Pharmacology & Pharmacy, 2012, 3, 462-467
http://dx.doi.org/10.4236/pp.2012.34063 Published Online October 2012 (http://www.SciRP.org/journal/pp) 1
Stability Test of Ampicillin Sodium Solutions in the
Accufuser® Elastomeric Infusion Device Using HPLC-UV
Method
Min-A. Kang1, Ju-Seop Kang2
1Department of Nursing, College of Nursing, Yonsei University, Seoul, South Korea; 2Department of Pharmacology & Clinical
Pharmacology Lab, College of Medicine and Division of Molecular Therapeutics Development, Hanyang Biomedical Research In-
stitute, Hanyang University, Seoul, South Korea.
Received May 30th, 2012; revised July 8th, 2012; accepted July 18th, 2012
ABSTRACT
The stabilities of two kinds of solutions (30 mg/mL) of Ampicillin sodium in 0.9% NaCl in water (NS, normal saline)
and in sterile water (SW) in the intravenous elastomeric infusion device (Accufuser®) were evaluated based on recom-
mended solu tions and storage periods. The injectable NS- and SW-Ampicillin so lutions in the Accufuser® device were
stored and evaluated at controlled temperature (room temperature, 25˚C ± 2˚C and cold temperature, 4˚C ± 2˚C) during
7 days. Effects of the periods of storage (from 0 to 7 days) and the temperatures of storage (RT and CT) on the physico-
chemical appearances and concentrations of active compounds were determined. The visual clarity, pH, and concentra-
tions of Ampicillin were determined by stability-indicating high-performance liquid chromatography (HPLC)-ultravio-
let (UV) detection. The results showed that the amount of Ampicillin in studied solutions gradually decreased with time.
The Ampicillin in NS, which was stored in CT, was relatively stable, retaining 94 % of its origin al amount up to 7 days.
The solution that showed least stability was Ampicillin in SW, which was stored in RT, retaining 80% of its original
amount. Generally, solutions that were stored in CT were more stable than the solutions that were stored in RT. No sig-
nificant changes in physical appearance or color of the solutions were observed during the study. Particles were not de-
tected in any solution samples. In summary, two kinds of solutions of Ampicillin sodium, in NS and SW, showed dif-
ferent chemical stabilities with time in intravenous infusion device without any significant physical changes and re-
tained about 94% vs 89% and 83% vs 80% of in itial con c entra tions after 7 days in CT and RT, respectiv ely. We suggest
that 30 mg/mL of Ampicillin sodium in NS solution in an Accufuser ® infusion device which is stored in CT can be ap-
plicable for 7 days in clinical situations.
Keywords: Ampicillin Sodium; Intravenous Elastomeric Infusion Device (Accufuser®); HPLC-UV Method; Solution
Stability
1. Introduction
The disposable silicon balloon infusion device (Ac-
cufuser®, Woo Young Medical Co. LTD., Seoul, South
Korea) is a well-established simplified silicon-based
elastomeric system for the administration of antibiotics
and other drugs or nutrients that are suitable for patients
as well as healthcare providers. An increasing number of
patients are being treated as outpatients, and for them,
drugs are often infused using portable pumps or infusion
devices outside the hospital. To be suitable for self-ad-
ministration by home-based patient, the an tibiotic should
be stable in the peritoneal dialysis solution for a number
of days under home storage conditions [1]. Therefore, it is
necessary to determine the physical and che mical stabili-
ties of the admixtures in the infusion system before they
appear in the clinical settings [2-8]. The purpose of this
study is to p rovide such information with commonly u sed
antibiotic solutions, Ampicillin sodium, into Accufuser®
elastomeric infusion device under recommended storage
conditions. The physical and chemical stabilities of Am-
picillin sodium (Figure 1) solutions (30 mg/mL, normal
Figure 1. Chemical structure of ampicillin sodium
(C16H19N3O4S·Na).

Stability Test of Ampicillin Sodium Solutions in the Accufuser® Elastomeric Infusion Device Using HPLC-UV Method 463
saline, NS and sterile water, SW) packed in sterile Ac-
cufuser® device were evaluated, in which each samples
were stored and evaluated at appropriate intervals up to 7
days under different storage conditions (room tempera-
ture, RT, 25˚C ± 2˚C and cold temperature, CT, 4˚C ±
2˚C). The study was done with Ampicillin sodium solu-
tions which were made with NS and SW since these are
the most available infusion solutions for Ampicillin so-
dium administrations in clinical situations [1,6,9].
