-------------------~
Antiviral Chemistry & Chemotherapy 1997 8(4): 353-362
Inhibition of human immunodeficiency virus type 1 reverse
transcriptase by degradation products of ceftazidime
SW Bcertschi!", AS CantrelP, MT Kuhfeld', U Lorenz',
DB Boyd2 and SR Jcskunos!"
1Lilly
Research Laboratories, Eli Lilly and Company,
Indianapolis, IN 46285-3811, USA.
2Department of Chemistry, Indiana University-Purdue
University at Indianapolis, Indianapolis, IN 46202-3274,
USA.
"Corresponding authors:
Tel: + 1 317276 1388 (SWB) or + 1 3172764679 (SRJ)
Fax: + 1 317 277 2154.
E-mail: baertschi@lilly.comorjaskunas_s_richard@lilly.com
Summary
Previous work by Hafkemeyer et a/. (1991) [Nucleic
Acids Research 19: 4059-4065] indicated that a
degradation product of ceftazidime, termed HP 0.35,
was active against the RNase H activity of human
immunodeficiency virus type 1 (HIV-1) and feline
immunodeficiency virus (FIV) reverse transcriptase (RT)
in vitro. Attempting to repeat these results, we isolated
HP 0.35 from an aqueous degradation of ceftazidime
and, after careful purification, we found HP 0.35 to be
essentially inactive against both the polymerase and
RNase H domains of HIV-1 RT (IC50 of> 100 I1g mL-1).
During the investigation we discovered that polymeric
degradation products of ceftazidime inhibited both
the polymerase and, to a greater extent, the RNase H
activities of HIV-1 RT in vitro (IC50 approximately 0.1
and 0.01 I1g mt', respectively). Subjecting HP 0.35 to
conditions under which it could polymerize induced
inhibitory activity similar to that of the polymeric cef-
tazidime degradation products. It is proposed that the
previously reported activity of HP 0.35 may have resulted
from the presence of low levels of polymeric material
either from incomplete purification or from polymeriza-
tion of HP 0.35 during storage or in vitro testing.
Keywords: Activity of HP 0.35; ceftazidime;
cephalosporins; HIV- 1 reverse transcriptase; RNase H
inhibition.
Received 17 November 1996; accepted 31 January 1997.
© 1997 International Medical Press ltd
Introduction
Reverse transcriptase (RT) of human immunodeficiency
virus type 1 (HIV-1) is the target for many antiviral com-
pounds in various stages of development as drugs for indi-
viduals infected with HIV-1 (for recent reviews, see
Hirsch & D'Aquila, 1993; Mayers, 1993; Sandstrom &
Oberg, 1993a,b). Two different types of anti-RT com-
pounds have received the most attention: nucleoside ana-
logues (Connolly & Hammer, 1992), such as AZT
(zidovudinc) (Mitsuya et al., 1985), ddC (zalcitabine) and
ddI (didanosine) (Mitsuya & Broder, 1986), and non-
nucleoside compounds, such as 9-Cl-TIBO (Pauwels et
al., 1990; Kukla et al., 1991; Debyser et al., 1991), HEPT
derivatives (Miyasaka et al., 1989), nevirapine (Merluzzi et
al., 1990; Hargrave et al., 1991; Hui et al., 1992), BHAP
compounds (Romero et al., 1991), pyridinone compounds
(Goldman et al., 1991; Saari et al., 1991), quinoline ana-
logues (Althaus et al., 1993a), and phenethylthiazole
thiourea (PETT) compounds (Ternansky et al., 1993;
Ahgren et al., 1995; Cantrell et al., 1996). However, the
clinical utility of many of these compounds has been ham-
pered by toxicity and limited overall clinical efficacy
(Sandstrom & Oberg, 1993a,b). The development of the
non-nucleoside compounds as antiviral agents has been
thwarted even more by the rapid emergence of resistant
viruses in cell culture (Richman et al., 1991; Nunberg et al.,
1991; Shih et al., 1991) and during therapy (Richman et
al., 1994; Saag etal., 1993).
In general, anti-R'T compounds have been found to
inhibit the DNA polymerase function ofRT (Connolly &
Hammer, 1992). On the other hand, they have much less
inhibitory activity for the RNase H function, to the extent
that it has been examined (Connolly & Hammer, 1992;
Hargrave et al., 1991; Althaus et al., 1993b; Hizi et al.,
1993). Nevertheless, the RNase H function is an essential
part of all retrovirus RT enzymes (Whitcomb & Hughes,
1992), including the HIV-1 enzyme (Boyer etal., 1992;
Hizi et al., 1990; Prasad & Goff, 1989). Furthermore, the
structural domain ofRT containing the RNase H function
is highly conserved (Johnson et al., 1986; Davies et al.,
1991; jacobo-Molina & Arnold, 1991).
Several inhibitors of RNase H enzymes have been
reported, including captan (Freeman-Wittig et al., 1986;
353
SW Baertschi et 0/.
Figure 1. Structure of ceftazidime and aldehyde degradation product (HP 0.35).
OY.-- COOH
/ H
~N---<s D
~ S
I ~
I o)=()·O
fN
~
0
1 Ceftazidime coo
Freeman-Wittig & Lewis, 1990), illimaquinone (Loya &
Hizi, 1993), sulphated polyanions (Moelling et al., 1989),
dAMP (Tan et al., 1991) and ribonucleoside vanadyl com-
plexes (Krug & Berger, 1989). An intriguing observation
over the last 5 to 10 years is that aged beta-lactam antibi-
otics, particularly ceftazidime and other cephalosporins,
inhibit viral RNase H (Hafkemeyer et al., 1991) as well as
eukaryotic DNA polymerase (Do et al., 1987; Neftel &
Hubscher, 1987; Hafkemeyer et al., 1990; Neftel et al.,
1993).
In an effort to identify the biologically active degrada-
tion productis) of ceftazidime (1) (Fig. 1), Hafkemeyer et
al. (1991) reported that a degradation product designated
HP 0.35 (2) (Fig. 1) inhibited the RNase H function of
the HIV-1 and feline immunodeficiency virus (FIV) RT
enzymes but not their polymerase functions, nor other
non-viral RNase H enzymes. Furthermore, they found
that HP 0.35 inhibited the replication of FIV in cell
culture at concentrations that did not inhibit cell
proliferation. These observations, plus our interest in
cephalosporins, prompted us to investigate the activity of
HP 0.35 in relation to the RNase H function ofHIV-1.
Materials and Experimental Procedures
Enzyme assays
HIV-1 RT was obtained from American Biotechnologies
(Cambridge, Mass., USA) and was assayed using standard
procedures. The RNA -dependent polymerase activity was
assayed with a poly(rC) template/oligo(dG IZ_18) primer
(20 ug mL-l; Pharmacia) in a buffer containing 50 mM
Tris-HCI pH 8.0, 75 mM KCI, 5 mM MgClz' 1 j..tM
[3H]dGTP (20 j..tCi mL-I), and 100 j..tg mL-I BSA. The
reaction (total volume 100 j..tL) was initiated by the addi-
tion of10 ul. enzyme (diluted with 50 mM Tris-HCI pH
8.0,1 mM EDTA,25 mM NaCI to give 4-25 ng per reac-
tion), incubated for 30 min at 3TC and stopped with 10
j..tL 0.5 M EDTA pH 8.0. Incorporation of the labelled
354
Y.--
/0 COOH
NlYl~H
N H 0
H2 N
---<
f
S
i 0 2
nucleotide into DNA was determined by adsorption onto
a DEAE Filtermat (Pharmacia) using a Skatron cell har-
vester (two 2xSSC washes and one 100% ethanol wash).
RNase H activity of HIV-1 RT was assayed as
described elsewhere (Starnes & Cheng, 1989). The stan-
dard reaction (50 j..tL) contained 50 mM Tris-HCI pH
8.0,50 mM KCI, 8 mM MgClz' 100 ug mL-I BSA, 2x10 4
c.p.m. of 3H-Iabelled substrate [poly(dC)·[3H]poly(rG)],
and 5 ul, of enzyme (10 ng) diluted as described for RT
polymerase assays.
Calf thymus DNA polymerase a and human DNA
polymerase p were obtained from Molecular Biology
Resources (Milwaukee, Wis., USA) and assayed essential-
ly as recommended by the manufacturer using salmon
sperm DNA as template, which had been activated by
digestion for 30 min at 3TC with 2 units mL-I DNase I
(Gibco BRL).
Analytical HPLC
The analytical scale reversed-phase (RP)-HPLC system
utilized a photo diode array detector with UV detection
from 200-400 nm at 4 nm resolution (Waters 991,
Milford, Mass., USA). The HPLC was run in a gradient
mode from 0 to 100% solvent B in 30 min and held at
100% solvent B for 10 min. Solvent A was an aqueous
solution containing 6.9 g L-I NH4HzP0 4, adjusted to pH
2.5 with H 3P04 . Solvent B was a mixture of 60% acetoni-
trile and 40% solvent A. The flow rate was 1.0 mL min-I.
A C-18 RP column was used [4.6x250 mm, YMC (5 j..tM,
120 A); YMC, Morris Plains, N.J., USA].
Preparative HPLC
The preparative HPLC system consisted of an automated
HPLC and fraction collection system. The HPLC was a
three-pump gradient system (Rainin HP; Rainin
Instruments, Woburn, Mass., USA); the third pump was
used to inject the sample onto the column. The column
was a semi-preparative C-18 RP column [20x250 mm
(5 j..tM, 120 A); YMC]. For the initial preparative cuts, the
© 1997 International Medical Press Ltd

HPLC was run in a gradient mode from 0 to 100% solvent
Bin 37 min. Solvent A was an aqueous solution of 0.1%
acetic acid and solvent B consisted of a mixture of 60%
acetonitrile and 40% water with 0.1% acetic acid. The flow
rate was 10 mL min-I. A fraction collector (Foxy 200;
ISCO, Lincoln, Nebr., USA) was used to automate sam-
ple collection, and the fractions were collected at O°C to
minimize post-column degradation. Fraction collection
was guided by observation of the UV response at 270 nm.
For subsequent purification steps, the sample was dis-
solved in solvent A and the sample was injected manually
onto the column using a 10 mL loop injector. The HPLC
was operated in an isocratic mode at 7 and 12% B with a
flow rate of20 mL min-I. The peak corresponding to alde-
hyde 2 was collected manually and cooled immediately to
O°C. For all preparative work, the solvent was removed by
lyophilization.
Liquid chromatography (LC)-mass
spectrometry (MS)
The LC-MS experiments were performed with a
Beckman System Gold liquid chromatograph (Beckman
Instruments, Palo Alto, Calif, USA) and a Sciex Model
API III mass spectrometer (Sciex, Thornhill, Canada). A
heated nebulizer atmospheric chemical ionization source
was used. The mobile phase for LC-MS was the same as
the preparative solvents described above; the gradient pro-
gramme was the same as described in the analytical HPLC
section. A Beckman model 168 diode array UV detector
was used between the column and mass spectrometer
interface to correlate any changes in retention order from
the phosphate eluent system described in the analytical
HPLC section. The source was held at 450"C with nitro-
gen used as the nebulizer gas at a pressure of 551 kPa.
Approximately 100 ilL of a 5 mg mL-I sample was inject-
ed onto the column.
NMR
The NMR spectra were collected on a Bruker 500 MHz
NMR system (Bruker Instruments, Billerica, Mass., USA)
in zHzO to which a small drop of zHCI had been added.
The chemical shifts were externally referenced to tetra-
methylsilane. Carbon-proton correlation assignments
were detected using two-dimensional heteronuclear exper-
iments designed to detect correlations due to coupling
through one (Derome, 1987) or more than one (Martin &
Zektzer, 1988) chemical bond.
MS
Fast atom bombardment (FAB) and FAB-MS/MS mass
spectra were generated from a VG ZAB-3F mass spec-
trometer and acquired by a Varian MAT SS200 data
system. Samples were dissolved in 'magic bullet' (dithio-
threitol:dithioerythritol, 5:1, in methanol) and delivered to
Antiviral Chemistry & Chemotherapy 8(4)
Inhibition by degradation products of ceftazidime
the ion source via a direct insertion probe. A Xenon fast
atom gun was used to deliver energetic particles (8 eV) for
bombardment of the sample. For MS/MS, the parent ion
(attenuated 50%) was selected by the first magnet and then
collisionally activated in the collision chamber with either
helium or air. Daughter ion mass spectra were then rnea-
sured by linked-scanning (B/E) of the ftnal two sectors.
Degradation of ceftazidime
Ceftazidime (pentahydrate, 32 g) was allowed to degrade
at pH 9 in 200 mM dibasic sodium phosphate buffer at
3TC (±3°C) for around 50 h, and at room temperature for
210 h. The pH of the solution dropped as degradation
occurred and was maintained between 6 and 9 by addition
of 5 M NaOH at several time points. HPLC analysis of
the resulting solution showed that ceftazidime was over
98% degraded; the largest single degradation product
(approximately 6% of total peak area, retention time
approximately 15 min; Fig. 2) was shown to have a mole-
cular weight of m/z 314 by LC-MS [atmospheric pressure
chemical ionization (APCI), positive ion mode], consis-
tent with that of aldehyde 2.
To simplify the degradation process for generation
of larger amounts of 2, the degradation of ceftazidime
was studied in 0.1 M NaOH. HPLC analysis of the
solution after 24 h at room temperature indicated a
profile of degradation peak similar to that described
immediately above. A higher level of aldehyde 2 was
present in the 0.1 M NaOH-degraded solution (>10%
total peak area). Thus, the 0.1 M NaOH degradation
was utilized to provide degraded material for prepara-
tive HPLC work in addition to the pH 9 degradation
described above.
Figure 2. HPLC analysis of a solution of ceftazidime after
degradation in a pH 9 buffer.
0.4
Aldehyde 2
1.5
/
0.3 AldehYd\
sc
a
-202
o
~ 10 15
Time[mln]
20 25 3D
0.1
OO--l...,--~""
10 15 20 25 30
Time (min)
The inset shows the HPLC chromatogram (UV detection,
210 nm) for the most highly purified sample of aldehyde 2.
355
SW Baertschi ef a/.

Fractionation for RNase H activity testing Table 1. NMR assignments for aldehyde 2.
The degraded solutions of ceftazidime were lyophilized
and reconstituted (10 mg mL-l) for preparative HPLC.
The solutions were fractionated by preparative RP- HPLC
(using the system described above with a gradient of
0-100% B over 35 min, 10 mL min"? flow rate) into eight
fractions and analysed for activity (see below). The results
of the RNase H activity testing showed that fractions 3
and 8 contained the highest activity. Analysis of fraction 3
by LC-MS (APCl, positive ion mode) showed the princi-
pal component to be aldehyde 2. Fraction 8 was presumed
to have significant levels of polymeric material, based upon
HPLC analysis of the sample that showed a 'broad hump'
Long-range
of peaks rather than one or a few well-defined peaks. The Site Multiplicity /) (carbon) s (proton) coupling
peak corresponding to fraction 3 (aldehyde 2) was chosen
for larger scale purification. 1 2 88.58 4.87 3.15
2 3 45.39 3.15
4 1 161.94 3.15
Isolation of aldehyde
5 1 144.21 6.81
Over 140 preparative injections of the reconstituted solu- 6 1 130.78 6.81
tions (20 mL per injection) were made using the prepara- 8 1 171.16 6.81
tive system described above, from which 1132 mg of alde- 10 2 112.16 6.81
hyde 2 was isolated. After two more HPLC purification 13 1 84.77 1.24
14 1 177.79 1.24
steps, the sample was analysed by HPLC for purity and
15,16 4 23.71 1.24 1.24
shown to be approximately 90% pure (Fig. 2). This sample
was used for spectroscopic characterization and was tested
for RNase H activity.
the solvent to 2H20PHCl resulted in a simplified spectrum
Spectroscopic characterization of aldehyde 2 with sharpened peaks. Analysis of the spectra revealed that
Analysis of2 by MS (FAB and APCl) showed a prominent the aldehydewas in the gem-diol form, consistent with that
ion (MH+) at m/z 315. Accurate mass FAB measurements previously described (Hafkemeyer et al, 1991). Carbon and
indicated a formula for MH+ of CllHlSN40SS (found m/z proton assignments are shown in Table 1.
315.0752; calcd 315.0763). The sample was chro-
matographed on a TLC plate (15:3:1, EtOAc:HOAc: Preparation of the ceftazidime high molecular
H 20, v/v/v); reaction with dinitrophenylhydrazine turned weight polymer (HMWP)
the main spot yellow, indicating the presence of a non-con- Bulk ceftazidime (around 1 kg) was heated at 60°C for
jugated aldehyde or ketone. NMR analysis in DMSO-d6 210 h in a covered container. The stressed material was
clearly showed the presence of an aldehyde (0 199.7 in dissolved in a stirred 0.1 M potassium phosphate solution
carbon and 0 9.52 in proton), but both the carbon and in which the pH was continuously adjusted to 7 with the
proton spectrawere complicatedby broad peaks (presumably addition ofN~C03' Following complete dissolution of the
due to the formation of multiple conformers). Changing material, the pH of the resulting solution was adjusted to 2

Table 2. Inhibition of RNase H and polymerase activities by partially polymerized samples of aldehyde 2.
Amount of 2 Anti-RNase H activity Anti-polyrnerose activity
Sample remaining (%) (IC 50 /lg mL-l) (IC 50 /lg mL-l)

2 and 0.1 M NaOH 6.7 0.08 0.2

2 and 0.05 M H2S04 5.6 0.06 0.2
2 and acetic acid 13 0.2 0.2
2 and water 18 1 1.8
2 and PO4 puffer pH 8 24 2 0.5
2 and heat only (no added water) 89 100 320
2 (starting material, >90% pure) 100 »100 »100
2* (partially purified) -75 45 »100
HMWP o 0.02 0.02

356 © 1997 International Medical Press Ltd

 Inhibition by degradation productsof ceftazidime

with the addition ofHCl. The precipitate that formed in this
step was filtered using Whatman number 1 filter paper.The
solid precipitate was redissolved in a 0.1 M potassium phos-
phate solution at pH 7. The resulting solution was placed in
dialysis bags with a cut-off of 10 kDa. The dialysis bags were
immersed in a flowing deionized water bath for 24 to 30 h.
The solutions remaining in the dialysis bagswere pooled and
the resulting solutions were dried via lyophilization. The
resulting solids served as the reference HMWP.
Polymerization of aldehyde 2
Aliquots (8 mg) of aldehyde 2 were placed in six separate 7
mL glass vials. To these vialswere added 200 I-lL of: (i) water;
(ii) acetic acid; (iii) 0.05 M sulphuric acid; (iv) 0.1 M phos-
phate buffer pH 8; (v)0.1 M NaOH; and (vi) nothing. These
vialswere capped and placed in a 60"C oven.After 3 days,the
lids were loosened to allow the liquid to evaporate slowly.
After 2 more days,the sampleswere removed from the oven
and assayed for percentage aldehydeby HPLC (Table 2).
Results
HIV-l RT inhibitory activity
Our initial goal was to assess the activity of a solution of
Figure 3. Inhibition of RNase H activity of HIV-l RT by
celtazidime, ceftazidime degradation products, captan,
and 9-chloro-TIBO.
• Ceftazidime HMWP
degraded ceftazidime (Fig. 2) and correlate the activity to
individual components of the solution. An initial RNase
H assay of the degraded ceftazidime solution confirmed
activity (Fig. 3). The solution was separated into frac-
tions by preparative RP-HPLC, and two fractions
were shown to have the highest activity. One fraction
was determined to contain aldehyde 2, while the other
fraction was determined to contain significant levels of
polymeric material (based on the appearance of the
HPLC chromatogram). Aldehyde 2 was then purified to
facilitate structure elucidation and activity testing. An
initial assessment of the RNase H inhibitory activity of a
partially purified sample of aldehyde 2 (approximately
75% pure by HPLC) showed activity (Fig. 3) similar to
that reported previously; ICso of 45 I-lg mL-l for RNase
H activity and >100 I-lg mL-l or polymerase activity
(Hafkemeyer eta!., 1991). Upon further purification of2
(>90% pure by HPLC; Fig. 2) we found very little RNase
H or polymerase inhibitory activity (IC so>100 I-lg mL-l
for both activities, Table 2).
To explore the origin of the activity in the partially
purified sample of aldehyde 2, the preparative RP-HPLC
ofthe degraded ceftazidime solution was repeated using an
optimized HPLC gradient. Five fractions close to the
retention time of aldehyde 2 were collected (Fig. 4).
Surprisingly, the anti-RNase H activity did not correlate
with the concentration of aldehyde 2 in the fractions, but
rather showed a maximum in a fraction that contained
only about 20% aldehyde 2 in the HPLC chromatogram
(fraction 4; Fig. 4). This result was confirmed in two
subsequent repeat fractionations (data not shown). These

..•
0 Ceftazidime DM observations suggested that 2 was not significantly active
Cellozidlrne aldehyde (2)
Captan against RNase H, but rather some closely eluting uniden-
f':" Ceftazidime tified impurity or impurities were responsible for the
X 9-eI-TIBO activity. Because ceftazidime is known to degrade to form
() Ceftazidime HMWP (RT)
polymeric material, and since we had observed significant
100 activity in fractions that appeared to contain polymeric
Q-l1 90 material, the possibility of 'polymeric' contamination of

:f ;~
0
aldehyde 2 was considered.

