248 Chem. Pharm. Bull. 63, 248–254 (2015) Vol. 63, No.

Regular Article

Prediction of the Stability of Meropenem in Intravenous Mixtures

Yuko Takasu, Miyako Yoshida, Mio Tange, Keiichi Asahara, and Takahiro Uchida*
School of Pharmaceutical Science, Mukogawa Women’s University; 11–68 Koshien 9-Bancho, Nishinomiya 663–8179,
Japan.
Received July 15, 2014; accepted February 2, 2015

The purpose of this study was to predict the stability of meropenem in a mixed infusion. The hydrolysis of
meropenem in aqueous solution was found to be accelerated by pH, and by increasing concentrations of sodium
bisulfite (SBS) and L -cysteine. Equations were derived for the degradation rate constants (kobs) of pH, SBS and
L -cysteine, and fractional rate constants were estimated by the nonlinear least-squares method (quasi-Newton
method using the solver in Microsoft Excel) at 25°C. The activation energy (Ea) and frequency factor (A) were
calculated using the Arrhenius equation. The pH of the mixed infusion was estimated using the characteristic
pH curve. From these results, an equation was derived giving the residual ratio (%) of meropenem at any time
after mixing an infusion containing SBS and/or L -cysteine at any temperature, and in the pH range 4.0–10.0. A
high correlation was shown to exist between the estimated and determined residual ratios (%).
Key words stability prediction; meropenem; pH; sodium bisulfite (SBS);  L-cysteine; degradation rate constant

Carbapenems are the most potent β-lactam antibiotics and
were  first  developed  in  the  1980s  to  enhance  resistance  to  teristic pH curve (PHC curve; a kind of pH titration curve),10)
β-lactamases. The early carbapenems were not very hydro-
lytically stable, however, limiting drug administration to con-
trolled intravenous infusions. In the search for a more stable
compound  with  better  toxicity  profile,  the  basic  nuclear  struc-
ture was maintained and a dimethyl carbamoyl-pyrrolidinyl-
thio group was added as a weakly basic C-2 side-chain. This
resulted in a reduction of central toxicity and nephrotoxicity,
while maintaining anti-pseudomonal activity. In addition, the
improvement of stability (DHP-I stability) in vivo and the
improvement of activity against Haemophilus influenzae, etc.,
were achieved by introducing 1-β-methyl group into the car-
bapenem frame, resulting in the creation of meropenem. The
improvement in nephrotoxicity was the key advance in allow-
ing the global development of meropenem as a single agent.1)
Meropenem is a broad-spectrum carbapenem, active against
several clinically relevant Gram-positive and Gram-negative
aerobes, anaerobic bacteria and Pseudomonas aeruginosa.
The bactericidal activity of meropenem results from the
inhibition of cell wall synthesis through the inactivation of
penicillin-binding proteins.
Sodium  bisulfite  (SBS),  used  as  a  stabilizer  in  injectable  Chemical  Industries,  Ltd.,  Osaka,  Japan.  SBS,  L-cysteine and
preparations, is known to degrade various drugs including
thiamine,2) epinephrine,3) gabexate mesilate,4) nafamostat me-
silate,5) urokinase,6) morphine7)  and  fluorouracil.8) However,
there are no reports of detailed kinetic studies on the degrada-
tion of meropenem in the presence of SBS.
The prediction of the stability of a drug in an intravenous
admixture (mixed infusion) is important for accurate and
safe drug administration. Generally, the stability of a drug
in  a  mixed  infusion  can  be  predicted  from  the  pH  profile  and  AMIGRAND® (Terumo Co., Ltd., Tokyo, Japan) were used as
Arrhenius equation of the degradation rate constants, if the
temperature and pH of the test solution are known. Although
meropenem is generally administered as an infusion in saline,
in some cases it may be mixed with other substances, such as
amino acids.9)
A method for predicting the pH of mixed infusions was
developed in order to be able to evaluate the compatibility of

