lm particles) precolumn (Alltech).
Chromatography software Kromex, ver

32S (Akrom-EX, Estonia) was used for

data acquisition.
Injection volume 40 lL and ow rate
0.7 mL min-1 in isocratic elution with a
mobile phase of 0.05 M phosphate buffer (pH 3.0 with 85% orthophosphoric
acid), ACN and methanol (86/12/2;
v/v/v). The autosampler temperature was
set at 4 C.
The absorbance of meropenem was
detected at 302 nm for plasma and at
320 nm for urine samples.

Validation
Method validation was performed
according to the FDA Guidance for
IndustryBioanalytical Method Validation [37].

Results and Discussion

Sample Preparation
Dierent sample preparation methods
were compared. Limiting sample preparation to just dilution and ltration of
plasma with syringe lters [7, 36] did not
give satisfactory results. Injecting such
samples caused the rise in chromatographic system pressure. The same
problems occurred with centrifugation
of plasma samples [20, 35]. Therefore,
ltration and centrifugation did not
separate compounds that caused clogging from plasma. Protein precipitation
with 12 and 1 M perchloric acid did not
transfer meropenem to the analysed
solution. When protein precipitation was
carried out with ethanol the meropenem
peak was small and asymmetric (AS =
2.5). Protein precipitation with ACN [1,
6] was not eective, even after addition
of methylene chloride, meropenem did
not transfer to the aqueous layer. This
discrepancy may be because plasma
samples were analysed in our work instead of serum as in the published study
[1]. The in-house developed protein precipitation with ACN was not eective as
meropenem transferred only partially to
the upper solution layer. Concentrating
the extracted sample layer by evaporation to dryness with air and a mixture of
gases caused meropenem degradation

Chromatographia 2009, 70, November (No. 9/10)

Full Short Communication

Table 1. Overview of conditions used for determination of meropenem in biological matrices

Sample

Sample preparation

Recovery (%)

Eluent and pH (all isocratic)

Reference

Human plasma

Plasma centrifugation
3,000 rpm, 30 min

96.1 2.9 for 0.1 lg mL-1 to

93.3 1.2 for 100.0 lg mL-1

Phosphate/ACN (pH = 7.4)

(90/10, v/v)
Phosphate/ACN/MeOH (pH = 2.8)
(84/12/4, v/v/v)
Isocratic phosphate/MeOH
(pH = 7.4) (82/18 for plasma
and 75/25 for urine, v/v)
Phosphate/MeOH(pH = 7.4)
(78/22, v/v)
Isocratic ammonium acetate/ACN
(pH = 4.0) (95/5, v/v)

[13]

Plasma, urine
Plasma,
ultraltrate
Serum
Serum, urine

Dilution with water 1/4 for

plasma (1/10 for urine) and
ltrated
Dilution with water 1/4 for
plasma and ltrated
Protein precipitation with
ACN and methylene chloride
Centrifugation 4,000 g for
10 min for serum
SPE Sep-Pak NH2 for urine

evidenced by peak shape change. Air

stream and mixture of gases gave the
same results.
The most suitable protein precipitation method for meropenem plasma
samples was extraction with methanol
[23]. Meropenem content in analysed
solution was high and constant between
dierent experiments. The meropenem
peak in chromatograms was high and
symmetric (As = 1.30). In the original
method a 15 min shaking was used to
mix methanol and plasma solution
before centrifugation. Our experiments
proved that similar results in terms of
meropenem peak height and area were
achieved when the aliquot of methanol
was added to plasma and the mixture
was vortexed for 30 s. The shorter
time is advantageous because of the low
stability of meropenem in extracted
samples.
In urine sample extraction by SPE
[20], meropenem was already partly
desorbed with water, cartridge washing
solutions, and partly with 0.1 N HCl.
Meropenem distribution between water
and 0.1 N HCl was not reproducible and
varied at dierent meropenem concentrations. The SPE stationary phase used
in the current work was from dierent
manufacturers Alltech NH2 as in the
literature [20] (Sep-Pak NH2, Waters).
The mass of stationary phase is not
mentioned in the article. SPE cartridges
with 500 mg of sorbent were used in the
current work. A possible dierence in
the stationary phase mass and manufacturer may explain the dierent results.

Full Short Communication

95.8 3.7 to 101.7 3.2

In serum ranged 98.1 1.17
to 98.7 1.12
In urine ranged 96.07 1.25
to 96.54 1.58

Isocratic phosphate/ACN/MeOH
(pH = 2.8) (84/12/4, v/v/v)

Filtration through syringe lters was

a fully satisfactory sample preparation
method for meropenem analysis in urine.
The ltrate was collected for LC analysis.