I
Q)
60 Polymer formation and activity
Z
o 50 Ceftazidi)lle (pentahydrate) is known to degrade in the
~ 40 solid-state to a non-homogeneous HMWP. A method for
0
c 30 the analysis of HMWP in ceftazidime has been previously
.., 0
described [Eli Lilly, unpublished results (USP XXIII-NF
.z 20
.:..c 18, Official Monographs, 1995, pp. 308-309)]. Testing of
.E 10
a the ceftazidime HMWP for RT inhibitory activity
0.001 0.01 0.1 1 10 100 1000 revealed that it strongly inhibited both the polymeras
Concentration (/1g rnl,"] and the RNase H activities of HIV-1 RT (Fig. 3). It also
inhibited the DNA polymerase activities of calf thymus
ceftazidime HMWP (RT) refers to inhibition of the RT DNA polymerase a (IC so 4 I-lg mL-l) and human DNA
polymerase function by the ceftazidimeHMWP; ceftazidime
polymerase ~ (IC so 75 I-lg mL-l) using DNase-nicked
aldehyde (2) corresponds to a partially purified sample (about
75% pure as judged by HPLC); ceftazidime DM is a ceftazidime salmon sperm DNA as a template (data not shown).
degradation mixture. Chromatographic and spectroscopic data obtained on

Antiviral Chemistry & Chemotherapy 8(4) 357

SW Baertschi ef 01.
Figure 4. Isolation and activity of ceftazidime degrada-
tion products.
(a)
Aldehyde 2
/ aldehyde 2 to the amino group of the thiazole, to form a
Inject
/ the samples. From the appearance of the HPLC chro-
12 3 4 5 22 min
Fraction
(0) Preparative HPlC chromatogram (UV detection, 260 nm)
and fractionation of a degraded solution of ceftazidime. HPlC
operated with a flow rate of 10 ml min- 1 in an isocratic mode
at 15% mobile phase B. (b) Inhibitory activity of the isolated
fractions shows that activity is not directly linked to relative
amount of aldehyde 2 in the sample. of the fractions shown in
this graph, fraction 2 was determined to have the greatest
relative amount of 2 and was defined as having 100% aldehyde
2 (even though it was not 100% pure) for purposes of normaliz-
ing the percentages of 2 present in the other fractions. Fraction
5 had no activity at either concentration, and there was no
detectable activity for the lower concentration of fraction 3.
358
the HMWP (UV, IR, FAB-MS and IH NMR; M
Kuhfeld,T-S Chou & R Lakin, unpublished results) led to
the proposal that the non-homogeneous ceftazidime poly-
mer consisted (in part) of a common repeating unit shown
in Fig. 5. The principal repeating unit of this polymer is
the result of a head-to-tail intermolecular condensation of
Schiff base; this structure is the same as would be predict-
ed from a polymerization of aldehyde 2. Thus, we exposed
2 to conditions that might be expected to induce polymer-
ization and perhaps RT inhibitory activity.
Polymerization of aldehyde 2
Aldehyde 2 was exposed to acidic, neutral and basic con-
ditions at 60°C. The samples were analysed by HPLC and
the amount of2 remaining was determined. These samples
were also assayed for RNase H and polymerase inhibition.
The results are summarized in Table 2.
As shown in Table 2, the RT activity was roughly
inversely proportional to the amount of 2 remaining in
matograms (broad hump in the baseline), a significant
amount of polymerization of 2 had occurred, presumably
through an amino-aldehyde condensation as shown in Fig.
5. The RT inhibitory activity of the 0.1 M NaOH- and
0.05 M H 2S04-exposed samples approached that of the
ceftazidime HMWP.
These observations are consistent with the following
hypotheses: (i) the ceftazidime HMWP is largely com-
posed of repeating units of the aldehyde that have been
polymerized head-to-tail as suggested above; and (ii) inac-
tive aldehyde 2 can be readily polymerized to an active
polymer,
Discussion
The aldehyde HP 0.35 is a relatively simple molecule that
would have been of interest, if it had the HIV-1 RT
inhibitory activity reported by Hafkemeyer et al. (1991).
This is particularly true because the activity was directed
against the RNase H function of RT, rather than the
polymerase function, which is unlike most anti-RT com-
pounds currently used for therapy or in development as
antiviral drugs. In an attempt to replicate and elaborate
these findings, HP 0.35 (2) was purified from degradation
products of ceftazidime by HPLC techniques essentially
as described by Hafkemeyer et al. (1991). The abilities of
peak fractions to inhibit the RNase H and polymerase
functions of HIV-1 RT were determined. Initially, we
observed that the partially purified peak fraction contain-
ing HP 0.35 inhibited the RNase H activity of HIV-I RT,
similar to the activity reported by Hafkemeyer et al.
(1991). However, as the material was purified further, both
© 1997 International Medical Press Ltd

Figure 5. Proposed degradation pathway to form HMWP.
OY--. COOH
rI H
__ A/~
"'N_<J ~ o)=(~o
1 ceftazidime
Amino-aldehyde
condensation
~ N
Aldehyde 2
the anti-RNase H and anti -polymerase activities were lost.
The most homogeneous material was essentially inactive
(IC so >100 I1g m.L").
The degraded solution of ceftazidime also contained
polymeric material, presumably from polymerization of
HP 0.35 via formation of a Schiff base (Fig. 5). All frac-
tions of the solution that had some anti-R'T activity also
appeared to contain variable amounts of polymeric mater-
iaL Because we found that HMWP from ceftazidime
inhibited both the RNase H and polymerase functions of
HIV-1 RT, it seemed likely that the anti-RT activity of
HP 0.35 resulted from the presence of polymeric or
oligomeric material.
To test this idea, the inactive purified aldehyde 2 was
allowed to polymerize under several conditions and was
then retested for anti-RT activity and the extent of poly-
merization. In each case, the polymerized material did
have anti-RT activity. Furthermore, the extent of activity
paralleled the extent of polymerization. These observa-
tions indicated that aldehyde 2, which has the structure
described for HP 0.35, had very little (if any) inhibitory
activity and that the anti-RT activity resulted from conta-
mination with polymeric material. It therefore seems like-
ly that the HP 0.35 material described by Hafkemeyer et
Antiviral Chemistry & Chemotherapy 8(4)
Inhibition by degradation products of ceftazidime
s
COo-
\ (c;~lid-state degradation
\,'dim, pentchydrote]
/:L-COOH
y l r N HI
I N~X
~N----< ION
S
Ceftazidime high molecular weight polymer
Non-homogeneous polymer, principal repeating unit
al. (1991) contained residual amounts of polymeric mate-
rial, or that it underwent polymerization prior to or during
the assays for anti-RT activity.
HMWP material obtained from degradation of cef-
tazidime is a potent non-specific inhibitor of several poly-
merases including the RNase H and polymerase activities
ofHIV-1 RT, calf thymus DNA polymerase a and human
DNA polymerase ~. The mechanism of inhibition by
HMWP is unknown. However, a polymer derived from 2
would be a polyanion at pH 7, owing to the presence of
carboxylate anions in oxime side chains (Fig. 5). While it
is conceivable that this material may inhibit RT and other
polymerases much like other polyanions (De Clercq, 1988;
Moelling et al., 1989), we have no evidence that this factor
is operative here.
Acknowledgements
The authors thank John L Occolowitz for the mass spec-
trometric analyses, Douglas E Dorman and Larry A
Spangle for the NMR studies, Ralph Riggin for providing
the LC-MS analyses, and T-S Chou and R Lakin for syn-
thetic help.
359
SW Baertschi et 0/.
References
Ahgren C, Backro K, Bell FW, Cantrell AS, Clemens M, Colacino
IM, Deeter]B, EngelhardtJA, Hogberg M,Jaskunas SR,
Johansson NG,Jordan CL, Kasher JS, KinnickMD, Lind P,
Lopez C, Morin Jr IM, Muesing MA, Noreen R, Oberg B,
Paget CJ, Palkowitz JA, Parrish CA, Pranc P, Rippy MK,
Rydergard C, Sahlberg C, Swanson S, Ternansky RM, Unge T,
VasileffRT, Vrang L, West SJ, Zhang H & Zhou X-X (1995)
The PETT series: a new class of potent nonnucleoside inhibitors
of human immunodeficiency virus type 1 reverse transcriptase.
AntimicrobialAgents and Chemotherapy 39: 1329-1335..
Althaus IW, Gonzales AJ, Chou JJ, Romero DL, Deibel MR, Chou
K-C, Kezdy FJ, Resnick L, Busso ME, So AG, Downey KM,
Thomas RC, AristoffPA, Tarpley WG & Reusser F (1993a)
The quinoline U-78036 is a potent inhibitor of HIV-1 reverse
transcriptase. Journal ifBiological Chemistry 268: 14875-14880.
Althaus IW, Chou JJ, Gonzales AJ, Deibel MR, Chou K-C, Kezdy
FJ, Romero DL, Palmer JR, Thomas RC, AristoffPA, Tarpley
WG & Reusser F (1993b) Kinetic studies with the non-nucleoside
HIV-1 reverse transcriptase inhibitor U-88204E. Biochemistry
32: 6548-6554.
Boyer PL, Ferris AL & Hughes SH (1992) Cassette mutagenesis of
the reverse transeriptase of human immunodeficiency virus type 1.
Journal if Virology 66: 1031-1039.
Cantrell AS, Engelhardt P, Hogberg M, Jaskunas SR, Johansson
NG,Jordan CL, KangasmetsaJ, KinnickMD, Lind P,
MorinIMJr, Muesing MA, Noreen R, Oberg B, Pranc P,
Sahlberg C, Ternansky RJ, VasileffRT, Vrang L, West SJ and
Hong Z (1996) Phenethylthiazolylthiourea (PETT) compounds
as a new class ofHIV-1 reverse transcriptase inhibitors. 2.
Synthesis and further structure-activity relationship studies of
PETT analogs.jouurnal ifMedicinal Chemist1y 39: 4261-4274.
Connolly KJ & Hammer SM (1992) Antiretroviral therapy: reverse
transcriptase inhibition. AntimicrobialAgents and Chemotherapy
36: 245-254.
Davies II JF, Hostomska Z, Hostomsky Z,Jordan SR & Matthews
DA (1991) Crystal structure of the ribonuclease H domain of
HIV-1 reverse transcriptase. Science 252: 88-95.
Debyser Z, Pauwels R, Andries K, Desmyer J,Janssen PAJ &
De Clercq E (1991) An antiviral target on reverse transcriptase of
human immunodeficiency virus type 1 revealed by tetrahydro-
imidazo-[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione
derivatives. Proceedings if the National Academy if Sciences, USA
88: 1451-1455.
De Clercq E (1988) Recent advances in the search for selective
antiviral agents. Advances in Drug Research 17: 1-59.
Derome AE (1987) Homonuclear shift correlation. In Modern
NMR Techniques fOr Chemist1y Research. Edited by JE Baldwin.
New York: Pergamon Press, pp. 183-243.
Do 00, Neftel KA, Spadari S & Hiibscher U (1987) Betalactam
antibiotics interfere with eukaryotic DNA-replication by
inhibiting DNA polymerase IX. Nucleic Acids Research
15: 10495-10506.
Freeman-Wittig M-J & Lewis RA (1990) Captan binding to avian
myeloblastosis virus reverse transcriptase and its effect on RNase
H activity. Molecular and Cellular Biochemistry 94: 9-18.
360
Freeman-Wittig M-J, Vinocour M & Lewis RA (1986) Differential
effects of captan on DNA polymerase and ribonuclease H
activities of avian myeloblastosis virus reverse transcriptase.
Biochemistry 25: 3050-3055.
Goldman ME, Nunberg JH, O'Brien JA, Quintero JC, Schleif
WA, Freund KF, Gaul SL, Saari WS, Wai JS, Hoffman IM,
Anderson PS, Hupe DJ, Emini EA & Stern AM (1991)
Pyridinone derivatives: specific human immunodeficiency
virus type 1 reverse transcriptase inhibition with antiviral
activity. Proceedings if the National Academy if Sciences, USA
88: 6863-6867.
Hafkemeyer P, Neftel K & Hiibscher U (1990) HIV-reverse
transcriptase and human DNA polymerase IX share amino acids
sequence homologies to bacterial penicillin-binding proteins.
Methods and Findings in Experimental and Clinical Pharmacology
12: 43-46.
Hafkemeyer P, Neftel K, Hobi R, Pfaltz A, Lutz H, Luthi K,
Focher F, Spadari S & Hubscher U (1991) HP 0.35, a
cephalosporin degradation product is a specific inhibitor of
lentiviral RNAses H. NucleicAcids Research 19: 4059-4065.
Hargrave KH, ProudfootJR, Grozinger KG, Cullen E, Kapadia SR,
Patel UR, Fuchs VU, Mauldin SC, Vitous J, Behnke ML,
Klunder IM, Pal K, Skiles JW, McNeil DW, Rose IM, Chow
GC, Skoog MT, WuJC, Schmidt G, Engel WW, Eberlein WG,
Saboe TD, Campbell SJ, Rosenthal AS & Adams J (1991) Novel
non-nucleoside inhibitors of HIV-1 reverse transcriptase. 1.
Tricyclic pyridobenzo- and dipyridodiazepinones. Journal if
Medicinal Chemistry 34: 2231-2241.
Hirsch MS & D'Aquila RT (1993) Therapy for human immuno-
deficiency virus infection. New EnglandJournal ifMedicine
328: 1686-1695.
Hizi A, Hughes SH & Shaharabany M (1990) Mutational analysis
of the ribonuclease H activity of human immunodeficiency virus 1
reverse transcriptase. Virology 175: 575-580.
Hizi A, Tal R, Shaharabany M, Currens MR, Hughes SH &
McMahon]B (1993) Specific inhibition of the reverse transcrip-
tase of human immunodeficiency virus type 1 and the chimeric
enzymes of human immunodeficiency virus type 1 and type 2 by
nonnucleoside inhibitors. AntimicrobialAgents and Chemotherapy
37: 1037-1042.
Hui PW,Jacober SP, Hargrave KD &AdamsJ (1992) Crystal
structure of nevirapine, a non-nucleoside inhibitor of HIV-1
reverse transcriptase, and computational alignment with a
structurally diverse inhibitor. Journal ifMedicinal Chemist1y
35: 201-202.
Jacobo-Molina A & Arnold E (1991) HIV reverse transcriptase
structure-function relationships. Biochemistry 30: 6351-6361.
Johnson MS, McClure MA, Feng D-F, Gray J & Doolittle RF
(1986) Computer analysis of retroviral pol genes: assignment of
enzymatic functions to specific sequences and homologies with
nonviral enzymes. Proceedings if the National Academy if Sciences,
USA 83: 7648-7652.
Krug MS & Berger SL (1989) Ribonuclease H activities associated
with viral reverse transeriptases are endonucleases. Proceedings if
the NationalAcademy if Sciences, USA 86: 3539-3543.
Kukla MI, Breslin HJ, Diamond CJ, Grous PP, Ho CY, Miranda
© 1997 International Medical Press Ltd

M, Rodgers]D, Sherrill RG, De Clercq E, Pauwels R, Andries
K, Moens LC, Janssen MAC &Janssen PAJ (1991) Synthesis
and anti-HIV-l activity of 4,5,6,7-tetrahydro-5-methylimidazo-
[4,5,1-jk] [1,4]benzodiazepin-2(1H)-one (TIBO) derivatives.
Part 2. Journal ofMedicinal Chemistry 34: 3187-3197.
Loya S & Hizi A (1993) The interaction of illimaquinone, a
selective inhibitor of the RNase H activity, with the reverse
transcriptases of human immunodeficiency and murine leukemia
retroviruses. Journal ofBiological Chemistry 268: 9323-9328.
Martin GE & Zektzer AS (1988) Long-range two-dimensional
heteronuclear chemical shift correlation. Magnetic Resonance
Chemistry 26: 640-652.
Mayers DL (1993) Zidovudine and other antiretroviral agents
including drug interactions and toxicities. Current Opinions in
Infectious Diseases 6: 210-217.
Merluzzi VJ, Hargrave KD, Labadia M, Grozinger K, Skogg M,
WuJC, Shih C-K, Eckner K, Hattox S, Adams J, Rosenthal AS,
Faanes R, Eckner RJ, Koup RA & Sullivan JL (1990) Inhibition
of HIV-l replication by a nonnucleoside reverse transcriptase
inhibitor. Science250: 1411-1413.
Mitsuya H & Broder S (1986) Inhibition of the in ouro infectivity
and cytopathic effect of human T-Iymphotrophic virus type
Ill/lymphadenopathy-associated virus (HTLV-Ill/LAV) by 2',
3'-dideoxynucleosides. Proceedings ofthe National Academy rf
Sciences, USA 83: 1911-1915.
Mitsuya H, Weinhold KJ, Furman PA, St Clair MH, Lehrmann
SN, Gallo RC, Bolognesi D, Barry DW & Broder S (1985)
3'-Azido-3'-deoxythymidine (BW AS09U): an antiviral agent
that inhibits the infectivity and cytopathic effect of human
T-Iymphotropic virus type IIIIlymphadenopathy-associated
virus in vitro. Proceedings ofthe National Academy rf Sciences,
USA 82: 7096-7100.
Miyasaka T, Tanaka H, Baba M, Hayakawa H, Walker RT,
Balzarini ] &De Clercq E (1989) A novel lead for specific anti-
HIV-l agents: 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-
thymine. Journal rfMedicinal Chemistry 32: 2507-2509.
Moelling K, Schulze T & Diringer H (1989) Inhibition of human
immunodeficiency virus type 1 RNase H by sulfated polyanions.
Journal rfVirology 63: 5489-5491.
Neftel K & Hubscher U (1987) Effects of ~-lactam antibiotics on
proliferating eucaryotic cells. AntimicrobialAgents and
Chemotherapy 31: 1657-1661.
Neftel K, Hafkemeyer P, Cittagnoud P, Eich G & Hubscher U
(1993) Did evolutionary forerunners of betalactam-antibiotics
bind to nucleic acid replication enzymes? In 50 Yean rfPenicillin
Application: Hist01Y and Trends. Edited by H Kleinkauf & H von
Dohrcn. Prague: Public, pp. 394-403.
Nunberg JR, SchleifWA, Boots EJ, O'Brien JA, Quintero JC,
Hoffman JM, Emini EA & Goldman ME (1991) Viral resistance
to human immunodeficiency virus type i-specific pyridinone
reverse transcriptase inhibitors. Journal rfVirology 65: 4887-4892.
Pauwels R, Andries K, Desmyter J, Schols D, Kukla MJ, Breslin HJ,
Raeymaeckers A, Van Gelder J, Woesenborghs R, Heykants J,
Schellekens K, Janssen MAC, De Clercq E &Janssen PAJ (1990)
Potent and selective inhibition of HIV-l replication in vitro by a
novel series ofTIBO derivatives. Nature 343: 470-474.
Antiviral Chemistry & Chemotheropy 8(4)
Inhibition by degradation products of ceftazidime
Prasad VR & Goff SP (1989) Linker insertion mutagenesis of the
human immunodeficiency virus reverse transcriptase expressed
in bacteria: definition of the minimal polymerase domain.
Proceedings rfthe National Academy rfSciences, USA 86: 3104-3108.
Richman D, Shih C-K, Lowy I, Rose J, Prodanovich P, Goff S &
Griffin J (1991) Human immunodeficiency virus type 1 mutants
resistant to nonnucleoside inhibitors of reverse transcriptase arise
in tissue culture. Proceedings rfthe National Academy rf Sciences,
USA 88: 11241-11245.
Richman DD, Havlir D, CorbeilJ, Looney D, Ignacio C, Spector
SA, Sullivan J, Cheeseman S, Barringer K, Pauletti D, Shih C-K,
Myers M & Griffin J (1994) Nevirapine resistance mutations of
human immunodeficiency virus type 1 selected during therapy.
Journal rfVirology 68: 1660-1666.
Romero DL, Busso M, Tan C-K, Reusser F, Palmer JR, Poppe SM,
AristoffPA, Downey KM, So AG, Resnick L & Tarpley WG
(1991) Nonnucleoside reverse transcriptase inhibitors that
potently and specifically block human immunodeficiency virus
type 1 replication. Proceedings rfthe National Academy rf Sciences,
USA 88: 8806-8810.
Saag MS, Emini EA, Laskin OL, Douglas J, Lapidus WI, Schleif
WA, Whitley RJ, Hildebrand C, Byrnes VW, Kappes JC,
Anderson KW, Massari FE, Shaw GM, Chopra P, Conway J,
DeLoach L, Hill S, Jiang SY, Maples V, Wang SR, Calandra GB,
Cnaan A, Gotlib L, Patterson P, Quintero JC, Rhodes A,
Schneider CL, Titus DL, Uncapher C & Waterbury JA (1993)
A short-term clinical evaluation ofL-697,661, a non-nucleoside
inhibitor ofHIV-l reverse transcriptase. New EnglandJournal rf
Medicine 329: 1065-1072.
Saari WS, Hoffman JM, Wai JS, Fisher TE, Rooney CS, Smith
AM, Thomas CM, Goldman ME, O'Brien JA, Nunberg JR,
Quintero JC, SchleifWA, Emini EA, Stern AM & Anderson PS
(1991) 2-Pyridinone derivatives: a new class of nonnucleoside,
HIV-l-specific reverse transcriptase inhibitors. Journal rf
Medicinal Chemistry 34: 2922-2925.
Sandstrom E & Oberg B (1993a) Antiviral therapy in human
immunodeficiency virus infections. Current status (part I). Drugs
45: 488-508.
Sandstrom E & Oberg B (1993b) Antiviral therapy in human
immunodeficiency virus infections. Current status (part II). Drugs
45: 637-653.
Shih C- K, Rose JM, Hansen GL, Wu JC, Bacolla A & Griffin JA
(1991) Chimeric human immunodeficiency virus type l/type 2
reverse transcriptases display reversed sensitivity to nonnucleoside
analog inhibitors. Proceedings ofthe National Academy of Sciences,
USA 88: 9878-9882.
Starnes MC & Cheng Y-C (1989) Human immunodeficiency virus
reverse transcriptase-associated RNase H activity. Journal of
Biological Chemishy264: 7073-7077.
Tan C, Zhang Z, Li Z, Tarpley WG, Downcy KM & So AG
(1991) Functional characterization of RNA -de~endent DNA
polymerase and RNase H activities of a recombmant HIV reverse
transcriptase. Biochemist1] 30: 2651-2655.
Ternansky RM, Morin Jr JM, Lopez C, Paget}r C]. Bell FW•.
Cantrell AS, Jaskunas SR, Jordan CL, Kml11~~ MD. Palk?WIt'L JA,
Parrish CA, Pranc P, VasileffRT, West S]. Hogberg M, Lmd p.
Noreen R, Sahlberg C, Zhou X-X, Vrang L. Rydergard C, Ahgren
361
 SW Baertschi et 0/.

C, Oberg B &Johansson NG (1993) The discovery and general

SAR studies of a novel class of potent non-nucleoside reverse tran- Whitcomb 1M & Hughes SH (1992) Retroviral reverse transcrip_
scriptase inhibitors. Antiviml Research 20: Supplement 1, 68. tion and integration: progress and problems. Annual Review 0/
Cell Biology 8: 275-306.