* To whom correspondence should be addressed.  e-mail: takahiro@mukogawa-u.ac.jp
the components of a mixed infusion. Employing the charac-
a method for pH estimation has been reported by Hirouchi et
al.11) based on computer simulations. However, the theoreti-
cal equations described by Hirouchi et al. were derived from
model preparations for injection and did not take into account 
the  influence  of  dilution  with  titrant.  The  PHC  curve  of  a 
mixed  infusion  could  not  therefore  be  fitted  to  the  equations 
of Hirouchi et al., but must be derived from observed values,
a painstaking and time-consuming exercise. In the present
study, a simple, theoretical, pH estimation method for mixed
infusions  was  derived,  using  a  fitted  PHC  curve  and  curve 
simulation by a computer (using Microsoft Excel).
In addition, we examined the degradation of meropenem
due  to  catalytic  hydrolysis  by  SBS  and  L-cysteine. From the
results, we derived an equation to predict the residual ratio
(%) of meropenem at any time after mixing, on the basis of
the simulated pH of the mixed infusion using the PHC curve,
SBS and  L-cysteine concentrations, and the temperature.
Experimental
Materials Meropenem was supplied by Wako Pure
other reagents were special grade commercial products. As
buffer solutions, 0.05 M acetate buffer (pH 4.0–5.0), 0.05 M
phosphate buffer (pH 6.0–8.0) and 0.05 M carbonate buffer
(pH  9.0–10.0)  were  adjusted  to  an  ionic  strength  of  0.15  with 
sodium chloride. Meropen® (Dainippon Sumitomo Pharma
Co., Ltd., Osaka, Japan), AMINOLEBAN® Injection, Fructlact 
Injection,  and  AMINOFLUID®  Injection  (Otsuka  Pharmaceu-
tical Factory, Inc., Tokushima, Japan), FULCALIQ® 2 and
injectable preparations.
Determination of Meropenem Meropenem concentration
was measured by high-performance liquid chromatography
(HPLC). Meropenem concentrations were determined 0, 1, 2,
3  and  4 h  after  mixing  with  SBS  or  L-cysteine. Each degrada-
tion rate constant (kobs) was calculated from the slope of the
relationship between the log residual ratio of meropenem (%)
© 2015 The Pharmaceutical Society of Japan
Vol. 63, No. 4 (2015) Chem. Pharm. Bull. 249

and time after mixing. Ca′n ⋅V0

Can = (Eq. 4)
Equipment V0 +Vt
 A  Shimadzu  LC-10AT  VP  high-performance  liquid  chro-
C ′⋅V0
matograph,  a  Shimadzu  SPD-10A  VP  UV-VIS  detector,  and  a  C= (Eq. 5)
Shimadzu C-R7A plus chromate pack were used. V0 +Vt
Measurement Conditions Ct′⋅Vt
 Preliminary  studies  confirmed  that  optimized  chromato- Ct = (Eq. 6)
V0 +Vt
graphic conditions were as described in the Pharmacopoiea.12)
Column, CAPCELL PAK C18 HPLC packed column type MG where Ca1, Ca2, Ca3, ..., Can are the concentrations of weak
5 µm (4.6 mm i.d.×150 mm);  flow  rate  1.0 mL/min;  column  acids 1, 2, 3, ..., n in the sample solution; K1, K 2, K3, ..., Kn
oven  set  at  40°C;  injection  volume  20 µL; detection wave- are the dissociation constants of the weak acids 1, 2, 3, ..., n,
length 300 nm. The isocratic mobile phase was a mixture respectively; C is the concentration of the strong acid (repre-
of 10 m M sodium phosphate buffer (pH 7.4) and acetonitrile sented as a positive value) or the base (negative value) present
(100 : 10, v/v). as an excipient in the sample solution; Ct is the concentration
Estimation of the pH of the Mixed Infusion Observed of HCl (represented as a positive value) or NaOH (negative
values from pH titration were used to plot the PHC curve of value), added as the titrant to the sample solution; C′an is the
the  mixed  infusion.  Based  on  the  general  equation  for  PHC  initial concentration of the acid (positive value) or base (nega-
curves,  the  PHC  curve  of  each  preparation  was  fitted  from  tive value) in the sample solution, respectively; C′t is the con-
the observed values, from which the pH of the mixed infusion centration of HCl or NaOH added to the sample solution as
could be estimated. titrant; V0 is the initial volume of the titration sample (500 mL)
pH Titration and Vt is the volume of the titrant added to the sample. [H+]