Chromatographic Conditions
To nd suitable chromatographic conditions, dierent mobile phases and ow
rates were compared. From dierent
mobile phases [2, 14, 20, 21, 23] a threecomponent eluent with the following
composition was chosen: phosphate
buer (pH = 3.0), ACN and methanol
(86/12/2; v/v/v). This separated meropenem peak from plasma components and
meropenem retention time was the
shortest (about 4.8 min). At a ow rate
of 0.7 mL min-1 good separation from
the preceding (RS = 2.35) and from the
following plasma component peak
(RS = 2.94) was achieved at higher meropenem concentrations (50 lg mL-1).
The same chromatographic conditions were used for plasma and urine
samples. The UV detector wavelength
was 302 nm for plasma samples and
320 nm for urine samples due to considerable absorbance of some urine
components at 302 nm.

Assay Validation
Calibration Curve and Selectivity

The calibration curves in concentration

ranges of 0.1 to 150 lg mL-1 (n = 9) in

[35]
[7]
[36]
[1, 6]
[20]

plasma and 1 to 250 lg mL-1 (n = 8) in

urine obtained from meropenem peak
area versus concentration were linear
with r2 > 0.998.
LOD and LOQ were estimated from
six replicates. LOD was 0.05 lg mL-1
for plasma and 0.5 lg mL-1 for urine
samples. The lowest point of the
calibration graph was set at LOQ, for
plasma samples 0.1 lg mL-1, with
acceptable accuracy 99.6% and precision
3.7%. For urine samples the LOQ was
1 lg mL-1 with accuracy 102% and
precision 3.2%.
Selectivity of the method was demonstrated by analysis of six plasma and
urine blank samples from various sources. None of the analysed blank plasma
or urine samples had peaks at the average meropenem retention time of
4.8 min. The closest peaks from the
plasma samples had average retention
times 3.6 and 6.6 min (Fig. 1a). The
closest peak from the urine samples had
an average retention time 3.4 min
(Fig. 2a).
Precision, Accuracy, and Recovery

Within-day accuracy for plasma calibration curve ranged from 92.9 to

105.8% (for urine 93.5 to 105.6%) of the
nominal concentration. Between-day
accuracy for plasma quality control
samples was 98.6% for 50 lg mL-1,
96.8% for 5 lg mL-1 and 99.6% for
0.5 lg mL-1 (for urine 98.7% for
163 lg mL-1, 96.9% for 25 lg mL-1
and 97.9% for 10 lg mL-1). The be-

Chromatographia 2009, 70, November (No. 9/10)

1425

Fig. 1. Chromatographic separation of plasma blank sample and meropenem plasma sample. a Plasma blank sample and b Meropenem sample
concentration 25 lg mL-1 in plasma

84 7% (CV 8%) over the calibration

curve in duplicate. The ltration of urine
samples gave the recovery for meropenem
100 3% (CV 3%) over the calibration
curve in duplicate. The accuracy and
precision of the method are comparable
to those described in the literature.

Stability

Fig. 2. Chromatographic separation of urine blank samples and meropenem sample in urine.
a Urine blank sample and b meropenem sample concentration 100 lg mL-1 in urine

Table 2. Urinary recovery of meropenem in patients with acute peritonitis after intravenous
administration of 1 g of meropenem every 8 h
Subject

04 h excretion
(g)

424 h excretion
(g)

024 h excretion
(g)

24 h urinary recovery
(%)

1
2
3
4

0.4
0.6
1.3
0.2

1.3
0.2
1.7
0.5

1.7
0.9
3.0
0.7

56
29
100
22

tween-day precision of ve replicates at

the three concentrations was <3.7% CV
(for urine <3.2% CV).
The recovery was determined by
comparing meropenem peak areas over

1426

the calibration curve for aqueous solutions (100%) and spiked plasma samples
after extraction. Liquidliquid extraction
with 0.5 mL methanol gave the mean
SD assay recovery for meropenem

After 3 months of storage at -80 C (the

solution was repeatedly thawed and
refrozen for preparation of calibration
solutions) the meropenem stock solutions
retained on an average 93% of their original concentrations. Freeze and thaw
stability for meropenem plasma samples
(with meropenem concentrations 25 and
1 lg mL-1) was 98%. Short-term stability at room temperature (20 C) in extracted plasma samples was 94% after
1 h. After 2, 3 and 4 h the retained values
were 85, 75 and 73%, respectively. After
12 h the concentration of meropenem in
the samples was only 17% of the original
concentration. The study showed that
after sample preparation with methanol
meropenem samples had low stability at
room temperature. Therefore autosampler temperature was set at 4 C and after
preparation samples were kept at -20 C
until analysis. Sample test tubes were
tightly closed to prevent evaporation of
methanol. The analysis showed that

Chromatographia 2009, 70, November (No. 9/10)

Full Short Communication

samples retained 95% of their meropenem contents during 3 h of storage at

4 C.
Samples retained on an average 95%
of their original meropenem contents
in long-term stability studies (45 days at
-80 C).