362

© 1997 International Medical Press Ltd

International Conference on Advanced Materials for Better Future 2018 IOP Publishing
IOP Conf. Series: Materials Science and Engineering 578 (2019) 012055 doi:10.1088/1757-899X/578/1/012055

Effect of Temperature and Length of Storage to

Chloramphenicol Eye Drop’s Concentration

A Yugatama1, R Nurmalinda1, S Rohmani1, D E Ermawati1, and F Prihapsara1

1
Pharmacy Department, Faculty of Mathematics and Natural Sciences, Sebelas Maret
University, Indonesia

Email: adiyugatama.apt@gmail.com; adi.yugatama@staff.uns.ac.id

Abstract. Chloramphenicol is a broad spectrum antibiotic that acts by inhibiting protein

synthesize. One of chloramphenicol dosage form which widely found in the marketplace is
eyedrop. Drug storage will affect drug stability. This research aimed to know the effect of
temperature and length of storage to chloramphenicol eye drop’s concentration. Analysis of
chloramphenicol concentration in eyedrops was done by UV spectrophotometry. This analysis
was done to eyedrops after stored 0; 7; 21; and 30 days unsealed with various storage
condition: under the sunlight, room temperature, and refrigerated. Data obtained was analyzed
statistically using one-way ANOVA with 95% CI by SPSS for windows software. The result
showed that the concentration of chloramphenicol eyedrop before stored was to meet the
requirements which were 95.96%. The result of chloramphenicol concentration in eyedrop
after stored 30 days in 3 storage condition was reduced significantly. While sunlight and
storage temperature did not affect the reduction of chloramphenicol concentration significantly.

1. Introduction
Chloramphenicol is a broad spectrum antibiotic that acts by inhibit protein synthesis. Chloramphenicol
having a bacterostatic activity against almost all gram positive and negative bacteria, and against
Spirochaeta, Chlamydia trachomatis and Mycoplasma. Chloramphenicol having a good bacterisid
activity against Str. pneumonia, Neiss. meningitides, and H. Influenzae [1].
One of chloramphenicol dosage form available in market is eyedrops, in 0.25–1% concentration.
Eyedrops is a sterile solution which combined and packaged for eyes purposes. Eyedrops have to be
sterile, isohydrile, and isotonic. This dosage form could not be used six weeks after being unsealed [2].
Drug storage affect drug stability utmost. Drug stability could be seen by concentration reduction
during storage [3]. Drugs have to be stored in room temperature and avoid direct sunlight to ensure
drug stability [4]. Temperature, light, moist and air condition are factors that induced drug
degradation. Although reduction 10% of prior concentration of active ingredients is acceptable [5].
There are some theories stated about relation of drug stability to temperature, light and length of
storage. Thus, it need to be studied about the effect of temperature and length of storage to
chloramphenicol concentration in eyedrops.

2. Experimental
2.1. Materials
Equipments used were UV-Vis Spectrophotometer (Genesys 10S UV-Vis), analytical balance
(Ohaus), micropipet (Joanlab), and glassware. Materials used were chloramphenicol standard,
chloramphenicol eyedrops brand X, aquadest and ethanol.

Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd 1
International Conference on Advanced Materials for Better Future 2018 IOP Publishing
IOP Conf. Series: Materials Science and Engineering 578 (2019) 012055 doi:10.1088/1757-899X/578/1/012055

2.2. Methods
2.2.1. Standard Solution. Standard solution was made by reconciling 100 mg chloramphenicol in 10
mL ethanol then diluted in aquadest until reached the concentration of 1000 ppm. From
chloramphenicol 1000 ppm solution, was taken 0.1; 0.5; 1; 1.5; 2; 2.5; 3 mL and added to aquadest
until 100 mL to generate standard solution of 1, 5, 10, 15, 20, 25, and 30 ppm chloramphenicol.

2.2.2. Sample Treatment. Samples were tested by stored in refrigerator temperature (3-7°C), room
temperature (27-30°C), and exposed to direct sunlight. The drugs were unsealed before stored.
Concentration of chloramphenicol in eyedrops then analyzed at day 0; 7; 21; and 30 after storage.

2.2.3. Determination of Chloramphenicol Concentration in Sample. As much as 5 mL samples were

added by 25 mL aquadest. The mixture was taken 1 mL and added by aquadest until 100 mL. The
solutions were measured its absorbance using spectrophotometer at 278 nm wavelength.

2.3. Data Analysis

Absorbance of samples were analyzed by linear regression until obtained chloramphenicol
concentration in eyedrops in various storage condition. The data then analyzed by one way Anova to
know the effect of temperature and length of storage to chloramphenicol concentration in eyedrops.

3. Results and Discussion

3.1. Determination of Maximum Wavelength
The result of 10 ppm chloramphenicol standard solution scanning in figure 1 showed that maximum
wavelength is 278 nm. Based on other research, maximum wavelength of chloramphenicol is 278 nm
[6].

Figure 1. Maximum wavelength of chloramphenicol

3.2. Determination of Chloramphenicol Standard Curve

Standard curve equation obtained was y= 0.0441x + 0.0115 and the correlation coefficient (r) was
0.9999. Based on requirement, r > 0.997 showed that the curve is having a good linearity [7]. This can
be concluded that standard curve equation obtained was having a good linearity. The chloramphenicol
standard curve could be seen in figure 2.

2
International Conference on Advanced Materials for Better Future 2018 IOP Publishing
IOP Conf. Series: Materials Science and Engineering 578 (2019) 012055 doi:10.1088/1757-899X/578/1/012055

Figure 2. Chloramphenicol standard curve

3.3. Determination of Chloramphenicol Concentration in Sample

The result of chloramphenicol concentration in eyedrops determination after storage showed that there
were reductions of chloramphenicol concentration both in refrigerator temperature, in room
temperature, and exposed to direct sunlight. The prior concentration of chloramphenicol in eyedrops
was 95.96%. This is in accordance with the requirements stated in USP standard that the concentration
of chloramphenicol in eyedrops is not less than 90% and not over 130%. In addition, the deadline for
using sterile preparation after unsealed is 28 days [8]. At 30 days length of storage after unsealed in
refrigerator temperature, room temperature, and direct sunlight exposure, the chloramphenicol
concentration in eyedrops were above 90%. This showed that chloramphenicol eyedrops brand X is
fulfill the standard and still be able to use until 30 days after unsealing. Chloramphenicol
concentration in various storage condition could be seen in figure 3.

Figure 3. Curve of chloramphenicol concentration in various storage condition

During storage, concentration of chloramphenicol in brand X eyedrops were reduced. The longer
the storage period, the lesser concentration of chloramphenicol found in eyedrops. This might be
resulted from hydrolysis reaction. This reaction occured because chloramphenicol in watery media
will be degraded by hydrolysis breakdown in amides circle and forming appropriate amides and
dichloracetic acid. Reaction rate is occured under the first order and independent from ionic power of
the media [9]. The hydrolysis reaction could be seen in figure 4.

3
International Conference on Advanced Materials for Better Future 2018 IOP Publishing
IOP Conf. Series: Materials Science and Engineering 578 (2019) 012055 doi:10.1088/1757-899X/578/1/012055

Cl Cl

O2N O2N
HN OH NH2
H 2O CHCl2COOH

OH OH OH OH

Figure 4. Chloramphenicol hydrolysis reaction

Besides, chloramphenicol in solution also sensitive to photodegradation reaction. Chloramphenicol

under direct sunlight slowly changed into yellow color and formed yellowish orange residu. In this
photodegradation reaction, chloramphenicol will changed into 4-nitrobenzaldehyde, 4-nitrobenzoic
acid, and 4,4’-azoxybenzoic acid through oxidation, reduction, and condensation reaction [9].
Storage in room temperature resulted in relatively lower concentration of chloramphenicol than in
direct sunlight exposure and refrigerator temperature. This might be caused by moist which commonly
found higher in room temperature. Factors inducing degradation reaction of a drug i.e. temperature,
light, moist, and air condition. Esters and amides compounds like chloramphenicol is easily hydrolized
in a presence of moisture [10].
Storage in refrigerator temperature resulting the least reduction of chloramphenicol concentration
compared to direct sunlight expossure and in room temperature storage. This showed that
chloramphenicol in eyedrops will be most stable if stored in refrigerator temperature. The temperature
of refrigerator used in this study is 3–7 °C, and included into cold temperature. The lower the
temperature, the slower the reaction rate will be. Reaction rate is linear to number of collision per unit
of time.
Result of statistic analysis using SPSS showed that there was a correlation between length of
storage, light, and temperature to chloramphenicol concentration. Based on normality test using
Kolmogorov-smirnov, the data were distributed normally by sig. 2 tailed value was 0.482. The value
of p > 0.05 showed that data were distributed normally.
Statistical analysis by one way anova resulted that significance value of length of storage, light, and
temperature to chloramphenicol concentration were 0.000; 0.925; dan 0.140. Based on these result,
significance value of length of storage was < 0.05. Thus, can be conclude that length of storage can
affect the reduction of chloramphenicol concentration. While sunlight and temperature were not
affecting chloramphenicol concentration significance.
Insignificance effect of light to chloramphenicol concentration might be caused by the package of
eyedrops that are white colored and not translucent so the effect of sunlight will be smaller, and the
incidence of photodegradation reaction also being lower.

4. Conclusion
Based on chloramphenicol concentration analysis in eyedrops for 30 days storage in 3 various
condition which are exposed directly to sunlight, room temperature, and refrigerator temperature using
UV Vis Spectrophotometer, could be concluded that length of storage having a significant effect to
chloramphenicol concentration reduction, while lights and storage temperature were not having a
significant effect to chloramphenicol concentration reduction.

Acknowledgement
The authors acknowledge to Sebelas Maret University which given the facilities for the research and
publication.

References
[1] Rahal JJ 1979 Antimicrob Agents Chemother 16(1) 13.

4
International Conference on Advanced Materials for Better Future 2018 IOP Publishing
IOP Conf. Series: Materials Science and Engineering 578 (2019) 012055 doi:10.1088/1757-899X/578/1/012055

[2] Virani S, Rewri P and Vyas A 2016 Del J Ophtalmol 26(3) 176.
[3] Bajaj S, Singla D and Sakhuja N 2012 J. Appl. Pharm. Sci. 2(3) 129.
[4] Shafaat K, Hussain A, Kumar B, Hasan R, Prabhat P and Yadav VK 2013 World J. Pharm.
Pharm. Sci. 2(5) 2499.
[5] Cantrell L, Suchard JR, Wu A and Gerona RR 2012 Arch Intern Med. 172(21) 1685.
[6] Karthikeyan S 2011 Arch. Phy. Res. 2(4) 72.
[7] Chan CC, Lam H, Lee YC and Zhang XM 2004 Analytical Method Validation and Instrument
Performance Verification (New Jersey: John Wiley & Sons, Inc.)
[8] US Pharmacopeial Convention, Inc. 2007 United States Pharmacopeia 30 (Rockville, MD: US
Pharmacopeial Convention, Inc.)
[9] Ali SL 1978 J. Chromatogr. A 154(1) 99.
[10] Honmane SM, Dange YD, Osmani RAM and Jadge DR 2017 Asian J. Pharm. 11(3) 479.

5
 Heliyon 10 (2024) e25433

Contents lists available at ScienceDirect

Heliyon
journal homepage: www.cell.com/heliyon

Research article

Photocatalytic degradation of ciprofloxacin from water with waste

polystyrene and TiO2 composites
Tugba Hayri-Senel *, Ebru Kahraman , Serhat Sezer , Nalan Erdol-Aydin ,
Gulhayat Nasun-Saygili
Istanbul Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Chemical Engineering, Turkey

A B S T R A C T

Ciprofloxacin (CIP) is one of the widely used antibiotics with a broad antimicrobial spectrum in the fluoroquinolone type. Its concentration in water
bodies has increased over the years due to its frequent use. Since ciprofloxacin in the aquatic ecosystem adversely affects human health as well as
other organisms, it must be removed from wastewater.
The aim of this study was to develop Polystyrene (PS) and Titanium dioxide (TiO2) composites that can be used as catalysts in CIP removal by
photocatalytic process. Waste PS (obtained from disposable cutlery) was used in the synthesis of these composites. In this study, PS-TiO2 composites
with different PS content (C20, C50, C80; the numbers in the names indicate the PS percentage in the composite by weight) were synthesized. This is
important in terms of the use of one waste in the removal of another waste.
This process was optimized using Box-Behnken design, one of the response surface method. Parameters such as the amount of polymer in the
composite, pH and initial CIP concentration were studied and their effects on CIP removal were found. The validity and adequacy of the selected
model were evaluated according to the relevant statistical data. These are R2 = 0.9751, adjusted R2 = 0.9565, the model’s p-value <0.05 and the
lack of fit-value 0.246. Optimum conditions for CIP removal were obtained when the C50 was used, with a pH value of 3 and an initial CIP
concentration of 5 mg/L.
In the process carried out under these conditions, the CIP removal was found to be 95.01 % at the end of 180 min. This value was predicted as
94.37 % in the model. According to these results, C50 composite synthesized with waste PS can be used effectively for CIP removal.

1. Introduction

Ciprofloxacin (CIP) is a fluoroquinolone antibiotic with a broad antimicrobial spectrum. CIP is one of the most widely used an­
tibiotics for both humans and animals. It can be used for many diseases such as digestive and urinary tract infections, various skin and
lung diseases [1,2].
Over the years, the concentration of antibiotics in water bodies has increased. Ciprofloxacin concentration varies according to
sources. In the literature, there are studies showing that the concentration of CIP in wastewater treatment plants, raw drinking water,
hospital wastewater, lakes, and discharges of the pharmaceutical industry has been reported as 11–99, 0.032, 150, 6.5 and 31–50 mg/
L, respectively [1].
CIP in the aquatic ecosystem adversely affects human health as well as other organisms there. In addition, ciprofloxacin in the
environment leads to the formation of antibiotic-resistant bacteria, resulting in higher dose antibiotic requirements in bacterial in­
fections [3].
Considering the significant detection of ciprofloxacin in aquatic environments, the necessity of removing it from wastewater

* Corresponding author.
E-mail address: hayri16@itu.edu.tr (T. Hayri-Senel).

https://doi.org/10.1016/j.heliyon.2024.e25433
Received 9 October 2023; Received in revised form 19 January 2024; Accepted 26 January 2024
Available online 29 January 2024
2405-8440/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

emerges. In the literature, there are studies on the removal of ciprofloxacin from wastewater [2–11]. One of the most common
techniques used for this purpose is the photocatalytic degradation of ciprofloxacin [1,12].
Titanium dioxide, TiO2, is an abundant, stable, economical, efficient photocatalyst that is widely used due to its high catalytic
activity, high oxidizing power, and high photocorrosion resistance. It serves many different fields, especially waste water treatment [1,
12,13].
In the photocatalytic degradation mechanism of CIP with TiO2, firstly e− and h+ pairs are formed by stimulating TiO2 under the
influence of light energy (1). Both reactive species participate in different reactions and contribute to the formation of (•OH) and (•O−2 )
radicals (2,3,4,5). All these reactive species and radicals (h+, •OH, •O−2 ) that may be present in the environment can effectively
degrade CIP molecules (6,7,8). Since the CIP structure contains regions suitable for attack, such as the piperazine ring, F atom,
carboxyl group and cyclopropyl group, they cause different degradation pathways to be exhibited. Which possibilities will occur varies
depending on the conditions of the environment, making it difficult to specify a definitive path. Studies have reported that solution pH
is one of the most effective parameters in determining the photocatalytic degradation pathway, as it affects the formation rates of
active species [3,14,15].

(1) TiO2 + hv → h+ + e-
(2) h+ + H2 O → • OH + H+
(3) e- + O2 → • O-2
(4) •O-2 + H+ + H2 O →H2 O2 + O2
(5) H2 O2 →2 • OH
(6) h+ + CIP →Degradation Products
(7) •O2- + CIP → Degradation Products
(8) •OH + CIP → Degradation Products

TiO2 can be used in wastewater studies in powder form, or it can be used in combination with suitable substrates. When used in
powder form, higher surface area and accordingly higher efficiency can be obtained. However, some problems arise when used in
powder form. One of these problems is that recovery of the catalyst after application is difficult, time consuming and expensive. Also,
there is a loss of catalyst at this stage. The most widely used way to overcome these disadvantages is the immobilization of TiO2 with
various substrates. Although the reaction surface area is reduced by immobilization, it is preferred because it provides advantages such
as both prolonging the life of the catalyst and increasing its reusability [3,9,13,16,17].
For this purpose, different support materials can be selected. Polymeric materials are widely used support materials. Polystyrene
(PS) is a support material that can be preferred due to its inert structure, ease of production, low cost and ability to float in water.
Plastic waste is a worldwide environmental problem. One of the commonly encountered plastic wastes is PS waste. Disposable PS
products cause serious environmental problems [16]. Although there are different techniques for recycling waste PS, it does not offer a
long-term solution [18]. It is very important to use these PS wastes for the removal of pollutants in different environments [16].
Photocatalytic degradation of PS and PS-TiO2 composite was investigated in a study by Shang et al. Although pure PS was observed
to degrade more slowly than PS-TiO2 composite, it was still reported that the degradation of PS-TiO2 composite remained below 5 %
even after continuous exposure for 24 h [18].
Various statistical and mathematical methods are used to analyze different chemical processes, to save time, to predict and simulate
the conditions in this process. One of the most common methods used for this purpose is Response Surface Methodology (RSM). RSM is
a mathematical tool used to design experiments in many fields such as chemical processes and to evaluate the results of these ex­
periments, which saves time by reducing the number of experiments. RSM has two design titles, Box-Behnken Design and Central
Composite Design.
RSM enables determination of optimum process conditions by examining the effects of independent variables on the process
through experimental design. In addition, it develops some empirical models of the process using the experimental data obtained from
the experimental design. Thus, it reveals the relationship between the parameters [10,19].
In this study, first of all, PS-TiO2 composites were synthesized and their removal performance was examined by using them as
catalysts in the photocatalytic degradation of CIP from wastewater. PS used in the synthesis of composites in the study was obtained
from disposable waste PS cutlery. One of the aims of this study is to ensure the reuse of disposable waste. It is also important to make PS
waste suitable for reuse after only a simple washing process, without applying a complex process that requires extra high energy and
cost. If this can be achieved, a more cost-effective product is expected to be obtained compared to when TiO2 is used alone. No study
has been found in the literature on CIP removal with a material similar to the one in this study (PS–TiO2 composite). Box-Behnken
Design was chosen to analyze the degradation process of Ciprofloxacin with PS-TiO2 composites and optimize the effective parame­
ters. MINITAB software version 21.0 was used for experimental design.

2. Materials and methods

2.1. Materials

Waste PS was collected from used waste spoons. TiO2 (anatase, powder 99.8 %), Ciprofloxacin, chloroform and ethanol were
purchased from Merck.

2
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

2.2. Characterization

X-Ray Diffraction (XRD) analysis was performed using the PANalytical XPert Pro device for the structural evaluation of anatase
TiO2, waste PS and PS-TiO2 composite. The samples were scanned by X-ray at a wavelength of 1.5406 Å for 2 theta ranging from 10◦ to
90◦ .
The surface morphology of waste PS and its composites with different polymer content formed with TiO2 were determined by
Scanning Electron Microscope (SEM, QUANTA FEG250 Field Emission). Elemental analysis was performed using Energy dispersive
analysis of X-ray (EDAX). Waste PS, TiO2 and PS-TiO2 composite were analyzed by FT-IR spectrophotometer (Bruker) in the range of
650 cm− 1 to 4000 cm− 1. Zeta potential measurements of the PS-TiO2 composite were carried using Malvern Zetasizer at different pH
values.

2.3. Polystyrene-TiO2 (PS–TiO2) composite preparation

The particles obtained by cutting the waste PS spoons were cleaned by washing with distilled water. To form the PS-TiO2 com­
posite, a known amount of the polymer particles and TiO2 were placed in a beaker containing 1:8 ethanol and chloroform. This mixture
was homogenized in a sonication water bath for 1 h. It was then stirred at 120 ◦ C for 2 h. The resulting solution was poured into a glass
Petri dish and kept in a fume hood for 24 h for solvent evaporation. The resulting product was washed several times with distilled water
in order to remove any impurities that may be on the product, and it was dried in an oven at 50 ◦ C for 24 h [20]. This procedure was
applied for all PS-TiO2 products (Cx) containing different ratios of polystyrene. Thus, C20, C50 and C80 composites were produced.
The numbers in the names refer to the percent by weight PS in the composite.

2.4. Evaluation of photocatalytic efficiencies

The efficiency of the PS-TiO2 photocatalysts was measured by the photocatalytic degradation of an aqueous ciprofloxacin solutions.
The schematic representation of the experimental system is shown in Scheme 1. Photodegradation studies were carried out under
magnetic stirring at room temperature. In these studies, two LED daylight lamps (metal halide lamp, λ = 400–800 nm) were used as the
light source. Ciprofloxacin solutions were prepared at different initial concentrations (5, 10 and 15 mg/L). In a typical study, 50 ml of
ciprofloxacin aqueous solution was analyzed to determine the initial CIP concentration (C0) and 20 mg of catalyst was added. The

Scheme 1. The schematic representation of the experimental system.

3
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

mixture was then stirred in the dark for 30 min. Thus, the adsorption and desorption equilibrium of the mixture was achieved. At the
end of this period, sample was taken from the experimental medium and analyzed. While the mixing was continuing, the LED lamps
were turned on and the concentration of the samples taken from the medium were measured at certain time intervals. The samples
were first filtered through a 0.20 mm PTFE membrane syringe filter and the absorbance values of the filtered solutions were deter­
mined at 277 nm with a UV–Vis spectrophotometer (Hach DR6000). The percent degradation of ciprofloxacin was calculated ac­
cording to Equation (1). Here, C0 and Ct represent the CIP concentration value at the start time (t0) and the concentration values at any
time (t), respectively.
( )
C0 − Ct
Ciprofloxacin removal (%) = x 100 (1)
C0

2.5. Experimental design

3-factor and 3-level Box-Behnken design was used to optimize ciprofloxacin removal conditions. Analysis of the results was per­
formed with Minitab software (Minitab 21.0). The effect of three factors (independent variables) on ciprofloxacin removal was
examined at three different levels, including the amount of polymer in the composite (wt%) (A), ambient pH (B), and ciprofloxacin
concentration in the initial solution (C). These are presented in Table 1. The percent ciprofloxacin removal (dependent variable, Y) was
determined as the response of the designed experiments. In this design used, 15 experimental studies were planned, including 3 center
points. The significance of the variables was evaluated by analysis of variance (ANOVA, p-value<0.05), while the fit of the model was
evaluated using R2 and adjusted R2 values. Data were fitted to obtain a quadratic polynomial. Contour charts were used to determine
the effect of independent variables on dependent variables. The conditions that achieved the highest Ciprofloxacin removal were
considered as optimized conditions for this system.