 Various  commercial  injections  were  diluted  to  500 mL  with  is the hydrogen ion concentration and Kw is the ion product
distilled water and titrated against 0.5 M hydrochloric acid and of water. For practical purposes, [H+] and Kw at 25°C were
sodium hydroxide. Similarly, 500 mL aliquots of various com- regarded as 10−pH obs and 1.0×10−14, respectively.
mercial infusions were titrated against 0.5 M hydrochloric acid The parameters of the general equations for the PHC curve
and sodium hydroxide. (i.e., concentrations and pKs of weak acids and concentra-
Fitting the PHC Curve of the Injection Preparation  tions  of  strong  acids  or  bases)  were  fitted  with  some  of  the 
 Injectable  drugs  are  generally  classified  as  weak  acids,  observed values. The parameters of the PHC curve were then
weak bases or salts. Taking a preparation of weak acid, with a obtained by solving the simultaneous equation Eq. 2. Next, the
total concentration of Ca1, the ionization of the weak acid may  parameters other than pK  were  fitted  by  the  nonlinear  least-
be represented as: HA→H++A−,  where  HA  is  the  non-ionized  squares method (simplex method). The PHC curves simulated
acid, A− is the ionized acid and H+ is hydrogen ion. Applying by these parameters and the observed titration values were
the law of mass action, material balance and charge balance drawn  graphically.  If  the  PHC  curve  only  partially  fitted,  the 
to the equilibrium of this weak acid, the general equation of parameters of the PHC curve were modified by the simultane-
the PHC curve for a preparation can be expressed as in Eq. ous equation Eq. 1 and the simplex methods.
1. Similarly, taking a preparation containing two kinds of Simulation of the PHC Curve of Mixed Infusions
weak acids of which the total concentrations are Ca1 and Ca2, The effect of dilution was an important factor when the
the general equation for the preparation can be expressed as parameters  of  the  PHC  curve  were  fitted  to  the  titration  data. 
in Eq. 2. If a preparation contains n kinds of weak acids, the Nevertheless, once these parameters had been obtained, the
general equation of the PHC curve for the preparation can be PHC curves of the preparations could be treated simply ac-
expressed as in Eq. 3. cording to Eqs. 1 to 3, without Eqs. 4 to 6. Therefore, assum-
ing  the  general  equations  of  the  PHC  curves  for  an  injection, 
Ca1 ⋅ K1 Kw
[H+ ] = + + C + Ct (Eq. 1) an infusion, a mixed infusion of these preparations and water,
[H+ ]+ K1 [H+ ] to  be  Eqs.  7  to  10,  respectively,  the  relationship  Eq.  9=Eq.
Ca1 ⋅ K1 Ca2 ⋅ K 2 Kw 7+Eq. 8−Eq. 10 held at a constant ion concentration. Each of
[H+ ] = + + + C + Ct (Eq. 2) the terms except CtA, CtB, CtW and CtM could then be eliminat-
[H+ ]+ K1 [H+ ]+ K 2 [H+ ]
ed from Eqs. 7 to 10 at a constant hydrogen ion concentration,
n
 Cai ⋅ Ki Kw  to obtain Eq. 11. Since each PHC curve represents the relation
[H+ ] =   [H +
+ + + C + Ct 
]+ Ki [H ] 
(Eq. 3) between the pH and the volume of the titrants (i.e., CtM), the
i=1
PHC curves of mixed infusions could be simulated by apply-
In order to improve solubility, preparations of weak bases ing Eq. 11 at all pHs. If n kinds of preparations were mixed,
often contain a strong acid as an excipient. The preparation of the PHC curve of the mixed infusion could be simulated from
the weak base can therefore be assumed to be a preparation the PHC curves of n−1 kinds of preparations and another ad-
of  a  conjugated  weak  acid.  Accordingly,  the  general  equation  ditive injection. 
of the PHC curve for a preparation of a weak base can be ex- CaA ⋅ K A Kw
pressed as in Eq. 1, and the PHC curve of the preparation of n [H+ ] = + + + + CA + CtA (Eq. 7)
[H ]+ K A [H ]
kinds of weak bases can be represented as in Eq. 3.
The general equation of the PHC curve for injectable prepa- CaB ⋅ K B Kw
[H+ ] = + + CB + CtB (Eq. 8)
rations can therefore, for practical purposes, be represented as [H+ ]+ K B [H+ ]
Eqs.  1  to  3,  and  the  influence  of  dilution  with  titrants  can  be 
corrected by Eqs. 4 to 6.
250 Chem. Pharm. Bull. Vol. 63, No. 4 (2015)