Application of the Meropenem

Assay in Biological Samples
Suitability of the method was demonstrated in a pharmacokinetic trial on
patients with acute peritonitis [27]. Plasma and urine samples were collected after
intravenous administration of 1 g of
meropenem over 20 min infusion every
8 h. Inter-day coecient of variation for
the determination of meropenem in
plasma and urine was 8 and 3%, respectively. Mean pharmacokinetic parameters were calculated from plasma data.
Table 2 shows that the 24 h urinary
recovery of meropenem in four patients
ranged from 22% (0.7 g) to complete
recovery (3 g).

Conclusion
A rapid and sensitive LC method was
developed for the determination of meropenem in human plasma and urine.
Dierent sample preparation methods
were compared. A number of SPE and
liquidliquid extraction methods published in the literature previously gave
untrustworthy results in sample preparation. Best results were obtained by the
protein precipitation method with methanol. Meropenem stability was examined
extensively under various handling and
storage conditions of samples and meropenem solutions. Meropenem had
low stability, but an acceptable stability
when autosampler temperature was set at
4 C and experiments were designed to
minimise the exposure of meropenemcontaining samples and solutions to
temperatures higher than 4 C.

Acknowledgments
The study was nancially supported by
the Estonian Science Foundation grant
GARFR 6691.
Full Short Communication

References
1. Elkhaili H, Niedergang S, Pompei D,
Linger L, Leveque D, Jehl F (1996) J
Chromatogr B 686:1926. doi:10.1016/
S0378-4347(96)00205-8
2. Mendez ASL, Steppe M, Schapoval EES
(2003) J Pharm Biomed Anal 33:947954.
doi:10.1016/S0731-7085(03)00366-2
3. Robatel C, Buclin T, Eckert P, Schaller
MD, Biollaz J, Decosterd LA (2002) J
Pharm Biomed Anal 29:1733. doi:
10.1016/S0731-7085(02)00022-5
4. Bax RP, Bastain W, Featherstone A,
Wilkinson DM, Hutchison M, Haworth
SJ (1989) J Antimicrob Chemother
24:311320. doi:10.1093/jac/24.3.311
5. Bedikian A, Okamoto MP, Nakahiro
RK, Farino J, Heseltine PNR, Appleman
MD, Yellin AE, Berne TV, Gill MA
(1994) Antimicrob Agents Chemother
38:151154
6. Bui KQ, Ambrose PG, Nicolau DP,
Lapin CD, Nightingale CH, Quintiliani R
(2001) Chemotherapy 47:153156. doi:
10.1159/000063216
7. Burman LA, Nilsson-Ehle I, Hutchison
M, Haworth SJ, Norrby SR (1991) J
Antimicrob Chemother 27:219224. doi:
10.1093/jac/27.2.219
8. Conte JE, Golden JA, Kelley MG, Zurlinden E (2005) Int J Antimicrob Agents
26:449456. doi:10.1016/j.ijantimicag.2005.
08.015
9. Kuti JL, Nightingale CH, Knauft RF,
Nicolau DP (2004) Clin Ther 26:493501.
doi:10.1016/S0149-2918(04)90051-3
10. Makino J, Motoko K, Shibasaki T,
Nakashima E, Kamata M, Ozawa S,
Maruyama H, Masuhara K, Kobayashi T
(2002) Jpn J Antibiot 55:7788
11. Mattoes HM, Kuti JL, Drusano GL,
Nicolau DP (2004) Clin Ther 26:1187
1198. doi:10.1016/S0149-2918(04)80001-8
12. Mouton JW, Touw DJ, Horrevorts AM,
Vinks AATMM (2000) Clin Pharmacokinet 39:185201. doi:10.2165/00003088200039030-00002
13. Niwa T, Nakamura A, Kato T, Kutsuna
T, Katou K, Morita H, Kojima Y, Itoh
M (2006) Respir Med 100:324331. doi:
10.1016/j.rmed.2005.05.009
14. Bompadre S, Ferrante L, MDe Martinis,
Leone L (1998) J Chromatogr A 812:249
253. doi:10.1016/S0021-9673(98)00249-0
15. Ehrlich M, Daschner FD, Kummerer K
(2001) J Chromatogr B 751:357363. doi:
10.1016/S0378-4347(00)00504-1
16. Granai F, Smart HL, Triger D (1992) J
Antimicrob Chemother 29:711718. doi:
10.1093/jac/29.6.711
17. Harrison MP, Haworth SJ, Moss SR,
Wilkinson DM, Featherstone A (1993)
Xenobiotica 23:13111323. doi:10.3109/
00498259309059441
18. Hikida M, Kawashima K, Yoshida M,
Mitsuhashi S (1992) J Antimicrob Chemother 30:129134. doi:10.1093/jac/30.2.129
19. Lee HS, Shim HO, Yu SR (1996) Chromatographia 42:405408. doi:10.1007/
BF02272131