3. Results and discussions

3.1. XRD analysis

Fig. 1a shows the XRD pattern of anatase TiO2. The sharp Bragg peaks indicate the highly crystalline nature of TiO2. All charac­
teristic peaks (2θ = 25.53◦ , 38.1◦ , 48.35◦ and 54.18◦ ) originating from the TiO2 anatase phase are observed [21,22]. Looking at the
XRD pattern for waste PS in Fig. 1b, an amorphous polymeric structure is seen. The XRD pattern of C50 composite is also shown in
Fig. 1c. This model confirms that amorphous phase is present in the compound due to PS. In addition, the broad peak at 2θ ≅ 20◦
corresponds to amorphous PS. The characteristic peaks in Fig. 1a are also seen here, confirming the presence of crystalline TiO2 in C50
[20,23].

3.2. SEM analysis

The morphologies of waste PS, C20, C50 and C80 composites were observed by SEM. SEM images are given in Fig. 2. Looking at the
waste PS images in Fig. 2a and b, it is seen that it exhibits a smooth and homogeneous structure. Again, the white dots appearing in the
same SEM images can be interpreted as the residual solvent (chloroform) thought to be trapped in the polymeric materials [24]. SEM
images of C80, C50 and C20 show that TiO2 is evenly distributed in the polymeric matrix [20].

3.3. EDAX analysis

Fig. 3 shows the elemental mapping results of waste PS and its composites with different PS content (C80, C50 and C20). In
addition, graphs showing the elemental density of the samples are embedded in the elemental mapping results of that sample. EDAX
measurements show the distribution of element C, element O and element Ti in the polymer matrix [25]. Elemental mapping results
confirmed that the composite with the highest TiO2 (C20) contained more Ti elements than the rest of the composites, as expected,
depending on its content.

Table 1
Factors and levels used in Box-Behnken design.
Factors Symbols Levels

Low Medium High

− 1 0 1

Amount of polymer (in composite, % by weight) A 20 50 80

pH B 3 7 11
Ciprofloxacin initial concentration (mg/L) C 5 10 15

4
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

Fig. 1. The XRD patterns of a) anatase TiO2, b) waste polystyrene, c) C50.

Fig. 2. SEM images of waste PS, C80, C50 and C20 a)all images for mag = 5000x b) all images for mag = 20000x.

3.4. FTIR analysis

FTIR spectra of waste PS, TiO2 and C50 are given in Fig. 4. Looking at the IR spectra of waste PS, it is known that the peaks between
2800 and 3100 cm− 1 originate from the C–H stretching vibration in the aromatic rings and the main chain. The peaks at 695, 748,
1027, 1451, 1492 and 1601 cm− 1 belong to the C–H skeletal vibrations in PS [26].The peak seen in the TiO2 curve at 721 cm− 1 is due to
the O–Ti–O bonding in the form of anatase. In addition, the peaks appearing at 1660 cm− 1 and 3375 cm− 1 indicate surface adsorbed
water and hydroxyl groups. The fact that all characteristic peaks appearing in both waste PS and TiO2 are also visible in the spectra of
C50 shows that TiO2 can be successfully embedded into the PS polymer matrix [27–30].

3.5. Zeta potential

Zeta potential measurements at different pH values of the C50 composite synthesized using PS and TiO2 are given in Fig. 5. It is

5
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

Fig. 3. EDAX mapping of waste PS, C80, C50 and C20.

Fig. 4. FTIR spectra of waste PS, TiO2 and C50.

known that CIP exists in different ionic forms at different pHs. In addition to this behavior of CIP, knowing the behavior of the
synthesized composite at different pHs plays an important role in better understanding the interaction between them and explaining
the removal process. In order to examine this important effect of pH on removal performance in more depth, it was desired to benefit
from zeta potential values. For this reason, zeta potential measurements of the C50 composite were carried out. The pH values of the
C50 suspensions were adjusted to 3.0, 7.0 and 11.0 using 1.0 M NaOH and 1.0 M HCl, respectively. It is seen that for all three pH values

6
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

Fig. 5. Zeta potential measurements of the C50.

used in the experimental design, the composite surface is negatively charged and its magnitude increases as the pH value increases
[31]. In a study where the zeta potential of the TiO2 anatase phase was measured, it was observed that the zeta potential value
decreased as the pH value increased [32]. In another study conducted for PS, it was reported that the zeta potential was adversely
affected by the increase in pH value, similar to TiO2 [33]. Considering the results in this study, the zeta potential behavior of the C50
composite containing PS and TiO2 is also compatible with these studies in the literature [31–33].

3.6. Experimental design and optimization studies

The experimental design results according to were presented in Table 2 with the experimental measurement results and their
predicted values. The minimum and maximum values of ciprofloxacin removal percentage were found to be 27 (R1) and 95 (R14),
respectively. The results of the ANOVA test were shown in Table 3. After eliminating insignificant terms (p-value>0.05) from the
model, the mathematical expression for the percent ciprofloxacin removal is as follows by Equation (2).
Y = 58, 29-(10, 5 ∗ A)-(20, 62 ∗ B)-(8, 37 ∗ C) + (7, 09 ∗ B ∗ B)-(5 ∗ A ∗ B)-(10 ∗ A ∗ C) (2)
The R2 value and the adjusted R2 value were found to be 97.51 % and 95.65 %, respectively, ensuring the integrity of data.
According to Table 3, the p value of the model is less than 0.05 and the p value calculated for the lack-of-fit is greater than 0.05 (F =
3.38, p = 0.246) indicates that the significance level of the model is acceptable.
The effect of independent variables on the CIP removal were presented by contour plots in Fig. 6. As seen in Fig. 6a, the change in
polymer content in the composite did not make a significant difference for constant pH values, while the increase in pH value caused a
significant decrease in drug removal in the case of constant polymer content. The highest removal result was achieved with the
composite with low pH value and medium polymer content. pH was observed as the most dominant factor. The fact that the coefficient
of the B term in the model equation is higher than the coefficients of the other parameters and the p value of the term was obtained as
<0.05 confirmed this situation (Table 3). It can be seen from Fig. 6b that at lower drug concentrations, the effect of polymer content
was reduced compared to higher drug concentrations. Fig. 6c shows that the CIP initial concentration factor does not make a significant
difference for constant pH. The elimination of the B*C term in the model equation is consistent with this result.

Table 2
Box-Behnken experimental design points and responses with their experimental and predicted values.
Runs Independent Variables Dependent Variables

A B C Y (experimental) Y(predicted)

R1 80 7 15 27 29
R2 50 7 10 59 58
R3 20 7 15 69 70
R4 50 7 10 57 58
R5 20 11 10 60 60
R6 20 3 10 90 91
R7 20 7 5 72 67
R8 80 7 5 70 66
R9 80 11 10 30 29
R10 80 3 10 80 80
R11 50 11 15 42 36
R12 50 11 5 47 53
R13 50 3 15 79 78
R14 50 3 5 95 94
R15 50 7 10 55 58

A = Amount of polymer (in composite, % by weight); B = pH; C= Ciprofloxacin initial concentration (mg/L); Y= Ciprofloxacin removal (%).

7
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

Table 3
ANOVA results for Box-Behnken experimental design.
Source DF Adj SS Adj MS F-Value P-Value

Model 6 5533.88 922.31 52.31 0.000

Linear 3 4846.25 1615.42 91.62 0.000
A 1 882.00 882.00 50.02 0.000
B 1 3403.13 3403.13 193.01 0.000
C 1 561.13 561.13 31.82 0.000
Square 1 187.63 187.63 10.64 0.011
B*B 1 187.63 187.63 10.64 0.011
2-Way Interaction 2 500.00 250.00 14.18 0.002
A*B 1 100.00 100 5.67 0.044
A*C 1 400.00 400 22.69 0.001
Error 8 141.05 17.63
Lack-of-fit 6 128.39 21.40 3.38 0.246
Pure Error 2 12.67 6.33
Total 14 5674.93

A = Amount of polymer (in composite, % by weight); B = pH; C= Ciprofloxacin initial concentration (mg/L); Y= Ciprofloxacin removal (%).

Fig. 6. Contour plots of CIP removal parameters showing the effect of independent variables (A = Amount of polymer (in composite, wt.%); B = pH;
C= Ciprofloxacin initial concentration (mg/L); Y= Ciprofloxacin removal (%)).

3.7. Effect of polymer amount in composite

It is the TiO2 content that shows the main photocatalytic effect in composites prepared using PS and TiO2. In this study, photo­
catalytic degradation of composites with different TiO2 and thus PS contents was investigated. Experimental design results showed that
the removal performance deteriorated with increasing PS content. Since the amount of composite used in the experiments is the same,
an increase in the polymer ratio means a decrease in TiO2, which is the catalyst effect in the composite. The decrease in the amount of
PS, that is, the increase in the amount of TiO2, causes an increase in the production rate of the electron/hole pair and accordingly the
hydroxyl radicals. This increase contributes to the improvement of photocatalytic degradation performance [20].

3.8. Effect of pH

When the experimental design results are evaluated, it is seen that the most influential parameter on the process is pH. The pH

8
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

change significantly affects the solution chemistry and, accordingly, the surface charge of the catalyst and the degree of ionization.
Accordingly, the interactions between the catalyst surface, charged radicals and solvent molecules also change. It mainly affects the
formation of hydroxyl radicals.
Due to a carboxyl group and an amine group in the CIP structure, it has two pKA values of 6.1 and 8.7, respectively. Therefore, its
solubility and hydrophobicity in water depend on pH. The solubility decreases as the pH increases. Below the value of pKA1 = 6.1, it is
found in cationic form, while above the value of pKA2 = 8.7 it is in the anionic form. Between these two values, it exists in the
zwitterionic form [3,34,35].
Likewise, when the zeta potential values of C50 are examined, it is seen that it changes depending on the pH. It is seen from the
results of the zeta potential that the composite surface is negatively charged for each pH value (3,7 and 11) where the experiments are
carried out, but the surface charge density increases as the pH increases.
It is thought that the reason for the dependence on pH in the removal process of CIP may be both the different speciation of CIP
depending on pH and the changes in the surface charge density of the C50 composite. At pH 3, electrostatic attraction occurs because
the cationic CIP and the composite surface are oppositely charged. At this pH, the adsorption of CIP is facilitated, thereby improving
photocatalytic degradation. When the pH reaches 7, cationic groups leave their place to the zwitterionic structure, according to the
first situation. Here the resolution of the CIP is also reduced. For these reasons, the interactions between the composite and CIP
decrease, and accordingly, the removal performance at this pH worsens. Anionic CIP increases at pH = 11. At this pH value, the surface
of the composite is also negatively charged. In other words, the removal process was adversely affected due to electrostatic repulsion
between the negatively charged surface composite and the anionic CIP. Similar results are also found in the literature. For the
aforementioned reasons, pH = 3 was chosen as the optimum pH value [3,34,35].

3.9. Effect of initial concentration

The initial drug concentration is one of the important parameters affecting the process. In order to examine its effect, it was chosen
as a parameter in the experimental design and worked as 5,10 and 15 mg/L. When the experimental design results are evaluated, it is
seen that the degradation efficiency decreases as the initial drug concentration increases. There are many possible reasons for this
situation. One is that increasing concentration shortens the photon path entering the solution, resulting in lower photon adsorption on
the catalyst particles. For this reason, the formation of hydroxyl radicals decreases and the photodegradation efficiency decreases. On
the other hand, the probability of reaction increases as the concentration decreases. While the active sites on the photocatalyst surface
can easily interact with the few CIP molecules in the medium, otherwise, only a part of the CIP can interact with the surface due to the
excess in the medium with an increase in concentration. Thus, increasing the concentration decreases the degradation efficiency [3,
34]. As expected due to the reasons mentioned above, the best removal result was obtained at the lowest drug concentration studied.

3.10. Effect of catalyst dosage

Catalyst dosage is also one of the important parameters in the photocatalytic degradation process. In this study, the effect of dosage
was not selected separately as a parameter for the experimental design, but experiments were carried out with different catalyst
dosages for the optimum conditions determined from the experimental design. Percentages of CIP removal against different catalyst
dosages (20 mg, 30 mg, 40 mg and 50 mg) for the same amount of solution (50 ml solution is used in the experiments) are given in
Fig. 7. While the photocatalytic degradation efficiency increased with the increase of the catalyst dosage up to a certain value, it
decreased after a certain value.
The reason for this increase in the beginning is that the increase in the amount of catalyst increases the number of active sites in the
environment and leads to an increase in the radicals that cause degradation. Despite that; when used above the critical amount of
catalyst, turbidity occurs in the solution, which blocks UV radiation and reduces degradation performance. Another reason is that
catalyst particles at high dosages undergo agglomeration during photodegradation. Due to agglomeration, the surface area is reduced

Fig. 7. CIP removal for different catalyst dosages.

9
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

and the drug removal performance is adversely affected [34,36,37].

3.11. Photocatalytic degredation kinetic

The photocatalytic degradation kinetics of CIP was investigated. For this purpose, the Langmuir-Hinshelwood model was used.
Langmuir-Hinshelwood is one of the most widely used kinetic models in catalyst studies [38]. In photocatalytic degradation studies
with TiO2-containing catalyst, it has been reported that the process conforms to the L-H kinetic model [3,39–41]. In these studies, the
formation or attack of hydroxyl radicals is generally considered as a rate limiting step. In addition, adsorption has a significant effect.
For this reason, it is suitable for the use of the L-H model [42].
The L-H model equation (Equation (3)) is given as follows:
dC kKC
− = (3)
dt 1 + KC
C is the concentration at any time of the degradation; t, illumination time; k is the reaction rate and K is the adsorption equilibrium
coefficient.
There are two possibilities in such a process. The first possibility is working with dilute solutions. In this case, KC ≪ 1 is assumed.
In condition KC ≪ 1 (usually valid if C is in the range of ppb and a few ppm), Equation becomes pseudo first order kinetic [38,39,43,
44] and Equation (3) can be written as follows:
( )
C0
In = kKt = kapp t (4)
C

Here (Equation (4)) kapp is pseudo first order rate constant (apparent rate constant). This rate constant is equal to the slope of the
( )
straight line when graphed In CC0 versus time.
The second possibility is when working at high concentration (Equation (5)), where KC ≫ 1 is valid and Equation (3) becomes:
(C0 – C) = kapp t (5)

The graphs according to Equation (4) and Equation (5) of the experiments performed with different initial drug concentrations at
constant pH using the same composite are given in Fig. 8(a–d). The R2 values to be used in the evaluation of the fit with the kinetic
model are also placed in the graphics. When the kinetic graphs of the experiment, which was carried out using 5 mg/L drug, are
examined, it is seen that the R2 value of the pseudo first order model is higher than the R2 value of the zero-order model. In other words,
it can be said that the results of 5 mg/L (dilute solution) fit the pseudo first order model as expected. The results of the experiments
carried out using 15 mg/L are similar. However, when the R2 values of the zero-order model graphs of both different initial con­
centrations are examined, it is seen that the R2 value of the process with higher concentration (15 mg/L) is higher than the R2 value of
the process with lower concentration (5 mg/L). This is thought to be caused by an increase in the initial concentration. The results are
consistent with the literature information [3,37–39].
The reaction kinetic graphs of the experiments performed with composites with the same initial concentration and pH value but
with different polymer content are given in Fig. 9(a–d). As can be seen from the graphs of both composites, the process complied with
the pseudo first order kinetics. As is known, the slope of the model equations given in these graphs is used to calculate the kapp value.
Looking at these graphs, the kapp values of C20 and C80 composites are 0.011 min− 1 and 0.007min− 1, respectively. Considering the
experimental design results, it was determined that the removal performance decreased with the increase of the polymer content in the
composites. Here, it is seen that the rate constant value of the composite with high polymer content is lower than the composite with
low polymer content. This can explain that the reaction takes place more slowly in the composite with high polymer content, and
therefore there is less removal in the same reaction time. These results are consistent with the results described in section 3.7.

4. Conclusions

In this study, composites were successfully synthesized using waste PS and TiO2 at different ratios (C20, C50 and C80). This has
been confirmed by XRD, FTIR, SEM and EDAX analysis. The usage of these composites as photocatalysts in the removal of CIP from
wastewater by photocatalytic degradation was investigated. The effect of the polymer amount in the composite, pH and initial CIP
concentration parameters on the removal process was observed. Box Behnken design, one of the experimental design methods, was
used to examine the effect of these parameters. According to the experimental design results, it was understood that the most effective
parameter on the process was pH and the optimum pH was pH = 3. At the same time, the most suitable composite for the process was
selected as C50 composite with 50 % polymer content, while an initial CIP concentration of 5 mg/L was found to be optimum. In the
experiment carried out under these conditions, maximum removal performance of 95 % was achieved. As a result of the experimental
design, the model equation was obtained and the accuracy of the model was examined. It is seen that the estimated data obtained using
the model equation and the results of the experimental trials are quite close to each other. In addition, the R2, adjusted R2 values of the
model were 97.51 %, 95.65 %, respectively, and the p value was <0.05 indicating that the model represents the process well. In
addition to the experimental design, the optimum catalyst amount was determined as 40 mg. Additionally, kinetic studies were
performed for the photodegradation of CIP. It has been determined that the photocatalytic degradation of CIP by the catalyst syn­
thesized and used in this study fits well with the Langmuir-Hinshelwood pseudo first order kinetic expression, with a high regression

10
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

Fig. 8. L-H kinetic model plots for C50, a. Pseudo first order (conditions: 15 mg/L, pH = 3), b. Zero order (conditions: 15 mg/L, pH = 3), c. Pseudo
first order (conditions: 5 mg/L, pH = 3) b. Zero order (conditions: 5 mg/L, pH = 3).

Fig. 9. L-H kinetic model plots for a. Pseudo first order (conditions: C20, 10 mg/L, pH = 3), b. Zero order (conditions: C20, 10 mg/L, pH = 3), c.
Pseudo first order (conditions: C80, 10 mg/L, pH = 3) b. Zero order (conditions: C80, 10 mg/L, pH = 3).

value. In the light of these results, it was concluded that C50 could be successfully used as a catalyst for CIP removal. With the addition
of PS, the difficulties encountered when TiO2 was used alone were overcome, and a catalyst with similar or even higher performance
(at relatively low drug concentrations) was synthesized using less TiO2. On the other hand, it is of great importance that wastes are
recycled and reused according to the circular economy. In this study, plastic spoon waste, which is frequently encountered and difficult

11
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

to dispose of, has been transformed into a new product and reused. In this context, a contribution was made to the circular economy by
using one waste to clean another waste.

CRediT authorship contribution statement

Tugba Hayri-Senel: Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Ebru Kahraman: Writing –
review & editing, Validation, Methodology, Investigation, Formal analysis. Serhat Sezer: Writing – review & editing, Validation,
Methodology, Investigation, Formal analysis. Nalan Erdol-Aydin: Resources, Investigation. Gulhayat Nasun-Saygili: Writing – re­
view & editing, Supervision, Resources, Methodology.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Acknowledgements

This study was supported by Istanbul Technical University Scientific Research Projects Unit (Grant: MGA-2021-43402).