CaA ⋅ K A CaB ⋅ K B Kw
[H+ ] = + + + CA + CB + CtM  (Eq. 9)
[H+ ]+ K A [H+ ]+ K B [H+ ]
Kw
[H+ ] = + CtW (Eq. 10)
[H+ ]

CtM = CtA + CtB − CtW (Eq. 11)

Revision of the PHC Curve for Increased Infusion Volumes

The  influence  of  dilution  with  additive  injections  is  not  in-
cluded in Eq. 11. If the infusion volume is increased with ad-
ditive  injections,  the  PHC  curves  of  the  mixed  infusion  must 
be corrected by Eq. 12. This equation was obtained following
the same process as for Eq. 11.

CtC = r ⋅ Ct − ( r − 1) ⋅ CtW (Eq. 12)

Fig.  1.  pH  Profile  of  the  Degradation  of  Meropenem  at  25°C  and 
μ=0.15
where r is the concentration ratio (initial infusion volume
Initial concentration of meropenem 500 µg/mL.
(500 mL)/increased  infusion  volume  with  additives),  CtC is the
corrected concentration of HCl or NaOH (added as the titrant)
in the mixed infusion at a constant pH, Ct is the concentration
of HCl or NaOH in a 500 mL aliquot of the mixed infusion at
the same pH, and CtW is the concentration of HCl or NaOH
in a 500 mL aliquot of distilled water at the same pH. At Ct
and CtW,  the  influence  of  dilution  with  additive  injections  was 
omitted.
Simulation of the PHC Curve of a Mixed Infusion
According to Eqs. 11 and 12, the PHC curve of the mixed
infusion can be simulated from the PHC curves of each con-
stituent preparation.
Estimated pH of a Mixed Infusion
 The  pHs  of  the  injections  (diluted  to  500 mL  with  distilled 
water) or infusions are represented by the pHs at the origin of
the PHC curve of the preparations. The estimated pH of the
mixed infusion was thus obtained as the pH at the origin of
the simulated PHC curve.
Fig. 2. Arrhenius-Type Relationship between the Degradation Rate
Kinetic Procedures The stability of the meropenem solu- Constant of Meropenem and the Temperature (4, 25 and 40°C) at pH
tion (500 µg/mL)  buffered  at  pH  4.0–10.0  in  the  presence  of  4.0–10.0 and μ=0.15
SBS  (0,  0.1,  0.5  and  1.0 m M) or L-cysteine (0, 0.25, 0.5, 1.0 Initial concentration of meropenem 500 µg/mL.  ◇ pH 4.0, * pH 5.0, ◆ pH 6.0,
and  2.0 mg/mL)  was  examined  at  4,  25  and  40°C.  The  sta- × pH 7.0, ▲ pH 8.0, ○ pH 9.0, ■ pH 10.0.
bility of Meropen®  in  AMINOLEBAN®  Injection,  Fructlact 
Injection,  AMINOFLUID®  Injection,  FULCALIQ® 2 and
AMIGRAND® was examined at 4, 25 or 40°C. hydrogen-ion-catalyzed  degradation  and  the  hydroxide-ion-
catalyzed  degradation  reaction,  respectively,  and  k H2O is the
Results and Discussion first-order  rate  constant  for  the  spontaneous  water-catalyzed 
Factors Affecting the Stability of Meropenem degradation reaction.
Degradation of Meropenem at pH 4.0–10.0 At 25°C, the residual meropenem concentration as de-
The degradation of mepenem, 500 µg/mL,  was  studied  at  termined by HPLC, and [H+], [OH−] calculated from pH,
25°C. The pH-profile of mepenem degradation in the pH range  were assigned to Eq. 13. The fractional rate constants, k H2O,
4.0–10.0 is shown in Fig. 1, where the rate constants are ex- kOH−, and k H+ were estimated by the nonlinear least-squares
pressed  on  a  logarithmic  scale.  It  was  concluded  that  specific  method (quasi-Newton method using the solver in Micro-
hydrogen-ion-catalyzed  degradation  occurred  at  pH  4.0–5.0,  soft Excel) and determined to be: k H2O, 1.15×10−3 h−1; kOH−,
water-catalyzed degradation at pH 5.0–8.0, and hydroxide-ion- 3.00×103 M−1 h−1 and k H+, 1.36×102 M−1 h−1.
catalyzed degradation at pH 8.0–10.0. The influence of temperature was also investigated. The Ar-
As  the  data  plot  in  Fig.  1  seemed  to  display  specific  acid– rhenius equation (Eq. 14) shows the relationship between the
base catalysis kinetics, Eq. 13 was used as the model kinetic degradation rate constant and the absolute temperature.
equation for the effect of pH on k0.
Ea 1
log kobs = − ⋅ + log A (Eq. 14)
2.303R T
k0 = kH2O + kOH− ⋅ [OH − ]+ kH+ ⋅ [H+ ] (Eq. 13)
where kobs is the degradation rate constant, Ea is the activation
where k H+ and kOH− are the second-order rate constants for the energy, R  is  the  gas  constant  (1.987 cal mol−1), T is absolute
 Chem. Pharm. Bull.
Vol. 63, No. 4 (2015)251

Fig. 3. Pseudo-First-Order Plot for Degradation of Meropenem in the Fig.  4.  Relationship  between  the  Total  Concentration  of  SBS  and  the 
Presence  of  SBS  (0,  0.1,  0.5  and  1.0 m M) in 0.05 M  Phosphate  Buffer  (pH  Degradation Rate Constant (kobs) of Meropenem in 0.05 M Phosphate
6.0, μ=0.15) at 25°C Buffer (pH 6.0, μ=0.15) at 25°C
Initial concentration of meropenem 500 µg/mL.  ◇  SBS  0 m M , ○  SBS  0.1 m M , Initial concentration of meropenem 500 µg/mL.
△ SBS 0.5 m M , □ SBS 1.0 m M.