2. Materials and Methods
2.1. Materials
Ampicillin Sodium was purchased from Fluka Co., USA.
Normal saline (NS, 0.9% NaCl in water) and sterile wa-
ter (SW, injectable distilled water) were purchased from
Choongwae Pharmaceutical Co., South Korea. Dispos-
able Silicon Balloon Infuser (Accufuser®, Figure 2) was
obtained from Woo Young Medical Co., LTD, South
Korea. Acetonitrile and potassium phosphate were pur-
chased from Sigma-Aldrich Co. (St. Louis, MO, USA)
and other chemicals for HPLC analysis were all HPLC-
grade and were prepared immediately before use. Milli-
pore’s Milli-Q system (MA, USA) was used throughout
the analysis.
2.2. Preparation and Sampling of Solution
To prepare the samples, the appropriate amounts of
Ampicillin sodium were added to the portion of the
infusion solution and were brought to a final volume of
20mL with NS and SW. The test solutions were pack-
aged in sterile Accufuser® system for testing. All mani-
pulations were performed in a biological safety cabinet.
The nominal concentration of Ampicillin sodium solu-
tion for the sample used in this testing was 30 mg/mL.
Triplicate test solutions under each storage conditions
were prepared. The test solutions were stored at RT
(25˚C ± 2˚C) and CT (4˚C ± 2˚C). Aliquots were
removed from each test solution initially and at the
intervals of each day during 7 days at 25˚C ± 2˚C and
4˚C ± 2˚C conditions and the concentrations of Ampicillin
sodium were quantified by the HPLC-UV method (Table
1).
2.3. Methods
The physical stabilities of the Ampicillin infusion solu-
tions were assessed by visual examination and HPLC
analysis. Visual examinations were performed in normal
diffuse fluorescent room light with unaided eye and
high-intensity monodirectional light. The pH of the solu-
tions was measured by a stainless electrode pH meter
Table1. Study designs for stability testing of ampicillin so-
dium solutions (30 mg/mL) in Accufuser® system.
˚C Day
Solutions 0 1 2 3 4 5 67
NS *oo o o o o o o
RT SW o o o o o oo o
NS o o o o o oo o
CT SW o o o o o oo o
*o: processed sample; RT: room temperature; CT: cold temperature; NS:
normal saline; SW: sterile water.
Figure 2. Disposable silicon balloon infusion system (Accufuser®, Woo Young Medical Co., LTD, South Korea).

Stability Test of Ampicillin Sodium Solutions in the Accufuser® Elastomeric Infusion Device Using HPLC-UV Method
464
(Thermo Scientific Co., MA, USA). The drug concentra-
tions were determined using a stability-indicating HPLC
assay method based on several references [10,11]. The
HPLC system [10,12-14] consisted of an isocratic sol-
vent delivery pump (Model 515, Waters Scientific Co.
USA) which pumped a mixture (v/v, 60/40, pH 3.0) of
acetonitrile (ACN, Sigma Co. USA) in 0.05M potassium
phosphate (Sigma Co. USA) through a Capsell Pak C18
UG120 (4.6 × 250 mm, 5 µm) column at 1.0 mL/min.
The ratio of ACN to 0.05 M potassium phosphate (60/40)
was held constant during the chromatographic run. The
samples of 2.0 µL were injected into the HPLC system
using an autosampler (Nanospace SI-2, Shiseido Co.,
Japan). The effluent from the column was monitored
with a variable wavelength ultraviolet detector (Nano-
space SI-1, Shiseido Co., Japan) at 220 nm. The integra-
tion of the chromatograms was performed by dsCHROM®
software (Do-Nam Instrumental Co., Seoul, Korea). The
method was validated for linearity, precision (inter-day
and intra-day), accuracy and selectivity. The standard
plot was constructed for Ampicillin in the range of 0 -
3.33 mg/mL. The experiment was repeated five times on
the same day and additionally on four consecutive days
to determine inter- and intra-day precisions [12]. Assays
of control solutions from Ampicillin sodium solutions
(3mg/mL) were undertaken to calculate th e intra-day and
inter-day variations using external standard method.