References

[1] S. Shehu Imam, R. Adnan, N.H. Mohd Kaus, Photocatalytic degradation of ciprofloxacin in aqueous media: a short review, Toxicol. Environ. Chem. 100 (2018)
518–539, https://doi.org/10.1080/02772248.2018.1545128.
[2] P. Martins, S. Kappert, H.N. Le, V. Sebastian, K. Kühn, M. Alves, L. Pereira, G. Cuniberti, M. Melle-Franco, S. Lanceros-Méndez, Enhanced photocatalytic activity
of au/TiO2 nanoparticles against ciprofloxacin, Catalysts 10 (2020), https://doi.org/10.3390/catal10020234.
[3] M. Malakootian, A. Nasiri, M. Amiri Gharaghani, Photocatalytic degradation of ciprofloxacin antibiotic by TiO2 nanoparticles immobilized on a glass plate,
Chem. Eng. Commun. 207 (2020) 56–72, https://doi.org/10.1080/00986445.2019.1573168.
[4] T. Suwannaruang, J.P. Hildebrand, D.H. Taffa, M. Wark, K. Kamonsuangkasem, P. Chirawatkul, K. Wantala, Visible light-induced degradation of antibiotic
ciprofloxacin over Fe–N–TiO2 mesoporous photocatalyst with anatase/rutile/brookite nanocrystal mixture, J. Photochem. Photobiol. Chem. 391 (2020),
https://doi.org/10.1016/j.jphotochem.2020.112371.
[5] W. Li, Y. Zuo, L. Jiang, D. Yao, Z. Chen, G. He, H. Chen, Bi2Ti2O7/TiO2/RGO composite for the simulated sunlight-driven photocatalytic degradation of
ciprofloxacin, Mater. Chem. Phys. 256 (2020), https://doi.org/10.1016/j.matchemphys.2020.123650.
[6] P.Y. Motlagh, S. Akay, B. Kayan, A. Khataee, Ultrasonic assisted photocatalytic process for degradation of ciprofloxacin using TiO2-Pd nanocomposite
immobilized on pumice stone, J. Ind. Eng. Chem. 104 (2021) 582–591, https://doi.org/10.1016/j.jiec.2021.09.007.
[7] M. Shi, W. Li, Q. Wang, H. Xu, Y. Zhao, G. He, Q. Meng, H. Chen, One-step hydrothermal synthesis of BiVO4/TiO2/RGO composite with effective photocatalytic
performance for the degradation of ciprofloxacin, Opt. Mater. 122 (2021), https://doi.org/10.1016/j.optmat.2021.111726.
[8] M. Chen, K. Zhuang, J. Sui, C. Sun, Y. Song, N. Jin, Hydrodynamic cavitation-enhanced photocatalytic activity of P-doped TiO2 for degradation of ciprofloxacin:
synergetic effect and mechanism, Ultrason. Sonochem. 92 (2023), https://doi.org/10.1016/j.ultsonch.2022.106265.
[9] A. Hassani, A. Khataee, S. Karaca, Photocatalytic degradation of ciprofloxacin by synthesized TiO2 nanoparticles on montmorillonite: effect of operation
parameters and artificial neural network modeling, J. Mol. Catal. Chem. 409 (2015) 149–161, https://doi.org/10.1016/j.molcata.2015.08.020.
[10] N. Parmar, J.K. Srivastava, Process optimization and kinetics study for photocatalytic ciprofloxacin degradation using TiO2 nanoparticle: a comparative study of
Artificial Neural Network and Surface Response Methodology, J. Indian Chem. Soc. 99 (2022), https://doi.org/10.1016/j.jics.2022.100584.
[11] S. Krishnan, A. Shriwastav, Chlorophyll sensitized and salicylic acid functionalized TiO2 nanoparticles as a stable and efficient catalyst for the photocatalytic
degradation of ciprofloxacin with visible light, Environ. Res. 216 (2023), https://doi.org/10.1016/j.envres.2022.114568.
[12] X. Hu, X. Hu, Q. Peng, L. Zhou, X. Tan, L. Jiang, C. Tang, H. Wang, S. Liu, Y. Wang, Z. Ning, Mechanisms underlying the photocatalytic degradation pathway of
ciprofloxacin with heterogeneous TiO2, Chem. Eng. J. 380 (2020), https://doi.org/10.1016/j.cej.2019.122366.
[13] S. Singh, H. Mahalingam, P.K. Singh, Polymer-supported titanium dioxide photocatalysts for environmental remediation: a review, Appl. Catal. Gen. (2013)
462–463, https://doi.org/10.1016/j.apcata.2013.04.039, 178–195.
[14] M. Chen, K. Zhuang, J. Sui, C. Sun, Y. Song, N. Jin, Hydrodynamic cavitation-enhanced photocatalytic activity of P-doped TiO2 for degradation of ciprofloxacin:
synergetic effect and mechanism, Ultrason. Sonochem. 92 (2023) 106265, https://doi.org/10.1016/j.ultsonch.2022.106265.
[15] X. Hu, X. Hu, Q. Peng, L. Zhou, X. Tan, L. Jiang, C. Tang, H. Wang, S. Liu, Y. Wang, Z. Ning, Mechanisms underlying the photocatalytic degradation pathway of
ciprofloxacin with heterogeneous TiO2, Chem. Eng. J. 380 (2020) 122366, https://doi.org/10.1016/j.cej.2019.122366.
[16] I. Altin, M. Sökmen, Preparation of TiO2-polystyrene photocatalyst from waste material and its usability for removal of various pollutants, Appl. Catal., B 144
(2014) 694–701, https://doi.org/10.1016/j.apcatb.2013.06.014.
[17] S. Das, H. Mahalingam, Dye degradation studies using immobilized pristine and waste polystyrene-TiO2/rGO/g-C3N4 nanocomposite photocatalytic film in a
novel airlift reactor under solar light, J. Environ. Chem. Eng. 7 (2019), https://doi.org/10.1016/j.jece.2019.103289.
[18] J. Shang, M. Chai, Y. Zhu, Solid-phase photocatalytic degradation of polystyrene plastic with TiO2 as photocatalyst, J. Solid State Chem. 174 (2003) 104–110,
https://doi.org/10.1016/S0022-4596(03)00183-X.
[19] I. Salehi, M. Shirani, A. Semnani, M. Hassani, S. Habibollahi, Comparative study between response surface methodology and artificial neural network for
adsorption of crystal violet on magnetic activated carbon, Arab J Sci Eng 41 (2016) 2611–2621, https://doi.org/10.1007/s13369-016-2109-3.
[20] S. Das, H. Mahalingam, Reusable floating polymer nanocomposite photocatalyst for the efficient treatment of dye wastewaters under scaled-up conditions in
batch and recirculation modes, J. Chem. Technol. Biotechnol. 94 (2019) 2597–2608, https://doi.org/10.1002/jctb.6069.
[21] P. Srinivasu, S.P. Singh, A. Islam, L. Han, Novel approach for the synthesis of nanocrystalline anatase titania and their photovoltaic application, Adv.
Optoelectron. (2011), https://doi.org/10.1155/2011/539382, 2011.
[22] J. Wang, J. Yu, X. Zhu, X.Z. Kong, Preparation of hollow TiO2 nanoparticles through TiO2 deposition on polystyrene latex particles and characterizations of
their structure and photocatalytic activity, Nanoscale Res. Lett. 7 (2012), https://doi.org/10.1186/1556-276X-7-646.
[23] A.D. Acharya, B. Sarwan, R. Sharma, S.B. Shrivastava, M.K. Rathore, Study of interfaces in polymer-metal oxide films and free-volume hole using low-energy
positron lifetime measurements, J. Sci.: Advanced Materials and Devices 4 (2019) 413–419, https://doi.org/10.1016/j.jsamd.2019.08.003.
[24] A.A. Ahmed, D.S. Ahmed, G.A. El-Hiti, M.H. Alotaibi, H. Hashim, E. Yousif, SEM morphological analysis of irradiated polystyrene film doped by a Schiff base
containing a 1,2,4-triazole ring system, Appl Petrochem Res 9 (2019) 169–177, https://doi.org/10.1007/s13203-019-00235-6.
[25] S. Wanjale, M. Birajdar, J. Jog, R. Neppalli, V. Causin, J. Karger-Kocsis, J. Lee, P. Panzade, Surface tailored PS/TiO2 composite nanofiber membrane for copper
removal from water, J. Colloid Interface Sci. 469 (2016) 31–37, https://doi.org/10.1016/j.jcis.2016.01.054.

12
T. Hayri-Senel et al. Heliyon 10 (2024) e25433

[26] B. Jaleh, M.S. Madad, M.F. Tabrizi, S. Habibi, R. Golbedaghi, M.R. Keymanesh, UV-degradation effect on optical and surface properties of polystyrene-TiO2
nanocomposite film, J. Iran. Chem. Soc. 8 (2011), https://doi.org/10.1007/BF03254293. S161–S168.
[27] Z. Liu, Z. Jian, J. Fang, X. Xu, X. Zhu, S. Wu, Low-temperature reverse microemulsion synthesis, characterization, and photocatalytic performance of
nanocrystalline titanium dioxide, Int. J. Photoenergy (2012) 1–8, https://doi.org/10.1155/2012/702503, 2012.
[28] G. Gohari, A. Mohammadi, A. Akbari, S. Panahirad, M.R. Dadpour, V. Fotopoulos, S. Kimura, Titanium dioxide nanoparticles (TiO2 NPs) promote growth and
ameliorate salinity stress effects on essential oil profile and biochemical attributes of Dracocephalum moldavica, Sci. Rep. 10 (2020) 912, https://doi.org/
10.1038/s41598-020-57794-1.
[29] S. Bagheri, K. Shameli, S.B. Abd Hamid, Synthesis and characterization of anatase titanium dioxide nanoparticles using egg white solution via sol-gel method,
J. Chem. 2013 (2013), https://doi.org/10.1155/2013/848205, 1–5.
[30] M. Shahshojaei, H. Behniafar, M. Shaabanzadeh, Preparation and characterization of polystyrene/TiO2 core-shell nanospheres via suspension technique, Adv
Mat Res 829 (2013) 120–125. https://doi.org/10.4028/www.scientific.net/AMR.829.120.
[31] M.-S. Chun, H. Il Cho, I.K. Song, Electrokinetic behavior of membrane zeta potential during the filtration of colloidal suspensions, Desalination 148 (2002)
363–368, https://doi.org/10.1016/S0011-9164(02)00731-2.
[32] H. He, Y. Cheng, C. Yang, G. Zeng, C. Zhu, Z. Yan, Influences of anion concentration and valence on dispersion and aggregation of titanium dioxide nanoparticles
in aqueous solutions, J. Environ. Sci. 54 (2017) 135–141, https://doi.org/10.1016/j.jes.2016.06.009.
[33] H. Xu, L.B. Casabianca, Probing driving forces for binding between nanoparticles and amino acids by saturation-transfer difference NMR, Sci. Rep. 10 (2020)
12351, https://doi.org/10.1038/s41598-020-69185-7.
[34] A. Hassani, A. Khataee, S. Karaca, Photocatalytic degradation of ciprofloxacin by synthesized TiO2 nanoparticles on montmorillonite: effect of operation
parameters and artificial neural network modeling, J. Mol. Catal. Chem. 409 (2015) 149–161, https://doi.org/10.1016/j.molcata.2015.08.020.
[35] X. Li, W. Wang, J. Dou, J. Gao, S. Chen, X. Quan, H. Zhao, Dynamic adsorption of ciprofloxacin on carbon nanofibers: quantitative measurement by in situ
fluorescence, J. Water Proc. Eng. 9 (2016), https://doi.org/10.1016/j.jwpe.2014.12.006 e14–e20.
[36] A. Kumar, A review on the factors affecting the photocatalytic degradation of hazardous materials, Material Science & Engineering International Journal 1
(2017), https://doi.org/10.15406/mseij.2017.01.00018.
[37] M.A. Rauf, S.S. Ashraf, Fundamental principles and application of heterogeneous photocatalytic degradation of dyes in solution, Chem. Eng. J. 151 (2009)
10–18, https://doi.org/10.1016/j.cej.2009.02.026.
[38] K.V. Kumar, K. Porkodi, F. Rocha, Langmuir–Hinshelwood kinetics – a theoretical study, Catal. Commun. 9 (2008) 82–84, https://doi.org/10.1016/j.
catcom.2007.05.019.
[39] I. Konstantinou, Photocatalytic transformation of pesticides in aqueous titanium dioxide suspensions using artificial and solar light: intermediates and
degradation pathways, Appl. Catal., B 42 (2003) 319–335, https://doi.org/10.1016/S0926-3373(02)00266-7.
[40] G.M. Neelgund, A. Oki, Photocatalytic activity of hydroxyapatite deposited graphene nanosheets under illumination to sunlight, Mater. Res. Bull. 146 (2022),
https://doi.org/10.1016/j.materresbull.2021.111593.
[41] C. Mounir, H. Ahlafi, M. Aazza, H. Moussout, S. Mounir, Kinetics and Langmuir–Hinshelwood mechanism for the catalytic reduction of para-nitrophenol over Cu
catalysts supported on chitin and chitosan biopolymers, React. Kinet. Mech. Catal. 134 (2021) 285–302, https://doi.org/10.1007/s11144-021-02066-w.
[42] O. Benhabiles, H. Mahmoudi, H. Lounici, M.F.A. Goosen, Effectiveness of a photocatalytic organic membrane for solar degradation of methylene blue pollutant,
Desalination Water Treat. 57 (2016) 14067–14076, https://doi.org/10.1080/19443994.2015.1061954.
[43] A. Houas, H. Lachheb, M. Ksibi, E. Elaloui, C. Guillard, J.-M. Herrmann, Photocatalytic Degradation Pathway of Methylene Blue in Water, 2001.
[44] M. El-Kemary, H. El-Shamy, I. El-Mehasseb, Photocatalytic degradation of ciprofloxacin drug in water using ZnO nanoparticles, J. Lumin. 130 (2010)
2327–2331, https://doi.org/10.1016/j.jlumin.2010.07.013.

13
Sci Pharm www.scipharm.at

Research article Open Access

Comprehensive Assessment of
Degradation Behavior of Aspirin and
Atorvastatin Singly and in Combination
by Using a Validated RP-HPLC Method
Omkar SHERIKAR, Priti MEHTA *

Department of Pharmaceutical Analysis, Institute of Pharmacy, Nirma University, Sarkhej-Gandhinagar

Highway, Ahmedabad, Gujarat, 382481, India.
* Corresponding author. E-mail: drpritimehta@nirmauni.ac.in (P. Mehta)
Sci Pharm. 2013; 81: 195–210 doi:10.3797/scipharm.1210-19
th
Published: December 11 2012 Received: October 18th 2012
Accepted: December 11th 2012
This article is available from: http://dx.doi.org/10.3797/scipharm.1210-19
© Sherikar and Mehta; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.

Abstract
A fixed-dose combination of atorvastatin and aspirin is widely used for the
treatment of myocardial infarction. The present work describes a compre-
hensive study of the stress degradation behavior of atorvastatin and aspirin
alone as well as in combination of 1:1 and 1:7.5 ratios, respectively, as per ICH
guidelines. The degradation products of aspirin as well as atorvastatin were
successfully separated by a developed simple, selective, and precise stability-
indicating reversed-phase HPLC method. Chromatographic separation was
achieved on the Phenomenex Luna analytical column, 150 mm x 4.6 mm, 5µm.
The mobile phase consisted of 0.1% glacial acetic acid in water and acetonitrile
in the ratio of 50:50 v/v at a flow rate of 1.0 ml/min. UV detection was performed
at 246 nm. The extent of degradation was significantly influenced when both of
the drugs were present in combination. Stress degradation behavior of
atorvastatin was highly influenced by aspirin under acid hydrolysis, thermal
degradation, and oxidative stress conditions. Similarly, the stress degradation
behavior of aspirin was affected by atorvastatin especially under neutral
hydrolysis, thermal degradation, and oxidative stress conditions. Additionally,
the combination ratio of aspirin and atorvastatin also influenced the percentage
degradation of each other. A mixture of aspirin and atorvastatin was also
analyzed after a one-month stability study at 40 °C and 75% RH. All the results
indicate chemical incompatibility of both aspirin and atorvastatin if present in
combination.
196 O. Sherikar and P. Mehta:

Keywords
Aspirin • Atorvastatin • Chemical incompatibility • Degradation profile • Drug combination•
HPLC • Acetylsalicylic acid

Introduction
The main cause of myocardial infarction (MI) is the interruption of the blood supply to the
heart, which is most commonly due to the blockage of a coronary artery due to a
hyperlipedemic condition. For the mitigation of such a condition, lipid lowering agents are
widely used alone or in combination with other antihypertensive agents or with aspirin.
Several fixed-dose combinations (FDCs) have been approved by the Food and Drug
Administration (FDA) along with a combination of ATR and ASP due to their effectiveness
against this condition [1, 2].

Atorvastatin calcium (ATR) is an antihyperlipidemic agent. It lowers lipid levels by inhibiting

HMG-CoA reductase, a rate-limiting enzyme in cholesterol biosynthesis in the liver, thus
reducing the cholesterol content in hepatocytes. ATR is chemically (3R,5R)-7-[2-(4-
fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-(propan-2-yl)-1H-pyrrol-1-yl]-3,5-dihydroxy-
heptanoic acid (Fig. 1a) [3, 4].

2+
- Ca
O OH OH O

N N O
H

F 2

1a

O 2+
- Ca
O OH OH O
O O
N N O
N N OH H
H

F 2
F

1b 1c

Fig. 1. Chemical structure of atorvastatin calcium (1a) and its major degradation
products atorvastatin lactone (1b) and atorvastatin diastereomer (1c).

Sci Pharm. 2013; 81: 195–210

 Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in… 197

Aspirin (ASP) is a cyclooxygenase inhibitor and is commonly used as an analgesic, anti-

inflammatory, and antipyretic. Additionally, it also has an antiplatelet effect, hence, it is
also used for the prevention of heart attacks. ASP is chemically 2-(acetyloxy)benzoic acid
(Fig. 2) [3, 4]. The physicochemical, pharmacological, and pharmacokinetic characteristics
of both ASP and ATR are well investigated [5–8].

O O O O

OH OH OH O OH

O OH O O O OH

O O O

2a 2b 2c 2d

Fig. 2. Chemical structure of aspirin (2a) and its degradation products salicylic acid
(2b), acetylsalicylsalicylic acid (2c), and salicylsalicylic acid (2d).

When ASP and ATR are used in combination, thromboxin-A2 (TXA2)-dependant aspirin
resistance is reduced. This mechanism is especially helpful to patients with acute
myocardial infarction and persistent platelet TXA2 production. In addition, ATR also
reduces lipid levels [9].

In the Indian market, FDC capsule formulations are available from various manufacturers
like Sun Pharmaceuticals Ltd. (AZTOR ASP 75), Zydus Healthcare Ltd. (ASP ATORVA
75), and Piramal Healthcare Ltd. (STATOR ASP 75) having strengths of 10 mg and 75 mg
of ATR and ASP respectively.

However, when FDCs are formulated, chemical incompatibility is an important factor

influencing drug stability. Interactions may take place either among different active
ingredients or active ingredients and excipients within the formulation. Chemical instability
always leads to the decomposition of actives present in the formulation [10, 11].

The chemical structure reveals that ASP is an ester moiety, and very susceptible to
hydrolysis under different hydrolytic conditions. Decomposition of ASP into salicylic acid
(SA) is well-known [5, 12, 13]. On the other hand, ATR is an organic acid with a pKa of
4.46 and it is an acid-labile moiety which gets converted into lactone under acidic
conditions [14–16].

Several analytical methods have been reported for the estimation of ASP and ATR in the
presence of each other as well as along with other drugs [17–23]. A literature survey
revealed that only one UPLC stability-indicating assay method is reported for the
combination of ASP and ATR in capsule dosage form [24].

So far, no reports are available regarding the comprehensive study on degradation

behavior of ATR and ASP in the presence of each other. From the reported data of the

Sci Pharm. 2013; 81: 195–210

198 O. Sherikar and P. Mehta:

degradation profile of both drugs, it was essential to evaluate the chemical stability of ASP
and ATR in combination. It was hypothesized that ATR may get degraded in the presence
of ASP, as ATR is an acid-labile moiety and ASP is acidic in nature.

Stress testing is an important tool for the prediction of stability-related problems in

pharmaceutical products. Therefore, in the present paper, a stress degradation study of
ATR and ASP was performed in combination of each other as per ICH guidelines and
degradation behavior of both drugs was monitored precisely. Additionally, a one month
stability test of a mixture of ATR and ASP was also evaluated to further confirm the
chemical incompatibility of these two drugs [25].

Materials and Methods

Drug and Chemicals
Pure ATR was kindly gifted by Dr. Reddys Ltd, (Hyderabad, India), certified to contain
99.1%, and used without further purification. Standard ASP was purchased from Sigma
Aldrich (India) with the stated purity of 99.8%. Salicylic acid was purchased from SD Fine
Chemicals, Mumbai, with the stated purity of 99.0%. Atorvastatin lactone was procured
from Varda Biotech Pvt. Ltd, Mumbai, India. FDC of ATR and ASP was purchased from
the local market with each capsule containing 10mg of ATR and 75mg of ASP
respectively.

HPLC grade acetonitrile was purchased from Merck (Mumbai, India). HPLC grade glacial
acetic acid and dimethyl sulfoxide (DMSO) were procured from CDH, New Delhi. 0.45 µm
nylon filters were used for sample filtration. Mili-Q water was used throughout the study.

Instrumentation and Chromatographic Condition

Analysis was carried out on the HPLC system (Jasco, Japan) consisting of the binary
pumps-Jasco PU-2080 and solvent mixing module-Jasco MX-2080, equipped with the
photodiode array (PDA) detector MD-2015 Plus (Jasco Japan). It also consisted of the
Rheodyne manual injector having a 20µL capacity. Data collection and data processing
were accomplished by using BORWIN-PDA software.

Separation was achieved on the Phenomenex Luna analytical column, 150 mm x 4.6 mm,
5µm. The mobile phase consisted of 0.1% glacial acetic acid in water and acetonitrile in
the ratio of 50:50 v/v. The mobile phase was filtered through a 0.45µm nylon filter and
degassed prior to use. An equal amount of acetonitrile and water was mixed and used as
a diluent throughout the study.

Experimental
Preparation of Standard Stock Solution
The standard stock solutions, 1000 µg/mL each of ATR and ASP, were prepared
individually in methanol.

Sci Pharm. 2013; 81: 195–210

 Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in… 199

Preparation of samples for the Forced Degradation Study

Forced degradation of ASP and ATR, individually as well as in combination, was carried
out as per ICH guidelines. Both ASP and ATR were forcibly degraded under different
stress conditions such as hydrolytic, oxidative, thermal, and photolytic degradation.

To perform the degradation study in combination, two ratios 1:1 and 1:7.5 were selected
for ATR and ASP, respectively. All degradation samples were prepared in DMSO and the
respective stressor in the ratio of 50:50 v/v. Stress samples were prepared by taking a
concentration of 2.5 mg/mL of each drug. Hydrolytic degradation was carried out in 0.1N
HCl, 0.1N NaOH, and distilled water by dissolving ASP and ATR in 25 mL of DMSO and
then the volume of this solution was made up to 50 mL with 0.1N HCl, 0.1N NaOH, and
distilled water respectively. All prepared samples were refluxed at 80 °C for 3 hours.
For oxidative stress, 3%v/v hydrogen peroxide was used. For both ASP and ATR, 2.5
mg/mL samples were prepared by dissolving the drug in 25 ml of DMSO and then the
volume was made with 3%v/v hydrogen peroxide solution and samples were kept at room
temperature for seven days. After completion of stress exposure, samples were
neutralized and further diluted to get a concentration of 50 µg/mL each of ATR as well as
ASP, respectively, for HPLC analysis. In the case of a 1:7.5 ratio, 50 µg/mL concentration
of ATR was selected and this correspondingly gave 375 µg/mL of ASP. Thermal
degradation was carried out in a solid state in a controlled temperature oven at 80 °C for
48 hours. For photodegradation, solid samples were spread uniformly in a petridish. Then
samples were exposed to bright, sunny days from 11 am to 5 pm for two days. So the
samples were exposed to sunlight for a total of 12hrs.
In addition, ASP and ATR, alone as well as in combination, were kept for solid-state
stability studies for one month at 40◦C and 75% RH in a stability chamber to extrapolate
the probable chemical incompatibility between these two drugs. Stock solutions of all solid
samples were prepared in methanol. Appropriate aliquots were further diluted to get 50
µg/mL each of ATR and ASP, respectively, for HPLC analysis.