temperature (K), and A is the frequency factor. As shown in where SBS concentration ([SBS]total) is represented by Eq. 17.4)
Fig. 2, Arrhenius plots revealed good linearity in the range
pH 4.0–10.0, with Ea values for mepenem of 10.7 kcal/mol (pH  [SBS]total = [H 2SO3 ]+[HSO3− ]+[SO32− ] (Eq. 17)
4.0),  16.1 kcal/mol  (pH  5.0),  15.9 kcal/mol  (pH  6.0),  12.9 kcal/
mol  (pH  7.0),  14.8 kcal/mol  (pH  8.0),  20.6 kcal/mol  (pH  9.0)  Based  on  the  dissociation  constant  of  SBS  at  25°C 
and 16.7 kcal/mol (pH 10.0). (100 kPa), kSBS1=1.72×10−2 (pKSBS1=1.8) and kSBS2=6.24×10−8
Degradation Rate of Meropenem in the Presence of SBS    (pKSBS2=7.2).14)  SBS  in  this  pH  range  (pH  4.0–10.0)  was  con-
SBS,  present  as  a  stabilizer  in  injectable  preparations,  is  sidered to be present as bisulfate ions (HSO3−) and sulfite ions 
known to degrade β-lactam antibiotics.13) However, the mecha- (SO32−), kSBS×[SBS]total can therefore be represented as Eq. 18:
nism by which the SBS concentration affects meropenem deg-
radation has not yet been elucidated. Therefore, kinetic experi- kSBS ×[SBS]total = kSO32− ×[SO32− ]+ kHSO3− ×[HSO3− ] (Eq. 18)
ments were performed, and the residual meropenem concen-
tration measured by HPLC. The degradation of meropenem, and  the  dissociation  constants  of  SBS  (KSBS1, KSBS2) can be
at an initial concentration of 500 µg/mL,  by  SBS  at  various  represented by Eqs. 19 and 20. 
concentrations was evaluated at 25°C and pH 4.0–10.0. The
[H+ ]×[HSO3− ]
degradation rate constant of meropenem in the presence of KSBS1=   (Eq. 19)
[H 2SO3 ]
SBS  (kobs) was measured at 25°C. A linear relationship was
observed between time and log residual ratio (%) at pH 6.0 [H+ ]×[SO32− ]
(Fig. 3), indicating that the degradation of meropenem fol- KSBS2 = (Eq. 20)
[HSO3− ]
lowed  pseudo-first-order  kinetics.  This  was  also  the  case  at 
other  pHs.  The  apparent  first-order  rate  constants  were  ob- From Eqs. 16 to 20, kSBS can be represented as shown in
tained from the slopes of the semi-log plots. The slope of the Eq. 21.
line increased with increasing SBS concentration.
The rate constant for the catalytic hydrolysis of meropenem kHSO3− ⋅ KSBS1 ⋅ [H+ ]+ kSO32− ⋅ KSBS1 ⋅ KSBS2
kSBS = (Eq. 21)
by SBS (kSBS) is obtained from Eq. 15. [H+ ]2 + KSBS1 ⋅ [H+ ]+ KSBS1 ⋅ KSBS2