Linearity was evaluated by serial dilutions of Ampicillin
sodium solutions with NS and SW for analysis. Linear
regression analysis of peak area and drug concentration
yielded a good correlation coefficient > 0.99 with range
from 0 to 3.33 mg/mL (Figure 3). The stability of Am-
picillin sodium infusion solutions was determined in
disposable infusion device (Accufuser®) during 7 days of
storage under RT and CT conditions [5]. Periodically,
samples were evaluated for appearances, visible particles,
pH and chromatographic analysis. We analyzed the con-
centration of Ampicillin sodium in two solutions at each
day during 7 days after the preparation of solutions by
HPLC-UV method [10,12,13]. On each day, 1.0 mL of
samples of Ampicillin sodium with a no minal concentra-
tion were drawn from Accufuser® infusion device for
chromatographic analysis and 2.0 µL were directly in-
jected into HPLC-UV system. The three aliquots of each
solution were processed. Statistical analysis was per-
formed using one-way ANOVA with the level of sig-
nificance set at 0.05 (PCS, version 4.0, Springer-Verlag,
New York, USA).
3. Results
There were no significant changes in physical appearances,
odors, or clarities of the solutions. The color of the
samples was transparent, with no color ch anges. Particles
were not detected in any samples. The pH of NS and SW
solutions slightly decreased from 9.20 to 8.49 and from
9.47 to 8.72 in the CT, but significantly (p < 0.05) de-
creased from 9.30 to 6.78 and 9.10 to 7.10 in the RT
during 7 days (Table 2). The linearity of calibration curves
of Ampicillin So d ium was es tablished which sho w ed good
linearity over the range concentration of 0 - 3.33 mg/mL
(r2 = 0.999, Figure 3).
Table 3 lists the relative standard deviation (R.S.D.)
data obtained from the analysis of the samples on the
same day (n = 5) and on consecutive days (n = 5). The
R.S.D values were <1.07 % and <0.92 % for intra-day
and inter-day results, respectively, meaning that the
method was sufficiently precise. The typical HPLC chro-
matogram of Ampicillin sodium (200 µg/mL) is shown
in Figure 4.
The retention time for Ampicillin sodium was about
Figure 3. Calibration curve for the determination of am-
picillin sodium concentrations (0 - 3.33 mg/mL) in study
solutions.
Table 2. The change of pH (Mean ± SD, n = 5) of ampicillin sodium solutions according to the time of storage.
˚C Time (day)
Solution 0 1 2 3 4 5 6 7
NS 9.30 ± 0.05 8.71 ± 0.048.52 ± 0.008.19 ± 0.017.81 ± 0.017.73 ± 0.04 7.20 ± 0.03 6.78* ± 0.02
RT
(25˚C ± 2˚C) SW 9.10 ± 0.01 8.65 ± 0.018.50 ± 0.028.14 ± 0.038.10 ± 0.017.90 ± 0.02 7.54 ± 0.01 7.10** ± 0.01
CT NS 9.20 ± 0.03 9.07 ± 0.078.98 ± 0.038.85 ± 0.038.69 ± 0.038.61 ± 0.04 8.53 ± 0. 0 2 8.49 ± 0. 03
(4˚C ± 2˚C) SW 9.47 ± 0.04 9.33 ± 0.049.26 ± 0.038.98 ± 0.018.82 ± 0.018.83 ± 0.01 8.79 ± 0.02 8.72 ± 0.01
RT: room temperature; CT: cold temperature; NS: normal saline; SW: sterile water; *p < 0.05 vs. 0 day and NS (CT); **p < 0.05 vs. 0 day and SW (CT).

Stability Test of Ampicillin Sodium Solutions in the Accufuser® Elastomeric Infusion Device Using HPLC-UV Method 465
Table 3. Intra-day and inter-day precision studies (n = 5).
RT CT RT CT RT
Condition
Validation NS SW NS SW NS
Accuracy (%) 90.86 ± 1.04 91.12 ± 0.32 90.67 ± 0.97 91.15 ± 0.79
Intraday (n = 5) R.S.D. (%) 0.87 0.35 1.07 0.87
Accuracy (%) 93.45 ± 0.86 101.32 ± 0.82 98.34 ± 0.87 92.56 ± 0.58
Interday (n = 5)
R.S.D. (%) 0.92 0.81 0.88 0.63
Figure 4. Typical chromatogram of standard solutions (NS and SW) containing ampicillin sodium of 200 µg/mL, Y- axis
(Absorbance units), X-axis (Retention time, min).
