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Background: The use of co-amoxiclav injectable form among neonates and infants necessitates the usage of a part of the reconstituted drug solution or suspension, based on the relative low weights of this group of patients, with the remaining part of the reconstituted form being discarded and not used for subsequent doses due to unreliability in dosage form stability. This practice increases the cost of the treatment. Objective: This study aims at evaluation of the stability of reconstituted co-amoxiclav injectable solutions in order to use it in the most cost-effective manner. Methods: Physical, chemical and microbiological stability of reconstituted co-amoxiclav injectable solutions were evaluated at 4 different storage conditions which assembled by iterating each of temperature and lighting condition at two possible levels. Responses measured were degradation rate, colour and pH changes, shelf life and sterility of reconstituted drug solutions. Results: Degradation of both Clavulanic acid and Amoxicillin was found to enhance with increasing of storage temperature whereas only the degradation of Clavulanic acid appeared to be affected by storage light conditions. Time dependent changes in colour and pH were observed in reconstituted solutions under all storage conditions, especially in samples stored at 30˚C in indoor room lighting. Best storage conditions for reconstituted co-amoxiclav injectable solutions was determined as refrigerated at 4-8˚C in dark with associated shelf life of 1 hour. All reconstituted solutions complied with the test for sterility. Conclusions: Reconstituted co-amoxiclav injectable solutions should either be used immediately after reconstitution or within a maximum of 1 hour of reconstitution if stored protected from light at 4-8˚C. Keywords: Co-amoxiclav, injectable solution, stability, shelf life, sterility.
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STABILITY OF CO-AMOXICLAV RECONSTITUTED INJECTABLE
SOLUTION
Abubakr O. Nur1*, Alharith A. A. Hassan1, Elrasheed A. Gadkariem2, Zuhair Osman1,
Gamal K. M. Ali3
1Department of Pharmaceutics, Faculty of Pharmacy, University of Khartoum, Sudan.
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Khartoum,
Sudan.
3Central Medical Supplies, Khartoum, Sudan.
Article Received on 23/02/2015 Article Revised on 16/03/2015 Article Accepted on 08/04/2015
ABSTRACT
Background: The use of co-amoxiclav injectable form among
neonates and infants necessitates the usage of a part of the
reconstituted drug solution or suspension, based on the relative low
weights of this group of patients, with the remaining part of the
reconstituted form being discarded and not used for subsequent doses
due to unreliability in dosage form stability. This practice increases the
cost of the treatment. Objective: This study aims at evaluation of the stability of
reconstituted co-amoxiclav injectable solutions in order to use it in the most cost-effective
manner. Methods: Physical, chemical and microbiological stability of reconstituted co-
amoxiclav injectable solutions were evaluated at 4 different storage conditions which
assembled by iterating each of temperature and lighting condition at two possible levels.
Responses measured were degradation rate, colour and pH changes, shelf life and sterility of
reconstituted drug solutions. Results: Degradation of both Clavulanic acid and Amoxicillin
was found to enhance with increasing of storage temperature whereas only the degradation of
Clavulanic acid appeared to be affected by storage light conditions. Time dependent changes
in colour and pH were observed in reconstituted solutions under all storage conditions,
especially in samples stored at 30˚C in indoor room lighting. Best storage conditions for
reconstituted co-amoxiclav injectable solutions was determined as refrigerated at 4-8˚C in
dark with associated shelf life of 1 hour. All reconstituted solutions complied with the test for
sterility. Conclusions: Reconstituted co-amoxiclav injectable solutions should either be used
ejpmr, 2015,2(3),109-123
SJIF Impact Factor 2.026
Research Article
ISSN 3294-3211
EJPMR
EUROPEAN JOURNAL OF PHARMACEUTICAL
AND MEDICAL RESEARCH
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*Correspondence for
Author
Abubakr O. Nur
Department of
Pharmaceutics, Faculty of
Pharmacy, University of
Khartoum, Sudan.
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immediately after reconstitution or within a maximum of 1 hour of reconstitution if stored
protected from light at 4-8˚C.