 2.303   r  where k HSO−3 is the second order degradation constant of

kobs =  −  ⋅ log  100  (Eq. 15)
 t    HSO3−, and kSO32− is the second order degradation constant
of SO32−. The fractional rate constants, k HSO−3 and kSO32− were
where kobs  is  the  pseudo-first-order  reaction  rate  constant,  t is estimated by nonlinear least-squares method (quasi-Newton
time after mixing, and r is the residual ratio (%). Typical plots method using the solver in Microsoft Excel), and values of
for  SBS  concentration  versus degradation rate constants of k HSO−3 (2.78 M−1 h−1) and kSO32− (2.75 M−1 h−1) were obtained.
meropenem (pH 6.0, 25°C) yielded a straight line as shown in These  results  show  that  the  accelerating  effect  of  sulfite  ions 
Fig. 4. The degradation rate of meropenem was proportional on the hydrolysis of meropenem was the same as that of bisul-
to  the  total  concentration  of  SBS  ([SBS]total). Therefore, the fate ions at pH 4.0–10.0 and 25°C.
rate  constant  of  meropenem  in  the  presence  of  SBS  (kobs) can The influence of temperature on the degradation of merope-
be represented by Eq. 16. nem by SBS was also investigated. As shown in Fig. 5, Arrhe-
nius plots revealed good linearity between SBS concentrations 
kobs = k0 + kSBS ×[SBS]total (Eq. 16) of 0, 0.1, 0.5 and 1.0 m M at pH 6.0, and the values obtained for
the Ea  of  meropenem,15.9 kcal/mol,  15.7 kcal/mol,  16.5 kcal/
252
Fig. 5. Arrhenius-Type Relationship between the Degradation Rate
Constant of Meropenem and Temperature (4, 25 and 40°C) in the Pres-
ence  of  SBS  (0,  0.1,  0.5,  1.0 m M) in 0.05 M  Phosphate  Buffer  (pH  6.0,
μ=0.15)
Initial concentration of meropenem 500 µg/mL.  ◇  SBS  0 m M , ○  SBS  0.1 m M ,
△ SBS 0.5 m M , □ SBS 1.0 m M.
mol, and 14.7 kcal/mol, respectively.
Degradation Rate of Meropenem in the Presence of L-
Cysteine
L-Cysteine is often added to infusions for purposes of
nutrition. It may be present as the free amino acid or as N-
acetylcysteine, which can be degraded by hydrolysis in the
body. L-Cysteine is known to degrade carbapenem antibiotics,
but the effect of the L-cysteine concentration on the rate of
degradation has not been elucidated. Kinetic experiments were
therefore carried out, with the residual meropenem concentra-
tions measured by HPLC. The degradation of meropenem at
an initial concentration of 500 µg/mL  by  various  concentra-
tions of L-cysteine or N-acetylcysteine was evaluated at 25°C
and pH 4.0, 6.0 and 8.0. The degradation rate constant of
meropenem in the presence of L-cysteine (kcys) was measured
at 25°C. Typical plots for L-cysteine concentration versus deg-
radation rate constants of meropenem yielded a straight line
as shown in Fig. 6. N-Acetylcysteine concentrations were con-
verted to L-cysteine concentrations. N-Acetylcysteine itself ap-
peared to have little effect on the degradation rate constants of
meropenem. The degradation rate of meropenem was directly
proportional to the total concentration of L-cysteine ([cys]total).
Based  on  the  dissociation  constant  of  L-cysteine at 25°C,
kcys1=1.20×10−2 (pKcys1=1.92),  kcys2=4.68×10−9 (pKcys2=8.33)
and kcys3=1.66×10−11 (pKcys3=10.78),15) the degradation con-
stant of meropenem in the presence of cysteine (kobs) can be
represented as Eq. 22, [cys]total as Eq. 23 and kcys×[cys]total as
Eq. 24.
kobs = k0 + kcys ×[cys]total
[cys]total = [COOH ⋅ NH+3 ⋅ SH]+[COO− ⋅ NH+3 ⋅ SH]
+ [COO − ⋅ NH+3 ⋅ S− ]+[COO − ⋅ NH 2 ⋅ S− ] (Eq. 23)
Chem. Pharm. Bull. Vol. 63, No. 4 (2015)
Fig. 6. Relationship between the Total Concentrations of L-Cysteine
(0,  0.25,  0.5,  1.0,  2.0 mg/mL)  and  N-Acetylcysteine (0, 0.25, 0.5, 1.0,
2.0 mg/mL;  Converted  to  L-Cysteine Concentration) and the Degradation
Rate Constant (kobs) of Meropenem in 0.05 M  Phosphate  Buffer  (pH  6.0, 
μ=0.15) at 25°C
Initial concentration of meropenem 500 µg/mL.  ○ N-acetylcysteine,
● L-cysteine.
 
kcys × [cys]total
(kCOO− ⋅ NH+3 ⋅SH ⋅ K cys1 ⋅ [H+ ]2
+ kCOO− ⋅ NH+3 ⋅S− ⋅ K cys1 ⋅ K cys2 ⋅ [H+ ]
+ kCOO− ⋅ NH2 ⋅S− ⋅ K cys1 ⋅ K cys2 ⋅ K cys3 )
= × [cys]total (Eq. 24)
[H+ ]3 + K cys1 ⋅ [H+ ]2 + K cys1 ⋅ K cys2 ⋅ [H+ ]
+ K cys1 ⋅ K cys2 ⋅ K cys3
The fractional rate constants, k HSO−3 and kSO32− were esti-
mated by the nonlinear least-squares method (quasi-Newton
method using the solver in Microsoft Excel) to obtain the
following values: kCOO−·NH+3 ·SH (2.48×10 M−1 h−1), kCOO−·NH+3 ·S−
(1.20×104 M−1 h−1) and kCOO−·NH2 ·SH+ (3.31 M−1 h−1). The value of
kCOOH·NH+3 ·SH was much smaller than the other fractional rate
constants, so kCOOH·NH+3 ·SH was omitted from the calculations,
as it seems to have little effect on the degradation rate con-
stants of meropenem.
The  influence  of  temperature  on  the  degradation  of  me-
ropenem by L-cysteine was also investigated. As shown in
Fig. 7, Arrhenius plots revealed good linearity at L-cysteine
concentrations  of  0,  0.25,  0.5,  1.0  and  2.0 mg/mL  at  pH  6.0. 
The values obtained for the Ea  of  meropenem  were  11.7 kcal/
mol  (0.25 mg/mL),  11.0 kcal/mol  (0.5 mg/mL),  10.4 kcal/mol 
(1.0 mg/mL)  and  10.6 kcal/mol  (2.0 mg/mL),  while  the  value 
obtained for the Ea  of  meropenem  was  16.0 kcal/mol  in  the 
absence of L-cysteine  (0 mg/mL).  There  were  no  differences 
between the values of Ea in presence of L-cysteine (0.25, 0.5,
1.0,  2.0 mg/mL).  However,  there  was  difference  between  the 
values of Ea in the presence of L-cysteine and the value of Ea
(Eq. 22) in the absence of L-cysteine. The dose-dependent effect of L-
cysteine on the Ea of meropenem in this reaction is suggested
to be small. The log kobs of meropenem showed a temperature-
dependent increase at any concentration of L-cysteine. From
these results, the degradation of meropenem is suggested to
increase in a temperature-dependent manner.
 Chem. Pharm. Bull.
Vol. 63, No. 4 (2015)253