2.27 min. The initial concen tration and the percentage of
the remaining concentration which were observed at
analytic time of each day during 7 days for each Am-
picillin sodium solutions and storage con ditions are listed
in Table 4. The concentration of Ampicillin Sodium
slightly changed. 94 % of initial concentration remained
in NS and 90 % remained in SW under CT and decreased
significantly (p < 0.05) to 83 % in NS and to 80 % in SW
under RT (Table 4).
4. Discussion and Conclusion
In order to verify the effects of mixing other drugs with
antibiotics in a range of intravenous media in hospital
pharmacy settings or home-based patient self-admini-
stration of peritoneal dialysis solution under home
storage conditions, the stability assays are essential for
the detection and measurement of interactions, which are
often unaccompanied by visual changes [1,2]. The expiry
date of IV medications after reconstitution or dilution is
often limited to about 24 hrs because of the potential for
Table 4. The changes of the amount (%) of ampicillin so-
dium in various solutions (NS and SW) according to each
storage temperature (RT and CT) and time (0 - 7 days).
RT (25˚C ± 2˚C) CT (4˚C ± 2˚C)
Condition
Time (day) NS SW NS SW
0 100 100 100 100
1 96 95 100 93
2 93 91 96 94
3 90 89 100 93
4 88 86 98 91
5 86 86 100 95
6 84 82 99 94
7 83 80 94 90
the breaks in sterility. However, when reconstitution and
dilution are carried out in an absolute aseptic environment,
according to USP Chapter 797 recommendations [15],

Stability Test of Ampicillin Sodium Solutions in the Accufuser® Elastomeric Infusion Device Using HPLC-UV Method
466
the expiry dates of many stable drugs can be extended
from 24 hrs to 14 days [15,16]. Extending the expiry date
could reduce the wastage of many drugs [17,18] and
might increase the convenience for ambulatory patient
and home care nurses by eliminating the need for fre-
quent visits to a clinic to obtain additional dose [18] It
could especially be convenient for patient who live in
regional areas where frequent travel to the local tertiary
hospital is difficult [1]. An increasing number of patients
are being treated as outpatients, and m a ny of these patients
receive their medications or intravenous nutrients by in-
fusion using portable pumps or devices [19]. For example,
many patients practice peritoneal dialysis-peritonitis and
intraperitoneal administration of antibiotics. This is su-
perior to other intravenous therapies [20] and allows on-
going outpatient treatment, which shortens hospital ad-
mission which is beneficial to both patients and to health
care system [1]. Therefore, reliable and widely available
stability information of antibiotics in the Accufuser® in-
fusion device is clinically important. This study was un de r-
taken to determine the stability of commonly used Ampi-
cillin sodium which was stored in the device according to
each recommended storage condition. Visual clarity, pH
and concentrations of Ampicillin sodium of the resultant
solutions were examined each day during 7 days under
different temperature conditions. No visible precipitation
or change in color or clarity was observed in both kinds
of Ampicillin sodium solutions (in NS and SW) under
CT and RT conditions during 7 days. When Ampicillin
sodium solutions were prepared under maker’s standar-
dized conditions to achieve concentrations of 10 mg/mL
in NS and SW, the concentrations of Ampicillin sodium
in NS and SW solutions which were stored in an Ac-
cufuser® device decreased to 83% and 80% during 7 days
in RT conditions, resp ectively. On the other hand, Ampi-
cillin sodium solutions that were made according to the
same conditions and stored in an Accufuser® device
decreased to 94% and 90% during 7 days in CT con-
ditions, respectively. Therefore, the Ampicillin sodium
solutions (in NS and SW) in an Accufuser® infusion
device were both more chemically stable and physically
compatible for 7 days at CT rather than RT storage con-
ditions. This result sugg ests that ready-to-u se solutio ns of
Ampicillin sodium in the Accufuser ® infusion device can
be more safely kept at CT than RT conditions up to 7
days regarding the change of potency and pH of the solu-
tions.
5. Acknowledgements
This study was supported by Division of Molecular T her a-
peutics Development, Hanyang Biomedical Research In-
stitute, Hanyang University and Woo Young Medical Co.
LTD., Seoul, South Korea.
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