20. Ozkan Y, Kucukguzel I, Ozkan SA,

Aboul-Enein HY (2001) Biomed Chromatogr 15:263266. doi:10.1002/bmc.68
21. Chang YL, Chou MH, Lin MF, Chen
CF, Tsai TH (2002) J Chromatogr A
961:119124. doi:10.1016/S0021-9673(02)
00078-X
22. Viaene E, Chanteux H, Servais H, Mingeot-Leclercq M, Tulkens PM (2002) Antimicrob Agents Chemother 46:23272332.
doi:10.1128/AAC.46.8.2327-2332.2002
23. Carlucci G, Biondi L, Vicentini C, Bologna M (1990) J Pharm Biomed Anal
8:283286.
doi:10.1016/0731-7085(90)
80038-Q
24. Mendez A, Chagastelles P, Palma E,
Nardi N, Schapoval E (2008) Int J Pharm
350:95102. doi:10.1016/j.ijpharm.2007.
08.023
25. Mendez ASL, Dalomo J, Steppe M,
Schapoval EES (2006) J Pharm Biomed
Anal 41:13631366. doi:10.1016/j.jpba.
2006.02.017
26. Cielecka-Piontek J, Zajac M, Jelinska A
(2008) J Pharm Biomed Anal 46:5257.
doi:10.1016/j.jpba.2007.08.024
27. Karjagin J, Lefeuvre S, Oselin K, Kipper
K, Marchand S, Tikkerberi A, Starkopf J,
Couet W, Sawchuk RJ (2008) Clin Pharm
Ther 83:452459. doi:10.1038/sj.clpt.
6100312
28. Dreetz M, Hamacher J, Eller J, Borner K,
Koeppe P, Schaberg T, Lode H (1996)
Antimicrob Agents Chemother 40:105
109
29. Kurihara Y, Kizu J, Hori S (2008) J Infect
Chemother 14:3034. doi:10.1007/s10156007-0576-x
30. Allegranzi B, Cazzadori A, Di Perri G,
Bonora S, Berti M, Framchino L, Biglino
A, Cipriani A, Concia E (2000) J Antimicrob Chemother 46:319322. doi:10.1093/
jac/46.2.319
31. Ikeda K, Ikawa K, Morikawa N, Miki M,
Nishimura S, Kobayashi M (2007) J
Chromatogr B 856:371375. doi:10.1016/
j.jchromb.2007.05.043
32. Denooz R, Charlier C (2008) J Chromatogr B 864:161167. doi:10.1016/j.
jchromb.2008.01.037
33. Legrand T, Chhun S, Rey E, Blanchet B,
Zahar J, Lanternier F, Pons G, Jullien V
(2008) J Chromatogr B 875:551556. doi:
10.1016/j.jchromb.2008.09.020
34. Farin D, Kitzes-Cohen R, Piva G,
Gozlan I (1999) Chromatographia
49:253255. doi:10.1007/BF02467552
35. Jaruratanasirikul S, Sriwiriyajan S (2003)
J Antimicrob Chemother 52:518521. doi:
10.1093/jac/dkg378
36. Thalhammer F, Schenk P, Burgmann H,
El Menyawi I, Hollenstein UM, Rosenkranz AR, Sunder-Plassmann G, Breyer S,
Ratheiser K (1998) Antimicrob Agents
Chemother 42:24172420
37. US Department of Health and Human
Services Food and Drug Administration
Center of Drug Evaluation and Research
(CDER) Center for Veterinarity Medicine
(CVM) (2001) Guidance for Industry
Bioanalytical Method Validation. Accessed
May 2004

Chromatographia 2009, 70, November (No. 9/10)

1427