Fig. 7. Arrhenius-Type Relationship between the Degradation Rate

Constant of Meropenem and Temperature (4, 25 and 40°C) in the Pres-
ence of L-Cysteine  (0,  0.25,  0.5,  1.0,  2.0 mg/mL)  in  0.05 M Phosphate
Buffer (pH 6.0, μ=0.15)
Initial concentration of meropenem 500 µg/mL. ◇ L-cysteine 0 mg/mL, ○ L-cys-
teine  0.25 mg/mL,  △ L-cysteine  0.5 mg/mL,  □ L-cysteine  1.0 mg/mL,  × L-cysteine
Fig. 8. Correlation between pH Estimated Using PHC Curve and the
2.0 mg/mL.
pH Values Determined after Mixing Meropen®  with  AMINOLEBAN®
Injection  ( ),  Fructlact  Injection  (×), AMINOFLUID®  Injection  ( ),
FULCALIQ® 2 ( ) or AMIGRAND® ( ) at 25°C
pH Estimation Method for a Mixed Infusion After y=1.00x, r2=0.98.
mixing Meropen®  with  AMINOLEBAN®  Injection,  Fructlact 
Injection,  AMINOFLUID®  Injection  or  FULCALIQ® 2 or
AMIGRAND®, pH was estimated at 25°C using the PHC
curve. Figure 8 shows the correlation between the estimated
and determined pH values in the mixed infusion (y=1.00x,
r2=0.98).  The  pH  of  each  mixed  infusion  could  be  estimated 
correctly using the PHC curve.
Stability Prediction of Meropenem in Practical Mixed
Infusions From the above results, the degradation rate con-
stant of meropenem after mixture with an infusion contained
SBS and  L-cysteine at pH 4.0–10.0, can be represented by Eq.
25.
kobs (T2 )
T1 − T2
= k0 (T1 ) ⋅ exp( − Ea1 / R ) ⋅
T1 ⋅ T2
(kHSO3− ⋅ KSBS1 ⋅ [H+ ]+ kSO32− ⋅ KSBS1 ⋅ KSBS2 )
+ [SBS]total ⋅
[H+ ]2 + KSBS1 ⋅ [H+ ]+ KSBS1 ⋅ KSBS2
T1 − T2
× exp(− Ea 2 / R ) ⋅
T1 ⋅ T2
Fig.  9.  Correlation  between  Estimated  and  Observed  Residual  Ratio 
+[cys]total (%) after Mixing Meropen®  with  AMINOLEBAN®  Injection,  Fructlact 
Injection, AMINOFLUID® Injection, FULCALIQ® 2 and AMIGRAND®,
(kCOO− ⋅ NH+3 ⋅SH ⋅ K cys1 ⋅ [H+ ]2 at 4, 25 and 40°C
+ kCOO− ⋅ NH+3 ⋅S− ⋅ K cys1 ⋅ K cys2 ⋅ [H+ ] Initial concentration of meropenem 500 µg/mL.  y=1.00x, r2=0.98. 
+ kCOO− ⋅ NH2 ⋅S− ⋅ K cys1 ⋅ K cys2 ⋅ K cys3 ) ○  AMINOLEBAN®  Injection  at  4°C,  —  Fructlact  Injection  at  4°C, 
× □ AMINOFLUID®  Injection  at  4°C,  ◇ FULCALIQ® 2 at 4°C, △ AMIGRAND®
[H+ ]3 + K cys1 ⋅ [H+ ]2 + K cys1 ⋅ K cys2 ⋅ [H+ ] at 4°C.   AMINOLEBAN®  Injection  at  25°C,  ×  Fructlact  Injection  at  25°C, 
+ K cys1 ⋅ K cys2 ⋅ K cys3 AMINOFLUID®  Injection  at  25°C,  FULCALIQ® 2 at 25°C,
AMIGRAND® at 25°C. ●  AMINOLEBAN®  Injection  at  40°C,  + Fructlact In-
T1 − T2 jection  at  40°C,  ■ AMINOFLUID®  Injection  at  40°C,  ◆ FULCALIQ® 2 at 40°C,
× exp(− Ea 3 / R ) ⋅ (Eq. 25) ▲ AMIGRAND® at 40°C.
T1 ⋅ T2
where Ea1 is the activation energy in the presence of the
acid–base catalytic effect, Ea2 is the activation energy in the (Ea1 to Ea3) are assigned to Eq. 25. The meropenem residual
presence  of  SBS,  Ea3 is the activation energy in the presence ratio (%) is represented by Eq. 26:
of L-cysteine; kobs at any temperature (T2) can be calculated if
298 K  (25°C)  is  assigned  to  T1 and the derived concentrations r =10{kobs ⋅ ( −t /2.303)} ×100 (Eq. 26)
and  constants  at  298 K  (25°C)  and  activation  energy  values 
254 Chem. Pharm. Bull.
From Eqs. 25 and 26, the meropenem residual ratio (%) can be
predicted at any time after mixing into an infusion containing
SBS and  L-cysteine at pH 4.0–10.0.
The correlation between the estimated and determined
residual ratio (%) of meropenem in Meropen® at 0, 1, 2, 3
and  4 h  after  mixing  with  AMINOLEBAN®  Injection,  Fruct-
lact  Injection,  AMINOFLUID®  Injection,  FULCALIQ® 2 or
AMIGRAND® at 4, 25 and 40°C (y=1.00x, r2=0.98) is shown  and L-cysteine concentration. Equations describing the deg-
in Fig. 9. The initial concentration of meropenem was 500 µg/
mL.  The  high  correlation  coefficient  q2  (0.99),  shows  a  high  been derived, and the activation energy (Ea) and frequency
correlation between the estimated and determined residual