Method Validation
The developed stability-indicating assay method was validated by assessing various
validation parameters such as linearity, precision, accuracy, specificity, limit of detection
and quantification, and solution stability, as prescribed by ICH guidelines [26].

Linearity
The calibration curves of ASP and ATR were constructed over a concentration range of
1–80 μg/mL and 1–60 μg/mL respectively. Different sets of six concentrations were
prepared and 20 μL of each solution were injected in triplicate under the operating
chromatographic conditions.

Method Precision
Repeatability (method precision) was assessed by analyzing the capsule samples six
times having a concentration of 60 μg/mL and 8 µg/mL for ASP and ATR respectively.

The intraday and interday precision of the proposed method was determined by estimating
the corresponding responses three times on the same day as well as on three different

Sci Pharm. 2013; 81: 195–210

200 O. Sherikar and P. Mehta:

days by taking three different concentrations of ASP and ATR (10, 20, and 40 μg/mL). The
result of the precision study was evaluated in terms of % RSD.

Specificity
The specificity of the method was assessed by checking the peak purity of both ASP as
well as ATR. Interference from degradation peaks in the estimation of ASP as well as ATR
was also checked.

Accuracy
The accuracy of the method was determined by estimating recoveries of ASP and ATR by
the standard addition method at three different levels (80,100, and 120 %) to the
preanalyzed capsule formulation. A known amount of standard ASP (24, 30, and 36 μg)
and ATR (3.2, 4, and 4.8 μg) were added to the preanalyzed sample solution containing 30
μg/mL of ASP and 4 μg/mL of ATR respectively. The accuracy of the method was
determined by comparing the amount recovered and the actual amount spiked.

Robustness
The robustness of the method was evaluated by analyzing the same samples of ASP and
ATR with deliberate changes in optimized chromatographic conditions. The changes in the
response of ASP and ATR as well as system suitability parameters were recorded. The
parameters which changed include flow rate (± 0.1 mL), detection wavelength (± 2 nm),
and organic component composition (±5%).

Solution Stability
Solution stability of the standard as well as the sample was performed at benchtop (room
temperature) and refrigeration temperature. The stability was checked by comparing the
peak area of the standard and sample each time with the initial concentration.

Limit of Detection and Quantification

The LOD and LOQ were determined from the standard deviation of the response and the
slope of the calibration curve. The equation mentioned in the ICH guidelines was used for
the determination of the LOD and LOQ.

Results and Discussion

To carry out a detailed stability study of ATR and ASP in combination, it is essential to
have a single stability-indicating assay method which can separate ATR and ASP along
with all of its the degradation products from each other. Thus, a novel RP-HPLC method
was developed and optimized using the Phenomenex C18 column (150x4.6 mm, 5μm
particle size) and a mobile phase consisting of acetonitrile and water with 0.1% glacial
acetic acid in the ratio of 50:50 v/v at a flow rate of 1.0 mL/min.

The amount of ASP is 7.5 times more than ATR in the combined marketed dosage form.
Therefore, to get the maximum response of analyte by this method, 246 nm was used as
the detection wavelength for both drugs, which is the absorption maxima of ATR, and a
promising response of ASP was also obtained. Fig. 3 shows the RP-HPLC chromatogram
of ATR and ASP in combination with a retention time of 9.86 and 2.60 min respectively.

Sci Pharm. 2013; 81: 195–210

 Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in… 201

Fig.3. Representative HPLC chromatogram of standard ASP and ATR

System suitability parameters of the developed method are summarized in Table 1.

Tab. 1. Results of System Suitability Parameters for ASP and ATR by the Proposed
RP-HPLC Method
Parameter ASP ATR
Rt (min) 2.60 9.86
Asymmetry factor 1.24 1.17
Theoretical plates 4939 9509
Resolution – 26.62

Validation of developed method

The developed method was successfully validated as per ICH guidelines by evaluating
various validation parameters like linearity, repeatability, interday and intraday precision,
LOD, and LOQ. Results of validation parameters are depicted in Table 2.

Tab. 2. Summary of Validation Parameters for the Proposed Method

Parameter ASP ATR
Linearitya (µg/mL) (n=3) 1–80 1–60
Intercept (c) −21282 −8019
Slope (m) 46251 36678
r2 value 0.999 0.999
LOD (µg/mL) 0.010 0.032
LOQ (µg/mL) 0.031 0.099
Precision
Intraday % RSD (n=3) 0.19–1.08 0.18–0.85
Interday % RSD (n=3) 0.03–0.5 0.15–042
Repeatability % RSD (n=6) 0.94 0.78
a
y = mx + c

Sci Pharm. 2013; 81: 195–210

202 O. Sherikar and P. Mehta:

The mean % recovery values of ASP and ATR were obtained in the range of 99.0 ± 0.04 –
99.9 ± 0.27 and 99.0 ± 0.71 – 99.8 ± 0.41 which proves that the method is accurate. The
results of the accuracy study are presented in Table 3.

Tab. 3. Results of Accuracy Studies of ASP and ATR by Proposed RP-HPLC Method
Amount of Amount of
drug taken standard Mean % Recoverya ± S.D
(µg) added(µg)
ASP ATR ASP ATR ASP ATR
30 4 24 3.2 99.0± 0.04 99.8 ±0.41
30 4 30 4 99.0 ±0.10 99.2±0.31
30 4 36 4.8 99.6±0.27 99.0±0.71
a
Average of three determinations.

The robustness of the methods was studied by analyzing the same samples of ASP and
ATR with deliberate variation in the method parameters. It was found that there was no
significant change in the system suitability parameters of both ASP and ATR. The results
obtained in the changed parameters were within an acceptable range.

Specificity of the method was confirmed by checking the peak purity of ASP and ATR. The
peak purity was found to be more than 990 for both ASP and ATR. The results obtained
from the degradation studies showed that the developed stability-indicating assay method
is specific towards ASP, ATR and their degradation products.

The prepared solution of ASP in diluent was found to be stable up to 6 hrs on the benchtop
as well as after refrigeration. After 6 hrs, there was formation of the major degradation
product of ASP i.e. salicylic acid. ATR was found to be stable for up to 24hrs in both
benchtop as well as after refrigeration. In the case of ASP, a decrease in area after 6 hrs
was more than 2% for both samples kept on the benchtop as well as samples kept in the
refrigerator. Conversely for ATR, there was no significant decrease in the area after 24 hrs
in both samples.

Forced Degradation study

Degradation behavior of ASP as well as ATR was studied individually and in combination
to check chemical compatibility in the presence of each other. Different stress conditions
were employed as suggested by ICH guidelines.

For the stress degradation study, a selection of co-solvents was a challenging task for
these poorly water-soluble drugs. ATR is very slightly soluble in water as well as
acetonitrile. Both the drugs have good solubility in methanol, but due to the presence of
the carboxylic acid functional group in both ATR and ASP, there is a chance that methyl
ester may form in the drugs. Degradation of the drugs by the formation of methyl ester
may lead to errors in the degradation study [27, 28]. DMSO is a comparatively inert solvent
[29-30] and both the drugs have good solubility in it. Hence, it was selected as the co-
solvent along with stressors for preparing solutions during the stress studies.

Sci Pharm. 2013; 81: 195–210

 Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in… 203

ATR and ASP individually

ASP, being an ester moiety, is susceptible to acid, neutral, base, and oxidative
degradation conditions. It primarily degrades into SA. SA was identified by analyzing
standard SA under the same chromatographic conditions. Confirmation was done on the
basis of retention time and PDA spectra. In photolytic and thermal stress conditions, ASP
alone is stable. Results are in accordance with the reported studies [11, 23].

ATR alone is susceptible to acidic hydrolytic conditions and is converted initially into the
major degradation product ATR lactone (ATR-DP). ATR-DP was identified by analyzing
standard ATR-DP under the same chromatographic conditions. Confirmation was done on
the basis of retention time and PDA spectra. Upon prolonged exposure, it further degraded
into two additional degradation products other than ATR-DP. Stress degradation of ATR is
also in accordance with the reported study [13, 23]. However, in neutral, base, oxidative,
photolytic, and thermal stress conditions, ATR does not show any degradation. Table 4
summarizes the degradation behavior of ASP and ATR alone.

ATR and ASP in the ratio of (1:1)

Except thermal degradation, no additional peak was observed in the HPLC chromate-
grams of the stress degradation study of ATR and ASP in combination (1:1 ratio)
compared to chromatograms of the individual degradation study in all conditions. This
indicates that no additional degradation product formed other than SA and ATR-DP when
ASP and ATR were present in combination. However, the percentage degradation of both
drugs affected by the presence of each other, depending on the stress conditions, were
employed for the study. The presence of ATR does not affect the stability of ASP in acidic
hydrolytic conditions, as the observed percentage degradation was 24.24% and 20.28% in
ASP alone and in combination, respectively.

In thermal degradation, three additional peaks of degradation products were observed.

PDA spectra of all three degradation products correlated with the PDA spectra of ASP.
This result indicates that additional degradation products may be generated from ASP.
This is further supported by the highest difference in % degradation of ASP alone and in
the presence of ATR under thermal degradation conditions.

Major influences of both drugs were observed on each other in the neutral, oxidative, and
thermal degradation study. ATR was highly stable in the above stress conditions when
present alone, but showed significant degradation when present in combination with ASP.
Table 4 summarizes the percentage degradation of both drugs in various stress
conditions.

ATR and ASP in the ratio of 1:7.5 (formulation ratio)

It was decided to perform the stress degradation study of ATR and ASP in the ratio of
1:7.5 respectively, as both drugs are present in this ratio in the marketed fixed dose
combination. Except for the thermal degradation study, no additional peak, other than SA
and ATR-DP, was observed in the HPLC chromatogram of the mixture of ATR and ASP in
1:7.5 ratios. In acid degradation, ATR showed slightly higher degradation as compared to
when it is present alone (Figure 4). But on increasing the amount of ASP up to 7.5 times,
the degradation rate of ATR was increased particularly in the neutral, oxidative, and
thermal stress degradation study as shown in figure 5, 6, and 7 respectively.

Sci Pharm. 2013; 81: 195–210

204 O. Sherikar and P. Mehta:

A summary of the degradation study of both drugs is furnished in Table 4. It was confirmed
that both drugs showed a significant influence on the percentage degradation of each
other if present together. From all of the results of the stress degradation studies, it can be
summarized that both ASP and ATR can interact chemically and degrade if present in
combination.

Tab. 4. Summary of Stress Degradation Behavior for ASP and ATR

Degra % Degradation % Degradation
Degradation
dation Sample Type observed for observed
Condition
Type ASP for ATR
ASP alone 24.25 –
0.1 N HCl
Acid ATR Alone – 29.6
3 Hrs reflux at
Hydrolysis ASP:ATR(1:1) Ratio 20.28 38
80 °C
ATR:ASP(1:7.5)Ratio 32.26 36.40
ASP alone 90 –
0.1 N NaOH
Alkaline ATR Alone – No Degradation
3 Hrs reflux at
Hydrolysis ASP:ATR(1:1) Ratio 90 No Degradation
80 °C
ATR:ASP(1:7.5)Ratio 93 No Degradation
ASP alone 19 –
Neutral 3Hrs reflux at ATR Alone – No Degradation
Hydrolysis 80 °C in water ASP:ATR(1:1) Ratio 39 6.28
ATR:ASP(1:7.5)Ratio 25.61 27.22
3% H2O2 ASP alone 54.49 –
Oxidative at room ATR Alone – No Degradation
Stress temperature ASP:ATR(1:1) Ratio 89 28
for 7 days ATR:ASP(1:7.5)Ratio 61 30
ASP alone No Degradation –
Thermal 80 °C for ATR Alone – No Degradation
Stress 48 Hrs ASP:ATR(1:1) Ratio 39.61 38.57
ATR:ASP(1:7.5)Ratio 15 50
ASP alone No Degradation No Degradation
Exposed to
Photolytic ATR Alone No Degradation No Degradation
sunlight for
Stress ASP:ATR(1:1) Ratio No Degradation No Degradation
12 hrs
ATR:ASP(1:7.5)Ratio No Degradation No Degradation

To get further scientific evidence, both of the drugs alone and in combination (in a ratio of
1:1 and 1:7.5, respectively, for ATR and ASP) were kept for one month stability studies at
40 °C and 75% RH. It was found that both ATR and ASP were stable if present alone.
Conversely, significant degradation was observed if present in combination. ASP was
found to be prone to degradation in the presence of ATR when both were in a 1:1 ratio.
The percentage degradation of ATR was increased when combined with 7.5 times ASP
(formulation ratio). The results indicated that the degradation of ATR is highly influenced
by the amount of ASP present in the sample. Results of the one month stability study are
furnished in Table 5.

Sci Pharm. 2013; 81: 195–210

 Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in… 205

Fig. 4. Representative HPLC Chromatogram for acid degradation of ATR and ASP in
1:7.5 combination.

Fig. 5. Representative HPLC Chromatogram for neutral degradation of ATR and ASP
in 1:7.5 combination

Sci Pharm. 2013; 81: 195–210

206 O. Sherikar and P. Mehta:

Fig. 6. Representative HPLC Chromatogram for oxidative degradation of ATR and

ASP in 1:7.5 combination.

Fig. 7. Representative HPLC Chromatogram for thermal degradation of ATR and ASP
in 1:7.5 combination showing three additional degradation products (A1, A2,
and A3).

Sci Pharm. 2013; 81: 195–210

 Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in… 207

Tab. 5. Summary of Degradation after One Month Stability Study at 40 °C and 75% RH.
% Degradation % Degradation
No Sample Type
observed for ASP observed for ATR
1 ASP alone No Degradation –
2 ATR Alone – No Degradation
3 ASP:ATR(1:1) Ratio 18 31
4 ATR:ASP(1:7.5)Ratio 5 54

Conclusion
The degradation behavior of ASP and ATR was described at length in the present study.
The stress study was considered as a tool to check the chemical incompatibility of both
drugs in the presence of each other. From the stress degradation studies, it can be
concluded that there is a significant difference in the % degradation of both of the drugs,
alone and in combination with each other. This chemical instability of ASP and ATR is
associated with the combined effect of temperature as well as humidity. Additionally, the
combination ratio of ASP and ATR also influenced their degradation rate. Therefore, while
formulating a fixed-dose combination of these two drugs, a proper formulation strategy is
very important which can ensure the stability of both ASP and ATR in formulation.

Acknowledgement
The authors are thankful to Dr. Reddy’s Laboratories Ltd. for providing a gift sample of
atorvastatin.

Authors’ Statement
Competing Interests
The authors declare no conflict of interest.

References
[1] Available from: http://www.who.int/cardiovascular_diseases/en/
[2] Available form: http://www.who.int/entity/bulletin/volumes/83/12/news.pdf
[3] Indian Pharmacopoeia.
Indian Pharmacopoeia Commission: Ghaziabad, India, Volume II, 2007: 745, 749.
[4] Martindale: The Complete Drug Reference.
36th ed., Pharmaceutical Press, London, 2009: 20, 1218.
[5] Gerald AH, John AS, Brian H, John RV.
Pharmacokinetics of aspirin and salicylate in relation to inhibition of arachidonate cyclooxygenase and
anti-inflammatory activity.
Proc Natl Acad Sci U S A. 1987; 84: 1417–1420.
http://www.ncbi.nlm.nih.gov/pubmed/3103135

Sci Pharm. 2013; 81: 195–210

208 O. Sherikar and P. Mehta:

[6] Frank EB, Peter RB.

The quantitative determination of aspirin and its degradation products in a model solution aerosol.
J Pharm Biomed Anal. 1995; 13: 111–119.
http://dx.doi.org/10.1016/0731-7085(94)00131-K
[7] Lennernas H.
Clinical pharmacokinetics of Atorvastatin.
Clin Pharmacokinet. 2003; 42: 1141–1160.
http://dx.doi.org/10.2165/00003088-200342130-00005
[8] Malhotra HS, Goa KL.
Atorvastatin: an updated review of its pharmacological properties and use in dyslipidaemia.
Drugs. 2001; 61: 1835–1881.
http://dx.doi.org/10.2165/00003495-200161120-00012
[9] Manjula Devi AS, Sriram S, Rajalingam B, Alfet Raju A, Varghese RS, Venkata Phani A.
Evaluation of the rationality of fixed dose combinations of cardiovascular drugs in a multispecialty
tertiary care hospital in Coimbatore, Tamilnadu, India.
Hygeia J Drugs Med. 2012; 4: 51–58.
[10] Sleight P, Pouleur H, Zannad F.
Benefits, challenges, and registerability of the polypill.
Eur Heart J. 2006; 27: 1651–1656.
http://dx.doi.org/10.1093/eurheartj/ehi841
[11] Panda J, Tiwari P, Uppal R.
Evaluation of the rationality of some FDCs: Focus on antihypertensive drugs.
Indian J Pharm Sci. 2006; 68: 659–653.
[12] Pfeiffer CD, Pankey JW.
Determination of related compounds in aspirin by liquid chromatography.
J Pharm Sci. 1982; 71: 511–514.
http://dx.doi.org/10.1002/jps.2600710508
[13] Verstraeten A, Roets E, Hoogmartens J.
Quantitative determination by high-performance liquid chromatography of acetylsalicylic acid and
related substances in tablets.
J Chromatogr A. 1987; 388: 201–216.
http://dx.doi.org/10.1016/S0021-9673(01)94480-2
[14] Shah RP, Kumar V, Singh S.
Liquid Chromatography/Mass Spectrometric studies on atorvastatin and its stress degradation
products.
Rapid Commun Mass Spectrom. 2008; 22: 613–622.
http://dx.doi.org/10.1002/rcm.3403
[15] Zahid Z, Farooqui MN, Mangle AA, Nikalje AG.
Stability-indicating high performance liquid chromatographic determination of atorvastatin calcium in
pharmaceutical dosage form.
Afr J Pharm Pharmacol. 2008; 2: 204–210.
[16] Khedr A.
Stability-Indicating high-performance liquid chromatographic assay of atorvastatin with fluorescence
detection.
J AOAC Int. 2007; 90: 1547–1553.
http://www.ncbi.nlm.nih.gov/pubmed/18193731
[17] Purushotam K, Sinha M, Damle C, Bothara KG.
A validated stability indicating HPTLC method for determination of Aspirin and Clopidogrel Bisulphate
in combined dosage form.
Eurasian J Anal Chem. 2009; 4: 152–160.

Sci Pharm. 2013; 81: 195–210

 Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in… 209

[18] Cemal A, Ismail TD, Sayal A, Aydin A, Yalcin O, Husamettin G.

Rapid and simultaneous determination of acetylsalicylic acid, paracetamol, and their degradation and
toxic impurity products by HPLC in pharmaceutical dosage forms.
Turk J Med Sci. 2008; 38: 167–173.
[19] Gupta KR, Askarkar SS, Wadodkar SG.
Stability indicating RP-HPLC method for simultaneous determination of atorvastatin and nicotinic acid
from their combined dosage form.
Eurasian J Anal Chem. 2009; 4: 294–303.
[20] Kadav AA, Vora DN.
Stability indicating UPLC method for simultaneous determination of atorvastatin, fenofibrate and their
degradation products in tablets.
J Pharm Biomed Anal. 2008; 48: 120–126.
http://dx.doi.org/10.1016/j.jpba.2008.05.018
[21] Patole SM, Potale LV, Khodke AS, Damle MC.
A validated HPLC method for analysis of atorvastatin calcium, ramipril and aspirin as the bulk drug
and in combined capsule dosage Forms.
Int J Pharm Sci Rev Res. 2010; 4: 40–45.
[22] Patel GF, Vekariya NR, Dholakiya RB.
Estimation of aspirin and atorvastatin calcium in combine dosage form using derivative
spectrophotometric Method.
Int J Pharm Res. 2010; 1: 62–66.
[23] Kumar V, Shah RP, Singh S.
LC and LC–MS methods for the investigation of polypills for thetreatment of cardiovascular diseases,
Part 1. Separation of Active Components and Classification of their interaction/degradation products.
J Pharm Biomed Anal. 2008; 47: 508–515.
http://dx.doi.org/10.1016/j.jpba.2008.01.041
[24] Vora DN, Kadav AA.
Validated Ultra HPLC method for the simultaneous determination of atorvastatin, aspirin and their
degradation products in capsules.
J Liq Chromatogr Related Technol. 2008; 31: 2821–2837.
http://dx.doi.org/10.1080/10826070802388482
[25] International Conference of Harmonization.
Stability Testing of New Drug Substances and Products, Q1A (R2).
International Conference of Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use. IFPMA, 2003, Geneva.
[26] International Conference of Harmonization.
Validation of Analytical Procedures: Text and Methodology, Q1A (R1).
International Conference of Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use. IFPMA, 2005, Geneva.
[27] Alsante KM, Martin L, Baertschi SW.
A stress testing benchmarking study.
Pharm Technol. 2003; 27: 60–72.
[28] Baertschi SW, ed.
Stress Testing: A Predictive Tool.
In: Pharmaceutical Stress Testing Predicting Drug Degradation.
1st ed., Taylor & Francis Group: Boca Raton, 2005: 24.
[29] Baertschi SW, ed.
Stress Testing: A Predictive Tool.
In: Pharmaceutical Stress Testing Predicting Drug Degradation.
1st ed., Taylor & Francis Group: Boca Raton, 2005: 25.

Sci Pharm. 2013; 81: 195–210

210 O. Sherikar and P. Mehta:

[30] Ahuja S, Scypinski S; eds.

Degradation and Impurity Analysis for Pharmceutical Drug Candidates.
In: Handbook of Modern Pharmaceutical Analysis.
2 nd ed., Academic Press: USA, 2011: 69.