KEYWORDS: Co-amoxiclav, injectable solution, stability, shelf life, sterility.
INTRODUCTION
The stability of a medicinal product relates to its resistance to the various chemical, physical,
and microbiological reactions that may change the original properties during transport,
storage, and use. The term "stability" is often expressed in quantitative term of shelf-life
which is the time during which the medicinal product is predicted to remain fit for its
intended use under specified conditions of storage.[1, 2]
Co-amoxiclav, which is known to enhance the activity of Amoxicillin due to the incorporated
Clavulanic acid as a beta-lactamase inhibitor,[3, 4] is among the widely used antibiotics in
hospitalized neonates and infants suffering from severe infectious diseases based on the
relative safety and wide spectrum of activity of the drug combination.[4, 5, 6]
In most neonates and infants, just a part of the reconstituted solution or suspension is used for
a single dose due to their relatively low weights with the remaining part of the solution or
suspension is usually discarded and not used for subsequent doses because of the unreliability
in dosage form stability.[7, 8] This practice increases the cost of the treatment on patient;
putting in the consideration this antibiotic is relatively expensive. So there is need for finding
out the most cost-effective manner for using this antibiotic.[8]
Many of the cited reports on stability of combined Amoxicillin and Clavulanic acid in
reconstituted drug preparations recommended the use of the reconstituted preparations within
four hours at ambient temperature or within eight hours if stored at 4°C which is in accord
with a published report of Medical and Healthcare products Regulatory Agency (MHRA)
dealing with the same matter.[9, 10, 11]
In Sudan, however, the use of this effective antibiotic faced challenges represented by the
medication cost in addition to the practice in paediatric hospital that results in increasing
further the medication cost as a consequence of using a new vial for each dose.
This work aims to investigate the effects of temperature, indoor light, and medium pH on
stability and sterility of reconstituted co-amoxiclave injectable solutions using the shelf-life
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criteria in order to determine the best storage conditions and, therefore, the most cost-
effective manner of using such drug combination.
MATERIALS AND METHODS
Materials
The following materials were utilized in the present study
Phosphoric acid(Bernd Kraft GmbH- Germany), hydrochloric acid(VMR International Ltd.
England), sodium dihydrogen orthophosphate anhydrous (Surechem Products Ltd. England)
and sodium hydroxide(Fisher Scientific Ltd. U.K.) were analytical grade and were used as
purchased. Tablets of phosphate buffer pH 7 and phthalate buffer pH 4 were products of
Fisher Scientific Ltd. UK and were donated by CMS. Sudan. HPLC-grade methanol was a
product of ScharlauChemie S.A. Spain. Other materials are different grades obtained from
different commercial sources.
Amoxicillin trihydrate and Clavulanate Potassium were working standard of Shin Poong
Pharmaceutical Co., Ltd. (Korea) with potency of 86% and 605Mg/ mg, respectively, and
were donated by GMC. Ltd. Sudan.Co-amoxiclav vials for injection of the brand name
Julmentine® were kindly provided by Gulf Pharmaceutical Industries, UAE. Labeled content
claim per vial is sterile 1g of Amoxicillin as Amoxicillin sodium and 200mg of Clavulanic
acid as potassium Clavulanate.
For sterility test materials, Thioglycollate Medium USP (culture medium Lot No. 967542),
Tryptone Soya Broth USP (Soybean-Casein Digest Medium, Lot No. 983099) and USP
Buffered 0.1% w/v solution of sodium chloride-peptone pH 7 were products of Oxoid Ltd.,
Hampshire, England. The first two medium were used as culture medium and prepared
according to manufacturer instructions while the later was utilized as a diluent.