ratios (%) of meropenem obtained in this study, which is rel-
evant for the drug combinations used in the clinical field.
These  results  confirm  that  an  equation  predicting  the  sta-
bility of meropenem, at any time and any temperature, after
mixing  with  an  infusion  containing  SBS  and  L-cysteine at pH
4.0–10.0 has been derived in this study.
We have previously derived an equation predicting the
stability of imipenem, at any time and any temperature, after
mixing  with  an  infusion  containing  SBS  and  L-cysteine at pH
4.0–10.0.16)  Both  the  kSBS and kcys of meropenem were smaller
than those of imipenem. This means that meropenem is more
stable than imipenem after mixing with an infusion contain-
ing  SBS  and  L-cysteine, probably because hydrolysis of the
β-lactam ring of imipenem occurs more easily than that of
meropenem, due to its chemical structure.
Itagaki et al.17)  reported  on  the  influence  of  L-cysteine on
the degradation of meropenem after mixing with an amino
acid infusion. In their report, N-acetyl cysteine had no effect
on the degradation of meropenem after mixing with an amino
acid infusion, thus supporting the result in the present paper.
It is assumed that N-acetyl cysteine is unable to mount a nu-
cleophilic attack on the β-lactam structure of meropenem be-
cause the electron density on the S atom of N-acetyl cysteine
is  significantly  lower  than  that  of  L-cysteine, due to the pres-
ence of the acetyl group, an electron-accepting substituent.
SBS  is  included  in  amino  acid  infusions  as  a  stabilizer.  In  8) Rock G. S., Pitman I. H., J. Pharm. Sci., 64, 216–220 (1975).
the present study, it is suggested that SBS affects the degrada-
tion of meropenem after mixing with the amino acid infusion.
Thus  the  influence  of  not  only  L-cysteine  but  also  SBS  must 
be considered when attempting to improve the precision of
prediction of the stability of meropenem in mixed infusions.
Our results indicate that, if antibiotics are administered as
an infusion using a bypass line, they may become unstable if
mixed  with  an  infusion  in  the  infusion  line  or  by  back-flow  102, 978–983 (1982).
from the bypass line to the main line. Yoshioka et al. reported
that the effectiveness of doripenem was decreased by a bypass
administration from amino acid infusion line.18) We suggest
that the effectiveness of meropenem may also be decreased
by a bypass administration from an amino acid infusion line.
The required minimum dosage and length of treatment should
therefore take account of this interaction between carbapenem
antibiotics and amino acid infusions, in order to maximise
the effectiveness of the antibiotics and avoid the appearance
of resistant bacteria. The equation derived in this study will
Vol. 63, No. 4 (2015)
enable prediction of the required concentration of meropenem
in mixed infusions and will therefore be useful for drawing up
an administration plan for this drug.
Conclusion
The  key  factors  influencing  the  stability  of  meropenem  in 
a  mixed  infusion  were  found  to  be  pH,  SBS  concentration, 
radation rate constants (kobs)  for  pH,  SBS  and  L-cysteine have
factor (A) calculated using the Arrhenius equation. The pH of
the mixed infusion could be estimated using the PHC curve.
Finally, an equation has been derived giving the residual ratio
(%) of meropenem at any time after mixing any infusion at
any temperature at pH 4.0–10.0. A high correlation was shown
between the estimated and the determined values for residual
ratio (%). The equation derived in this study will enable us to
predict the concentration of meropenem in mixed infusions
and will therefore improve the correct use of this drug.
Conflict of Interest The  authors  declare  no  conflict  of 
interest.
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