Sci Pharm. 2013; 81: 195–210

 antibiotics
Article
Stability of Ampicillin plus Ceftriaxone Combined in
Elastomeric Infusion Devices for Outpatient Parenteral
Antimicrobial Therapy
Beatriz Fernández-Rubio 1 , Laura Herrera-Hidalgo 1,2, * , Rafael Luque-Márquez 2 , Arístides de Alarcón 2 ,
Luis E. López-Cortés 3,4 , Sonia Luque-Pardos 5 , José María Gutiérrez-Urbón 6 , Aurora Fernández-Polo 7 ,
María V. Gil-Navarro 1,2,4,† and Alicia Gutiérrez-Valencia 2,†
Citation: Fernández-Rubio, B.;
Herrera-Hidalgo, L.; Luque-Márquez,
R.; de Alarcón, A.; López-Cortés, L.E.;
Luque-Pardos, S.; Gutiérrez-Urbón,
J.M.; Fernández-Polo, A.; Gil-Navarro,
M.V.; Gutiérrez-Valencia, A. Stability
of Ampicillin plus Ceftriaxone
Combined in Elastomeric Infusion
Devices for Outpatient Parenteral
Antimicrobial Therapy. Antibiotics
2023, 12, 432. https://doi.org/
10.3390/antibiotics12030432
Academic Editor: Jeffrey Lipman
Received: 24 January 2023
Revised: 20 February 2023
Accepted: 21 February 2023
Published: 22 February 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Antibiotics 2023, 12, 432. https://doi.org/10.3390/antibiotics12030432
1 Unidad de Gestión Clínica de Farmacia, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de
Sevilla (IBiS), 41013 Seville, Spain
2 Unidad de Gestión Clinica de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital
Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBiS), 41013 Seville, Spain
3 Infectious Diseases and Microbiology Clinical Unit, University Hospital Virgen Macarena, Departament of
Medicine, School of Medicine, University of Sevilla, Biomedicine Institute of Sevilla (IBiS)/CSIC,
41009 Seville, Spain
4 Centro de Investigación en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III,
28029 Madrid, Spain
5 Unidad de Gestión Clínica de Farmacia, Hospital del Mar, 08003 Barcelona, Spain
6 Unidad de Gestión Clínica de Farmacia, Complexo Hospitalario Universitario de A Coruña, 15006 A Coruña, Spain
7 Unidad de Gestión Clínica de Farmacia, Hospital Universitari Vall d’Hebron, 08035 Barcelona, Spain
* Correspondence: lauraherrerahidalgo@gmail.com
† These authors contributed equally to this work.
Abstract: Currently, ampicillin plus ceftriaxone (AC) is one of the preferred treatments for
Enterococcus faecalis infective endocarditis. However, there is a lack of stability data for the combination
of both drugs in elastomeric devices, so the inclusion of AC in Outpatient Parenteral Antimicrobial
Therapy (OPAT) programs is challenging. The objective of the study was to determine the stability
of AC in elastomeric pumps when stored at 8 ± 2 ◦ C, 25 ± 2 ◦ C, 30 ± 2 ◦ C and 37 ± 2 ◦ C using
LC-MS/MS. The combination was diluted in 0.9% sodium chloride and the final concentrations were
ampicillin 24 g/L plus ceftriaxone 8 g/L. Physical and chemical stability were evaluated at 12, 20, 24,
36 and 48 h after preparation. Stability was met at each time point if the percentage of intact drug
was ≥90% of its respective baseline concentration and color and clearness remained unchanged. The
drug combination was stable for 48 h when it was kept at 8 ± 2 ◦ C. At 25 ± 2 ◦ C and 30 ± 2 ◦ C, they
were stable for 24 h of storage. At 37 ± 2 ◦ C, the stability criterion was not met at any time point.
These results prove that AC could be included in OPAT programs using elastomeric infusion devices
for the treatment of E. faecalis infections.
Keywords: stability; outpatient parenteral antimicrobial therapy; elastomers; ampicillin; ceftriaxone;
infective endocarditis
1. Introduction
Infective endocarditis is a rare but life-threatening condition associated with high rates
of morbidity and mortality. Staphylococcus aureus is the most prevalent cause of infective
endocarditis, followed by viridans group streptococci, other streptococci and enterococci.
Therefore, Enterococcus species are the third most common cause of infective endocarditis,
excluding cases associated with injection drug use, and they represent around 10–15% of
the total amount. Enterococcus faecalis is the leading agent of all enterococcal endocarditis,
representing more than 90% of all cases, followed by Enterococcus faecium (5%) and other
species [1]. Treatment of enterococcal endocarditis is known to be challenging due to the
https://www.mdpi.com/journal/antibiotics
Antibiotics 2023, 12, 432 2 of 10

ability of enterococci to develop antibiotic resistance, with increasing high-level amino-

glycoside resistance. Current American and European guidelines for the management
of infective endocarditis caused by E. faecalis recommend dual beta-lactam therapy with
ampicillin (AMP) and ceftriaxone (CRO) for 4–6 weeks as the first-line therapy [2,3]. Af-
ter 2–3 weeks, most patients are clinically and hemodynamically stable, but they have to
remain at hospital for another 3–4 weeks due to the antimicrobials’ intravenous administra-
tion [4]. In this context, Outpatient Parenteral Antimicrobial Therapy (OPAT) programs
have been established as a high-quality and cost-effective alternative [5].
OPAT is a successful and safe care modality for patients who require intravenous
treatment and have sufficient clinical stability to be at home [6]. Benefits of OPAT pro-
grams include a reduced length of hospital stay, readmission avoidance, a reduced risk of
healthcare-associated infections, improved patient satisfaction and significant healthcare
cost savings compared to inpatient care [7]. Outpatient clinic model, nurse home visits and
self (or carer) administration are the OPAT modalities that have been explored [8]. Various
drug administration strategies are safely employed in these programs, including infusion
by gravity, portable programmable devices or elastomeric pumps. Elastomeric devices
present numerous advantages, such as ease of management, since the manipulation of these
devices only requires connection and disconnection from the catheter. Additionally, they
are light, silent and do not require an external power supply for their functioning, allowing
the complete mobility of the patient [9,10]. Among the limitations, the long-term use of
elastomeric devices is more expensive than the one-time purchase of an electric infusion
pump and the associated disposables, although continuous elastomeric pump infusion
reduces the number of daily nurse visits [11,12]. Moreover, environmental factors such as
the external temperature need to be taken into account, as they could affect the stability of
the antimicrobials [13].
Consequently, the utilization of elastomeric pumps in OPAT programs for the treat-
ment of E. faecalis infective endocarditis appears to be valuable and useful [14]. At the
hospital, the standard dosage of AMP and CRO used is 2 g every 4 h and 2 g every 12 h to
treat this infection, respectively. In the OPAT setting, continuous administration through
elastomeric devices could be an interesting alternative, given the fact that both antimi-
crobials are beta-lactams, so continuous infusion would achieve higher plasma antibiotic
concentrations than intermittent administration and would improve the clinical cure. Our
study group has already proven that the combination of both antimicrobials at the same
daily dose (12 g of AMP and 4 g of CRO per day) is stable for up to 30 h at 25 ◦ C and 30 ◦ C
when they are diluted in 0.9% sodium chloride polypropylene infusion bags [15]. Therefore,
it is no longer necessary to use two simultaneous electronic devices, since daily doses of
both antibiotics can be dissolved in the same solution and they can be administered through
a single electronic pump. Nevertheless, it was demonstrated that the storage container is
a factor that can have an impact in drug absorption, so it is necessary to study the stability
data for this antimicrobial combination in elastomeric devices [16]. Additionally, the com-
bination of AMP plus CRO could also be suitable in other infections, such as osteomyelitis
caused by E. faecalis, or even against other microorganisms such as Listeria monocytogenes in
invasive infections, especially when the central nervous system is compromised [17,18].
The purpose of the present study was to determine the stability of AMP plus CRO
in elastomeric pumps when stored under refrigerated conditions and at three different
room temperatures. This information will provide guidance regarding the stability and the
potential use of this combination for the treatment of E. faecalis infective endocarditis in
OPAT programs.

2. Materials and Methods

Materials. Ampicillin sodium was obtained from Normon Laboratories (Madrid,
Spain) and ceftriaxone sodium for injection was purchased from Medochemie Limited
(Limassol, Cyprus). The internal standard cefixime was supplied by Alsachim (Illkirch,
France). Dosi-fuser elastomeric pumps and tube clamps were supplied by Leventon
Antibiotics 2023, 12, 432 3 of 10

(Barcelona, Spain). Normal saline 0.9% sodium chloride bags were purchased from Baxter
Healthcare, Inc., Deerfield, Illinois. Liquid chromatography–mass spectrometry (LC-MS)-
grade (reagent-grade, >98% pure) acetonitrile was obtained from Merck KGaA (Darmstadt,
Germany), and formic acid was purchased from Scharlab (Barcelona, Spain). Ammonium
acetate was obtained from Fisher Scientific (Newington, NH, USA). Purified water was
prepared in-house with a Milli-Q water system from Millipore (Bedford, MA, USA).
Preparation of solutions. AMP and CRO were reconstituted with water for injection
(10 mL per each gram of antibiotic), resulting in a concentration of 100 g/L. According
to the current guidelines for the treatment of endocarditis caused by E. faecalis, the daily
dosages of AMP and CRO (12 g and 4 g, respectively) were diluted together in 0.5 L of 0.9%
sodium chloride and introduced into the polyisoprene elastomeric pumps. Therefore, the
final concentrations obtained were AMP 24 g/L and CRO 8 g/L. Three different elastomeric
pumps were prepared for each temperature condition.
Storage conditions. Antibiotic stability was tested at 8 ± 2 ◦ C, 25 ± 2 ◦ C, 30 ± 2 ◦ C
and 37 ± 2 ◦ C. Once initial (0 h) samples were collected, the elastomeric pumps were
stored in an air thermostat oven for the duration of the study (48 h). In-line filters and
flow restrictors were removed from the elastomeric devices for the study and the outlet
was clamped using a suitable tube clamp. At each time point (0, 12, 20, 24, 36 and 48 h),
duplicate 1 mL samples were taken and frozen at −80 ◦ C until the analysis.
Sample processing. Before the analysis, samples were diluted in Milli-Q water, vor-
texed and aliquoted in autosampler vials. Five microliters of this solution were injected
into the HPLC-MS/MS device. The color and clarity of each elastomeric pump solution
were visually assessed at each sampling time point.
LC-MS/MS quantification. Antibiotics concentrations were measured by a liquid
chromatography–tandem mass spectrometry (LC-MS/MS) assay using a method previously
described [15]. Validation of the method was performed following FDA guidelines, and
the results met the acceptance criteria [19].
Briefly, samples were analyzed by using an Agilent 1290 Infinity liquid chromatograph
(Agilent Technologies, Palo Alto, CA) equipped with a 1260 Bin Pump VL binary pump
and a HiP-ALS autosampler pump coupled to an AB SCIEX API 4000 mass spectrometer
(AB Sciex, Darmstadt, Germany) operating in electrospray positive ionization mode. Moni-
tored transitions were 350.178 → 106.100 (AMP), 555.1 → 359.9 (CRO) and 454.0 → 285.0
(cefixime, internal standard) m/z. The column and auto-sampler tray temperatures were
set at 40 ◦ C and 4 ◦ C, respectively. Separation was performed on a Phenomenex Luna C18
analytical column (5 mcm, 150 × 2.0 mm) with isocratic elution (70/30). The mobile phase
consisted of 10 mM ammonium acetate and 1% formic acid in Milli-Q water (phase A) and
0.1% formic acid in acetonitrile (phase B). Ten microliters were injected into the column
and both drugs were eluted at a flow rate set at 0.5 mL/min.
Chemical stability. Drug stability was calculated as the percentage (P) of the initial
drug remaining in the elastomeric device at each analyzed time point (Ct), in relation to the
baseline concentration (C0) (P = Ct/C0 × 100). Stability was met at each time point if the
mean of the triplicate assays contained ≥ 90% of its respective baseline concentration [16].
Data are expressed as means and their 90% confidence intervals (CIs).
Physical stability. Precipitation, transparency and color of each elastomeric pump
were visually assessed at each sampling time point. For the evaluation of the appearance of
the solutions, the data were evaluated based on “no significant change” from the initial
time point solution to the hold solutions.

3. Results
Chemical stability. Percentages and 90% CIs of the remaining concentrations that
were observed at each analytic time point during 2 days for each storage condition are
listed in Table 1.
Antibiotics 2023, 12, 432 4 of 10

Table 1. Stability at 8 ± 2 ◦ C, 25 ± 2 ◦ C, 30 ± 2 ◦ C and 37 ± 2 ◦ C.

Concentration Remaining (90% CI)

Temperature Antibiotic
12 h 20 h 24 h 36 h 48 h
102.50 97.32 100.32 96.46 96.23
AMP
(106.62–98.37) (103.96–90.68) (105.30–95.35) (100.12–92.79) (99.07–93.40)
8 ± 2 ◦C
107.27 93.88 96.19 101.52 100.17
CRO
(112.00–102.53) (97.44–90.32) (99.81–92.57) (106.89–96.14) (106.13–94.21)
93.96 95.67 93.17 88.52 84.81
AMP
(97.82–90.09) (100.26–91.08) (95.15–91.19) (90.42–86.62) (88.03–81.59)
25 ± 2 ◦ C
99.46 94.11 94.31 95.59 98.28
CRO
(102.39–96.52) (97.26–90.97) (96.05–92.56) (99.65–91.53) (104.49–92.06)
98.75 92.44 95.59 87.89 74.96
AMP
(102.91–94.58) (94.87–90.01) (99.86–91.33) (92.01–83.76) (75.33–74.58)
30 ± 2 ◦ C
97.41 102.14 97.16 91.97 87.36
CRO
(104.18–90.64) (109.35–94.93) (102.19–92.14) (93.79–90.16) (91.06–83.65)
82.49 77.05 73.66 64.16 62.35
AMP
(85.40–79.59) (80.41–73.69) (77.41–69.92) (67.70–60.62) (65.28–59.41)
37 ± 2 ◦ C
80.72 81.14 85.07 73.11 77.78
CRO
(85.55–75.90) (85.73–76.56) (89.60–80.54) (76.11–70.11) (81.47–74.09)

At refrigerated temperature (8 ± 2 ◦ C), AMP and CRO attained the stability criterion
of >90% of the original concentration for the whole experiment. On the contrary, at
25 ± 2 ◦ C, ampicillin attained the stability criterion of >90% until 24 h of storage. At the
same temperature, CRO remained stable after 48 h of storage in the elastomeric infusion
device. At 30 ± 2 ◦ C, AMP was stable for 24 h and CRO attained the stability criterion of
>90% of the original concentration for 36 h. Before 12 h at 37 ± 2 ◦ C, both concentrations of
Antibiotics 2023, 12, x FOR PEER REVIEW
AMP and CRO dropped below 90%. 6 of 11
AMP and CRO solution stability data are depicted in Figures 1 and 2, respectively.

Figure 1. AMP stability expressed as mean percentage of original concentration over time at 8 °C ±
Figure 1. AMP stability expressed as mean percentage of original concentration over time at 8
2 °C, 25 °C ± 2 °C, 30 °C ± 2 °C and 37 °C ± 2 °C. Dashed line indicates 90% of residual ratio.
± 2 ◦ C,
25 ± 2 ◦ C, 30 ± 2 ◦C and 37 ± 2 ◦ C. Dashed line indicates 90% of residual ratio.
Antibiotics 2023, 12, 432 5 of 10
Figure 1. AMP stability expressed as mean percentage of original concentration over time at 8 °C ±
2 °C, 25 °C ± 2 °C, 30 °C ± 2 °C and 37 °C ± 2 °C. Dashed line indicates 90% of residual ratio.

Figure 2. CRO
Figure stability
2. CRO expressed
stability as mean
expressed percentage
as mean of original
percentage concentration
of original over
concentration time
over at at
time 8 °C
8 ±± 2 ◦ C,
2 °C, 25 °C ±◦ 2 °C, 30 °C◦ ± 2 °C and 37◦ °C ± 2 °C. Dashed line indicates 90% of residual ratio.
25 ± 2 C, 30 ± 2 C and 37 ± 2 C. Dashed line indicates 90% of residual ratio.

Physical stability.
Physical There
stability. werewere
There no observed changes
no observed in clarity
changes or color
in clarity and no
or color andvisible
no visible
precipitation appeared in any sample during the whole experiment (48 h).
precipitation appeared in any sample during the whole experiment (48 h).

4. Discussion
4. Discussion
The The
present study
present has has
study evidenced that that
evidenced AC combined
AC combinedin elastomeric devices
in elastomeric couldcould
devices be be
included in OPAT programs, although the external temperature needs to be controlled:
included in OPAT programs, although the external temperature needs to be controlled: in in
◦
warmer environments, where 37 C can be reached, the combination of both drugs should
not be used, since its concentration drops below 90% in the first 12 h after its preparation.
Nonetheless, temperatures between 25 ◦ C and 30 ◦ C are more common in elastomeric
infusion devices, and we have demonstrated the stability of AC at both conditions until
24 h of storage [20]. Additionally, both drugs attained the stability criterion of >90% of the
original concentration for 48 h at refrigerated temperatures.
Currently, AMP 2 g every 4 h combined with CRO 2 g every 12 h is one of the recom-
mended therapies for the treatment of E. faecalis infective endocarditis [2,3]. This double
beta-lactam combination has been supported by preclinical data proving its synergistic
activity and clinically good results in many cohorts [21–23]. However, the administration
of this regimen via OPAT is complex due to the requirement of two venous accesses and
the need for the twice-daily administration of CRO, since its delivery in a single daily
dose of 4 g has been associated with insufficient drug concentrations throughout the entire
administration interval, with a greater number of relapses reported in a clinical cohort [24].
For decades, AMP plus gentamycin was the first election, even though it was set aside due
to the high rate of toxicity, especially in frail elderly patients [25,26]. Different alternatives
for the treatment of E. faecalis infective endocarditis suitable in the outpatient setting have
been proposed, such as teicoplanin or dalbavancin through OPAT [27,28]. In the first case,
the main advantage is its long elimination half-life, which permits a once-daily dose, and
it is not associated with renal toxicity [27]. Regarding dalbavancin, it is approved in the
USA and Europe to treat adults with skin infections caused by several Gram-positive cocci
isolates, including vancomycin-susceptible E. faecalis. One of the most important benefits
of dalbavancin is its exceptional pharmacokinetic profile, which allows its administration
Antibiotics 2023, 12, 432 6 of 10

every several days. However, the available evidence for the treatment of E. faecalis infective
endocarditis in both cases is still limited, so further studies are needed [28]. In the last
few years, new alternatives such as daptomycin or oral treatment have emerged [29,30].
Daptomycin is a cyclic lipopeptide antibiotic with highly concentration-dependent bacteri-
cidal activity against Gram-positive bacteria and has been used in the setting of resistant
enterococci. However, the conclusions obtained in the studies that assessed this therapeutic
approach in E. faecalis infective endocarditis were radically different, so more investigations
are needed before clear recommendations can be made [29]. With regard to oral antibiotic
treatment, it seems attractive since it avoids the need for intravenous catheters. Moreover,
it shows normal gastrointestinal uptake and good adherence. Amoxicillin-based regimens
at high doses and linezolid are the two oral therapies that have been explored, but, in the
two cases, further studies are needed to safely decide which patients could complete their
antibiotic treatment using oral alternatives [30].
To our knowledge, the stability of AC combined in elastomeric devices has not been
previously studied. Nevertheless, several articles have reported data about the stability of
both antibiotics separately in elastomeric pumps [31–34]. Regarding AMP, the conditions
for each measurement were different in terms of the elastomer composition (latex, silicon or
polyisoprene), concentration (20, 30 or 50 g/L) and diluent (0.9% sodium chloride, sterile
water or acetate ringer solution) [31–33]. Therefore, the reported results are considerably
different: at 20 g/L using 0.9% sodium chloride as a diluent and latex as the material
of the elastomeric reservoir, AMP was chemically stable for 3 days when it was stored
refrigerated for 8 h at 25 ◦ C [31]. When the concentration was 30 g/L using a silicon-
based elastomeric system, AMP preserved > 90% of the initial concentration after 3 days
stored at 25 ◦ C in 0.9% sodium chloride and 2 days in sterile water [32]. However, it was
chemically stable for 7 days using both diluents at refrigerated temperatures. At the highest
concentration, 50 g/L, diluted in acetate ringer solution and contained in a polyisoprene
reservoir, AMP was chemically unstable at 25 ◦ C and 31.1 ◦ C, probably due to its high
concentration [33]. In relation to CRO’s chemical stability in elastomeric pumps, two
studies have been carried out [31,34]. The first one found it to be stable at 20 g/L for
10 days at refrigerated temperatures and 3 days at 25 ◦ C in a latex reservoir using 0.9%
sodium chloride or 5% dextrose as diluents [31]. Similarly, at a concentration of 5 g/L and
40 g/L diluted in 0.9% sodium chloride or 5% dextrose solution and contained in silicon,
the stability was 14 days at 4 ◦ C and 2 days at room temperature [34]. The stability of
CRO in elastomeric devices at temperatures above 30 ◦ C had not been assessed prior to
this study.
Thus, our data provide sufficient evidence to allow the administration of AC combined
through an elastomeric pump when the temperature is under 30 ◦ C for 24 h. Only two
previous investigations studied the stability of AC combined, although both the methodol-
ogy and the results were remarkably different from our study [15,35]. In the first case, it
was a simulation of Y-site administration using glass test tubes over 4 h, with uncertain
results [33]. Regarding the other investigation, it was carried out by our research group but
using a different infusion device, polypropylene infusion bags, and it was widely demon-
strated that the composition of the delivery devices, as well as the antibiotic concentration
and temperature, were significant modifiers of the stability of drugs [15,16]. Consequently,
this study shows the stability of AC combined in elastomeric infusers at the usual condi-
tions of OPAT programs, providing essential information to enhance its use. Given the fact
that our results corroborate the safety of AC combined in terms of drug stability for 48 h at
refrigerated temperatures and for 24 h at 25 ◦ C and 30 ◦ C, the mixture can be administered
through continuous infusion at home. Therefore, patients who are clinically stable can be
discharged and complete their treatment at home using elastomeric pumps. In addition, as
the elastomeric devices need to be replaced only once a day by a nurse or by the patient
himself, it avoids multiple daily interventions, so there is a significant improvement in the
quality of life of the patient [36]. Moreover, it is established that the pharmacodynamic
parameter best related to AMP activity, as with the rest of the beta-lactam antibiotics, is the
Antibiotics 2023, 12, 432 7 of 10

time above the MIC (T > MIC), so prolonged plasma concentrations are necessary for an
optimal treatment. AMP is poorly bound to plasma protein (10%) and has a half-life of 2 to
4 h, and this is the reason that it usually requires multiple and frequent administrations,
which can be avoided by using continuous infusion [37]. Furthermore, considering the re-
frigerated stability proven in this study, 48 h, it seems reasonable to prepare simultaneously
two elastomers, one for immediate use at a maximum temperature of 30 ◦ C, and the other
to be stored refrigerated for 24 h until usage. This strategy would improve OPAT resource
management, since it could reduce costs associated with nursing visits and pharmacy drug
preparation to every two days. Nonetheless, before general recommendations are made,
further studies are required to prove the stability of both antibiotics after sequential storage
at two temperatures—for example, 24 h at refrigerated temperatures, followed by another
24 h at 25 ◦ C or 30 ◦ C.
In order to further improve the quality of life of patients included in OPAT programs,
the utilization of elastomeric pumps is growing. However, it has been demonstrated that
the temperature reached in these devices may increase much higher than 25 ◦ C due to
direct exposure to sunlight [38,39]. Additionally, when the pump stays in direct contact
with the patient’s body—for example, around the waist—and is kept under a blanket
during nighttime, the temperature may rise up to 32 ◦ C. Moreover, in warmer climates,
the solution temperature might occasionally reach temperatures around 38 ◦ C [40]. In this
scenario, the National Health System (NHS) in the UK established a document named the
“Standard Protocol for Deriving and Assessment of Stability”, commonly known as the
Yellow-Covered Document (YCD), that describes the minimum data required from a study
to be applicable for use in the OPAT context [41]. In this document, warm temperature
data are considered essential. In the same way, a recent systematic review concluded that
there is an urgent need for studies that verify the stability and appropriate use of many
antibiotics used in OPAT at standard room temperature and in warmer climates [42].
Among the strengths that can be found in our study, it is remarkable that we included
a range of temperatures similar to those achievable due to real-life temperature variations
at home. Regarding the technique employed, we used HPLC coupled to tandem mass spec-
trometry (MS/MS), which, compared to other detectors, such as ultraviolet, is more robust,
sensitive and specific [43]. Lastly, the composition of the elastomeric devices that we tested
in our study included polyisoprene, the most commonly material used in the elastomeric
pumps applied in OPAT programs [44]. Our study has also some limitations: although 90%
chemical stability has been established as defined in the European and US Pharmacopoeias,
recently, a 95–105% limit has been proposed [19,41]. Secondly, pH variations were not mea-
sured. However, since pH changes are related to AMP degradation and its concentrations
were measured using LC-MS/MS, we did not consider it necessary [45,46]. In the third
place, the effect of additives on the stability has not been investigated [47,48]. Despite the
fact that each generic or name-brand drug may not contain the same additives, the number
of brands available in the market made it unfeasible to test them all. Impurities or others
degradation products were not measured either.