Methods
Experimental design
Based on the study objectives, 22 full factorial design was selected where two factors,
namely, light condition and temperature were investigated at two possible levels each for
their influence on stability of reconstituted Co-amoxiclav injections. Dark and indoor room
light were set as levels for light factor whilst 30°C and 4-8°C were selected as levels for
temperature factor. The design composed of four possible storage conditions (refrigerated at
4°C-8°C in indoor light, refrigerated at 4°C-8°C in dark, at 30°C in indoor light and at 30°C
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in dark) and within each experimental run, four vials were examined. Selection of vials and
the order of runs were done randomly.[12]
Samples preparation and processing
For each storage condition, vials were reconstituted with 20 ml of sterile water for injection
(according to the manufacturer’s instructions). Directly after reconstitution, 200 µl from each
of the four vials of the nominal concentration 50 mg/ ml of Amoxicillin and 10 mg/ ml of
Clavulanic acid were withdrawn using micropipette, transferred into 50 ml volumetric flasks
and completed to volume using distilled water. 20 µl of the last formed solution were used in
duplicates for the HPLC based drug assay. Vials were then introduced into a cooled incubator
(ES110 nÜve, Turkey) adjusted to the required conditions of temperature and light.
The source of light consists of five cool white fluorescent lamps of one foot length (Tazen,
China) that fixed inside two cooled incubator at a distance of about 17 cm from vials to give
the highest possible illumination in order to simulate lighting of pharmacies, especially
hospital pharmacies. Both light brightness (measured in Lux unit), wavelength and energy
(measured in watt.hrs/ m2) were determined at the beginning and at the end of the storage
periods (4 hrs) for samples stored in light at either 4-8˚C or 30˚C by using photometer (Model
450-1 with multiprobe 550-2, USA) and spectrometer (Ocean Optics Inc. USB 2000 VIS/IR,
with Ocean Optics software, USA) apparatus.
The pH of the Co-amoxiclav solutions in the vials was measured (Inolab®,level1,TÜV,
Germany) immediately after the reconstitution and at the end of study. The vials in the other
groups were manipulated by the same way with difference in the storage conditions in the
cooled incubator and the process of samples collection for analysis was repeated at every
predetermined time interval following the same manner.
HPLC method for drug assay
Chromatographic analyses of drug components in the combination was performed following
USP official HPLC method[13] with some modifications in the flow rate. The HPLC system
(Shimadzu, Kyoto, Japan) was equipped with Inertsil® ODS-SP column (4.6 mm x 15 cm,
5µm, GL Sciences Inc. Tokyo, Japan), a pump (LC-20AB, Prominence), CTO-20A,
Prominence Column Oven, SIL- 20A, Prominence Auto sampler and a UV-VIS detector
operating at 220 nm (SPD-20A, Prominence).
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Mobile phase used consists of mixture of Methanol and pH 4.4 Sodium dihydrogen
Orthophosphate buffer (5:95). This mixture was passed through a filter having 0.5 µm
porosity and then degassed using the Ultrasonic device (Retsch, UR-275 D, GS, Type: T570,
Germany). The buffer was prepared following USP method.(1) For equilibration of mobile
phase with stationary phase, the flow rate was increased gradually from 0 to 1 ml/min in 0.1
ml increments. The equilibration needs approximately 10 column volumes (around 20
minutes).
Eluted peaks and corresponding specifications of Calvulanic acid and Amoxicillin were
traced on a PC using LC-Solution software and terminal HP LaserJet printer.
Validation of the HPLC assay method
Since the official HPLC method for drug components analysis was performed with some
modifications, the method was validated for linearity, selectivity, accuracy, reproducibility
and suitability as stability indicating.
For linearity, 20 µl samples of standard solutions ranging in concentration of Amox:Clav
from 20:2 to 500:100 µg/ml were injected directly into the chromatographic system using the
autoinjector (SIL- 20A). Peak responses were integrated and calibration curves relating peak
areas to the corresponding component concentrations were generated and inspected for
linearity.
Concerning method selectivity, different solutions of standard Amoxicillin, Clavulanic acid
and a mixture of both were prepared and subjected to acid hydrolysis with 1M HCl, thermal
degradation on a water bath and photodegradation using a UV lamp at short wave length (254
nm). Samples of degraded solutions were chromatographed and the resulting chromatograms
were inspected for appearance of additional peaks and changes in concentration, retention
time and peak shape of the drug components peaks in relation to a fresh, non degraded
samples.