5. Conclusions
In summary, the importance of the present study resides in the possibility of including
AC in OPAT programs using elastomeric infusion devices for the treatment of E. faecalis
infections. This drug combination is stable for 48 h when it is kept at 8 ± 2 ◦ C and it
can be used for 24 h if the temperature reached is 25 ± 2 ◦ C or 30 ± 2 ◦ C. However, at
37 ± 2 ◦ C, the mixture should not be used, since it becomes unstable in the first 12 h after
its preparation.

Author Contributions: B.F.-R. wrote the manuscript and conducted the experiments. B.F.-R. and
L.H.-H. analyzed the data. L.H.-H., M.V.G.-N. and A.G.-V. supervised the project. L.H.-H., M.V.G.-N.,
A.G.-V., R.L.-M., A.d.A., L.E.L.-C., S.L.-P., A.F.-P. and J.M.G.-U. reviewed and contributed to the final
manuscript. All authors have read and agreed to the published version of the manuscript.
Antibiotics 2023, 12, 432 8 of 10

Funding: This work was supported by the Sociedad Española de Farmacia Hospitalaria and the
AFinf Working Group for the project “Stability study of antimicrobials under conditions analogous to
the outpatient parenteral antibiotic therapy program (OPAT)”. A.G.-V. was supported by the Instituto
de Salud Carlos III, co-financed by the European Development Regional Fund (“A way to achieve
Europe”), Subprograma Miguel Servet (grant CP19/00159). L.H.-H. was supported by the Instituto
de Salud Carlos III, co-financed by the European Development Regional Fund (“A way to achieve
Europe”), Subprograma Juan Rodés (grant JR22/00049).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank Leventon for their kind supply of the dosi fuser elas-
tomeric devices.
Conflicts of Interest: L.E.L.-C. has served as a scientific advisor for Angelini, a speaker for Angelini,
ViiV, Gilead, and Correvio, and as a trainer for ViiV. A.d.A. has served as a scientific advisor for
Angellini, Novartis, Roche, and Cook Medical, a speaker for MSD, Pfizer, Angellini, Novartis, Roche,
and ViiV, and as a trainer for MSD and Cook Medical. The remaining authors have no conflict of
interest to declare.

References
1. Beganovic, M.; Luther, M.K.; Rice, L.B.; Arias, C.A.; Rybak, M.J.; LaPlante, K.L. A Review of Combination Antimicrobial Therapy
for Enterococcus Faecalis Bloodstream Infections and Infective Endocarditis. Clin. Infect. Dis. 2018, 67, 303–309. [CrossRef]
2. Habib, G.; Lancellotti, P.; Antunes, M.J.; Bongiorni, M.G.; Casalta, J.-P.; Del Zotti, F.; Dulgheru, R.; El Khoury, G.; Erba, P.A.;
Iung, B.; et al. 2015 ESC Guidelines for the Management of Infective Endocarditis: The Task Force for the Management of Infective
Endocarditis of the European Society of Cardiology (ESC). Endorsed by: European Association for Cardio-Thoracic Surgery
(EACTS), the European Association of Nuclear Medicine (EANM). Eur. Heart J. 2015, 36, 3075–3128.
3. Baddour, L.M.; Wilson, W.R.; Bayer, A.S.; Fowler, V.G.; Tleyjeh, I.M.; Rybak, M.J.; Barsic, B.; Lockhart, P.B.; Gewitz, M.H.;
Levison, M.E.; et al. Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications:
A Scientific Statement for Healthcare Professionals From the American Heart Association. Circulation 2015, 132, 1435–1486.
[CrossRef]
4. Herrera-Hidalgo, L.; de Alarcón, A.; López-Cortes, L.E.; Luque-Márquez, R.; López-Cortes, L.F.; Gutiérrez-Valencia, A.; Gil-
Navarro, M.V. Is Once-Daily High-Dose Ceftriaxone plus Ampicillin an Alternative for Enterococcus Faecalis Infective Endocardi-
tis in Outpatient Parenteral Antibiotic Therapy Programs? Antimicrob. Agents Chemother. 2020, 65, e02099-20. [CrossRef]
5. Pericàs, J.M.; Llopis, J.; González-Ramallo, V.; Goenaga, M.Á.; Muñoz, P.; García-Leoni, M.E.; Fariñas, M.C.; Pajarón, M.;
Ambrosioni, J.; Luque, R.; et al. Outpatient Parenteral Antibiotic Treatment for Infective Endocarditis: A Prospective Cohort
Study From the GAMES Cohort. Clin. Infect. Dis. 2019, 69, 1690–1700. [CrossRef]
6. López-Cortés, L.E.; Luque, R.; Cisneros, J.M.; DOMUS Outpatient Antimicrobial Therapy Group. Next Step, Outpatient
Antimicrobial Therapy Programs as a Tool of Stewardship Programs. Clin. Infect. Dis. 2019, 68, 2155. [CrossRef]
7. Mitchell, E.D.; Czoski Murray, C.; Meads, D.; Minton, J.; Wright, J.; Twiddy, M. Clinical and Cost-Effectiveness, Safety and
Acceptability of Community Intravenous Antibiotic Service Models: CIVAS Systematic Review. BMJ Open 2017, 7, e013560.
[CrossRef]
8. Hamad, Y.; Dodda, S.; Frank, A.; Beggs, J.; Sleckman, C.; Kleinschmidt, G.; Lane, M.A.; Burnett, Y. Perspectives of Patients on
Outpatient Parenteral Antimicrobial Therapy: Experiences and Adherence. Open Forum. Infect. Dis. 2020, 7, ofaa205. [CrossRef]
9. Bugeja, S.J.; Stewart, D.; Vosper, H. Clinical Benefits and Costs of an Outpatient Parenteral Antimicrobial Therapy Service. Res.
Soc. Adm. Pharm. 2021, 17, 1758–1763. [CrossRef]
10. Diamantis, S.; Longuet, P.; Lesprit, P.; Gauzit, R. Terms of Use of Outpatient Parenteral Antibiotic Therapy. Infect. Dis. Now 2021,
51, 14–38. [CrossRef]
11. Skryabina, E.A.; Dunn, T.S. Disposable Infusion Pumps. Am. J. Health-Syst. Pharm. 2006, 63, 1260–1268. [CrossRef] [PubMed]
12. Diamantis, S.; Dawudi, Y.; Cassard, B.; Longuet, P.; Lesprit, P.; Gauzit, R. Home Intravenous Antibiotherapy and the Proper Use
of Elastomeric Pumps: Systematic Review of the Literature and Proposals for Improved Use. Infect. Dis. Now 2021, 51, 39–49.
[CrossRef] [PubMed]
13. Fernández-Rubio, B.; Del Valle-Moreno, P.; Herrera-Hidalgo, L.; Gutiérrez-Valencia, A.; Luque-Márquez, R.; López-Cortés, L.E.;
Gutiérrez-Urbón, J.M.; Luque-Pardos, S.; Fernández-Polo, A.; Gil-Navarro, M.V. Stability of Antimicrobials in Elastomeric Pumps:
A Systematic Review. Antibiotics 2021, 11, 45. [CrossRef] [PubMed]
14. Gil-Navarro, M.V.; Lopez-Cortes, L.E.; Luque-Marquez, R.; Galvez-Acebal, J.; de Alarcon-Gonzalez, A. Outpatient Parenteral
Antimicrobial Therapy in Enterococcus Faecalis Infective Endocarditis. J. Clin. Pharm. Ther. 2018, 43, 220–223. [CrossRef]
[PubMed]
Antibiotics 2023, 12, 432 9 of 10

15. Herrera-Hidalgo, L.; López-Cortes, L.E.; Luque-Márquez, R.; Gálvez-Acebal, J.; de Alarcón, A.; López-Cortes, L.F.; Gutiérrez-
Valencia, A.; Gil-Navarro, M.V. Ampicillin and Ceftriaxone Solution Stability at Different Temperatures in Outpatient Parenteral
Antimicrobial Therapy. Antimicrob. Agents Chemother. 2020, 64, e00309-20. [CrossRef] [PubMed]
16. Raby, E.; Naicker, S.; Sime, F.B.; Manning, L.; Wallis, S.C.; Pandey, S.; Roberts, J.A. Ceftolozane-Tazobactam in an Elastomeric
Infusion Device for Ambulatory Care: An in Vitro Stability Study. Eur. J. Hosp Pharm. 2020, 27, e84–e86. [CrossRef] [PubMed]
17. Euba, G.; Lora-Tamayo, J.; Murillo, O.; Pedrero, S.; Cabo, J.; Verdaguer, R.; Ariza, J. Pilot Study of Ampicillin-Ceftriaxone
Combination for Treatment of Orthopedic Infections Due to Enterococcus Faecalis. Antimicrob. Agents Chemother. 2009, 53,
4305–4310. [CrossRef]
18. Lepe, J.A.; Rodríguez-Villodres, A.; Martín-Gutiérrez, G.; Luque, R.; Aznar, J. In Vitro Study of Synergy of Ampicillin with
Ceftriaxone against Listeria Monocytogenes. Rev. Esp. Quimioter. 2019, 32, 465–468.
19. U.S. Department of Health and Human Services Food and Drug Administration. Guidance for Industry: Drug Stability Guidelines;
U.S. Department of Health and Human Services Food and Drug Administration: Silver Spring, MD, USA, 2008.
20. Docherty, T.; Montalto, M.; Leslie, J.; King, K.; Niblett, S.; Garrett, T. Temperature Profiles of Antibiotic-Containing Elastomeric
Infusion Devices Used by Ambulatory Care Patients. Am. J. Health-Syst. Pharm. 2017, 74, 992–1001. [CrossRef]
21. Gavaldà, J.; Torres, C.; Tenorio, C.; López, P.; Zaragoza, M.; Capdevila, J.A.; Almirante, B.; Ruiz, F.; Borrell, N.; Gomis, X.; et al.
Efficacy of ampicillin combined with ceftriaxone and gentamicin in the treatment of experimental endocarditis due to Enterococcus
faecalis with no high-level resistance to aminoglycosides. J. Antimicrob. Chemother. 2003, 52, 514–517. [CrossRef]
22. Gavaldà, J.; Torres, C.; Tenorio, C.; López, P.; Zaragoza, M.; Capdevila, J.A.; Almirante, B.; Ruiz, F.; Borrell, N.; Gomis, X.; et al.
Efficacy of ampicillin plus ceftriaxone in treatment of experimental endocarditis due to Enterococcus faecalis strains highly resistant
to aminoglycosides. Antimicrob. Agents Chemother. 1999, 43, 639–646. [CrossRef]
23. Pericàs, J.M.; Llopis, J.; Muñoz, P.; Gálvez-Acebal, J.; Kestler, M.; Valerio, M.; Hernández-Meneses, M.; Goenaga, M.Á.; Cobo-
Belaustegui, M.; Montejo, M.; et al. A Contemp. Pict. Enterococcal Endocarditis. J. Am. Coll. Cardiol. 2020, 75, 482–494.
[CrossRef]
24. Herrera-Hidalgo, L.; Lomas-Cabezas, J.M.; López-Cortés, L.E.; Luque-Márquez, R.; López-Cortés, L.F.; Martínez-Marcos, F.J.; de
la Torre-Lima, J.; Plata-Ciézar, A.; Hidalgo-Tenorio, C.; García-López, M.V.; et al. Ampicillin Plus Ceftriaxone Combined Therapy
for Enterococcus Faecalis Infective Endocarditis in OPAT. J. Clin. Med. 2021, 11, 7. [CrossRef]
25. Fernández-Hidalgo, N.; Almirante, B.; Gavaldà, J.; Gurgui, M.; Peña, C.; de Alarcón, A.; Ruiz, J.; Vilacosta, I.; Montejo, M.;
Vallejo, N.; et al. Ampicillin plus Ceftriaxone Is as Effective as Ampicillin plus Gentamicin for Treating Enterococcus Faecalis
Infective Endocarditis. Clin. Infect. Dis. 2013, 56, 1261–1268. [CrossRef]
26. Grau, S.; De Antonio-Cuscó, M.; Ortonobes-Roig, S.; Los-Arcos, I.; Jiménez, M.J.; Soy, D. Personalized Antimicrobial Therapy in
Critical and Elderly Patients. Farm. Hosp. 2021, 45, 64–76.
27. Escolà-Vergé, L.; Fernández-Hidalgo, N.; Rodríguez-Pardo, D.; Pigrau, C.; González-López, J.J.; Bartolomé, R.; Almirante, B.
Teicoplanin for Treating Enterococcal Infective Endocarditis: A Retrospective Observational Study from a Referral Centre in
Spain. Int. J. Antimicrob. Agents 2019, 53, 165–170. [CrossRef]
28. Hidalgo-Tenorio, C.; Vinuesa, D.; Plata, A.; Martin Dávila, P.; Iftimie, S.; Sequera, S.; Loeches, B.; Lopez-Cortés, L.E.; Fariñas,
M.C.; Fernández-Roldan, C.; et al. DALBACEN Cohort: Dalbavancin as Consolidation Therapy in Patients with Endocarditis
and/or Bloodstream Infection Produced by Gram-Positive Cocci. Ann. Clin. Microbiol. Antimicrob. 2019, 18, 30. [CrossRef]
29. Herrera-Hidalgo, L.; de Alarcón, A.; López-Cortes, L.E.; Luque-Márquez, R.; López-Cortes, L.F.; Gutiérrez-Valencia, A.; Gil-
Navarro, M.V. Enterococcus Faecalis Endocarditis and Outpatient Treatment: A Systematic Review of Current Alternatives.
Antibiotics 2020, 9, 657. [CrossRef]
30. Iversen, K.; Ihlemann, N.; Gill, S.U.; Madsen, T.; Elming, H.; Jensen, K.T.; Bruun, N.E.; Høfsten, D.E.; Fursted, K.; Christensen, J.J.; et al.
Partial Oral versus Intravenous Antibiotic Treatment of Endocarditis. N. Engl. J. Med. 2019, 380, 415–424. [CrossRef]
31. Allen, L.V.; Stiles, M.L.; Prince, S.J.; Smeeding, J. Stability of 14 Drugs in the Latex Reservoir of an Elastomeric Infusion Device.
Am. J. Health Syst. Pharm. 1996, 53, 2740–2743. [CrossRef]
32. Kang, M.A.; Kang, J.-S. Stability Test of Ampicillin Sodium Solutions in the Accufuser&#174; Elastomeric Infusion Device Using
HPLC: UV Method. Open Pharmacol. J. 2012, 3, 462–467.
33. Nakamura, T.; Enoki, Y.; Uno, S.; Uwamino, Y.; Iketani, O.; Hasegawa, N.; Matsumoto, K. Stability of Benzylpenicillin Potassium
and Ampicillin in an Elastomeric Infusion Pump. J. Infect. Chemother. 2018, 24, 856–859. [CrossRef]
34. Walker, S.E.; Iazzetta, J.; Law, S.; Biniecki, K. Stability of Commonly Used Antibiotic Solutions in an Elastomeric Infusion Device.
Can. J. Hosp. Pharm. 2010, 63, 212–224. [CrossRef] [PubMed]
35. Trissel, L. Compatibility of ampicillin sodium with common paren- teral medications. In Compatibility Data; TriPharma: Kennesaw,
GA, USA, 2007.
36. Saillen, L.; Arensdorff, L.; Moulin, E.; Voumard, R.; Cochet, C.; Boillat-Blanco, N.; Gardiol, C.; de Vallière, S. Patient Satisfaction in
an Outpatient Parenteral Antimicrobial Therapy (OPAT) Unit Practising Predominantly Self-Administration of Antibiotics with
Elastomeric Pumps. Eur. J. Clin. Microbiol. Infect. Dis. 2017, 36, 1387–1392. [CrossRef] [PubMed]
37. Gatti, M.; Tedeschi, S.; Trapani, F.; Ramirez, S.; Mancini, R.; Giannella, M.; Viale, P.; Pea, F. A Proof of Concept of the Usefulness of
a TDM-Guided Strategy for Optimizing Pharmacokinetic/Pharmacodynamic Target of Continuous Infusion Ampicillin-Based
Regimens in a Case Series of Patients with Enterococcal Bloodstream Infections and/or Endocarditis. Antibiotics 2022, 11, 1037.
[CrossRef]
Antibiotics 2023, 12, 432 10 of 10

38. Voumard, R.; Van Neyghem, N.; Cochet, C.; Gardiol, C.; Decosterd, L.; Buclin, T.; de Valliere, S. Antibiotic Stability Related
to Temperature Variations in Elastomeric Pumps Used for Outpatient Parenteral Antimicrobial Therapy (OPAT). J. Antimicrob.
Chemother. 2017, 72, 1462–1465. [CrossRef]
39. Keller, S.C.; Cosgrove, S.E.; Kohut, M.; Krosche, A.; Chang, H.-E.; Williams, D.; Gurses, A.P. Hazards from Physical Attributes of
the Home Environment among Patients on Outpatient Parenteral Antimicrobial Therapy. Am. J. Infect. Control 2019, 47, 425–430.
[CrossRef]
40. Perks, S.J.; Robinson, N.; Pain, T.; Franklin, R. Extended Duration Infusion Temperatures in the Tropics: 2 (EDITT2). J. Pharm.
Pract. Res. 2018, 48, 423–430. [CrossRef]
41. NHS Pharmaceutical Quality Assurance Committee. Standard Protocol for Deriving and Assessment of Stability, Part 1: Aseptic
Preparations (Small Molecules), 5th ed, ©NHS Pharmaceutical Quality Assurance Committee, UK, 2019. Available online:
https://www.sps.nhs.uk/wp-content/uploads/2013/12/Stability-part-1-small-molecules-5th-Ed-Sept-19.pdf (accessed on 20
February 2023).
42. Perks, S.J.; Lanskey, C.; Robinson, N.; Pain, T.; Franklin, R. Systematic Review of Stability Data Pertaining to Selected Antibiotics
Used for Extended Infusions in Outpatient Parenteral Antimicrobial Therapy (OPAT) at Standard Room Temperature and in
Warmer Climates. Eur. J. Hosp. Pharm. 2020, 27, 65–72. [CrossRef]
43. Adaway, J.E.; Keevil, B.G. Therapeutic Drug Monitoring and LC–MS/MS. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012,
883–884, 33–49. [CrossRef]
44. Patel, R.P.; Jacob, J.; Sedeeq, M.; Ming, L.C.; Wanandy, T.; Zaidi, S.T.R.; Peterson, G.M. Stability of Cefazolin in Polyisoprene
Elastomeric Infusion Devices. Clin. Ther. 2018, 40, 664–667. [CrossRef] [PubMed]
45. Allwood, M.C.; Brown, P.W. Stability of Ampicillin Infusions in Unbuffered and Buffered Saline. Int. J. Pharm. 1993, 97, 219–222.
[CrossRef]
46. Raffanti, E.F.; King, J.C. Effect of p H on the Stability of Sodium Ampicillin Solutions. Am. J. Health-Syst. Pharm. 1974, 31, 745–751.
[CrossRef]
47. Tange, M.; Yoshida, M.; Nakai, Y.; Uchida, T. Comparison between Original and Generic Versions of Ceftriaxone Sodium
Preparation for Injection: Compatibility with Calcium-Containing Product. Chem. Pharm. Bull 2012, 60, 429–434. [CrossRef]
48. Maher, M.; Jensen, K.J.; Lee, D.; Nix, D.E. Stability of Ampicillin in Normal Saline and Buffered Normal Saline. Int. J. Pharm.
Compd. 2016, 20, 338–342.

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.