The accuracy of the method was tested using standard addition method and the average %
recovery was calculated. In addition, the reproducibility of the standard curves were tested
over 5 days and system suitability criteria (theoretical plates, tailing, and resolution) were
developed and evaluated to ensure consistent chromatographic performance, according to
accepted analytical guidelines.[13, 14]
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Sterility studies
Immediately after the end of stability study the remaining solutions of each group of vials
were tested for their microbiological stability. Sterility test was performed using Membrane
Filtration as a technique suitable for the product.[13]
Thioglycollate USP culture medium USP was used primarily for the culture of anaerobic
bacteria, but also detects aerobic bacteria and was prepared by suspending 15 g in 1L of
distilled water and boiling on water bath to dissolve completely. Five screw capped
borosilicate glass bottles were filled each with about 80 ml of the medium and tightly closed.
15 g of USP Tryptone Soya Broth was dissolved in 500 ml of distilled water, mixed well,
and distributed into 5 screw capped borosilicate glass bottles as final containers. The medium
was used for the culture of aerobic bacteria and fungi.
14.63 g of Peptone diluent was dissolved in one liter of purified water, mixed well to form
0.1% w/v solution and distributed into 6 screw capped borosilicate glass bottles as final
containers. The diluent was used for washing the membrane to remove any residue of the
antibiotic.
Prepared culture media, Sartorius filtration apparatus (Sartorius AG 37070 Goettingen,
Germany) and the membranes were autoclaved (LS-B50L, China) either at 121°C for 30
minutes (for the culture medium) or at 121 °C for 15 minutes (for filtration apparatus and
filter).
Solutions of the co-amoxiclav vials were filtered aseptically under a Class A laminar-air-flow
(BIOAIR Instruments, AURA VF 48, Siziano (PV), Italy) located within a Class B clean-
room. The membranes were washed with the diluent (0.1 % w/v Peptone).The membranes
were removed and cut aseptically into two equal parts. A one half was transferred to
Thioglycollate Medium and the other to Tryptone Soya Broth. Then both media were
incubated for 14 days at 33°C± 2 for Thioglycollate Medium and at 22.5°C ± 2.5 for
Tryptone Soya Broth.
At intervals during the incubation period and at its conclusion, the media were examined for
macroscopic evidence of microbial growth (turbidity).
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Statistical data analysis
Mean, standard deviation and relative standard deviation (coefficient of variation) were
employed to describe the HPLC assay method validation data. The calibration plots and the
assay data were subjected to regression statistics for fitting and predicting product-moment
correlation coefficient (r) and coefficient of determination (r2) for model fitting and
predicting regression coefficients in determination of order of degradation kinetics.
Inferential statistics based on two-way analysis of variance (ANOVA) was performed to
analyze data derived from the 22 factorial design in order to estimate the effects of
temperature and lighting condition on degradation of drug components. Computations were
aided by software computer package STATISTICA 8 (Statsofts Inc., USA) and in all
analysis, a probability p=0.05 was considered as a cutoff point for significant measures.
RESULTS AND DISCUSSION
Validation of HPLC stability- indicating assay method
HPLC chromatogram for simultaneous determination of Clavulanic acid (10 µg/ml) and
Amoxicillin (50 µg/ml) shows corresponding sharp and well separated peaks for the two
drugs at Rt of 3.98 and 6.88 min, respectively (Figure 1).
Figure 1. HPLC chromatogram for simultaneous analysis of Clavulanic acid (10µg/ml)
and Amoxicillin (50µg/ml)
Calibration curves relating the different concentrations of standard Amox: Clav solutions to
their respective peak areas were found linear with significant high correlation coefficients for
both drugs at different concentration levels investigated (r ranged 0.9985-0.9995, Fcal>>Fcrit).
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HPLC chromatograms of different solutions of standard Amoxicillin and Clavulanic acid that
subjected to acid hydrolysis and thermal degradation show sharp peaks of drugs separated
well from their degradation products (Fig. 2a and b) and the same was observed with samples
that subjected to photo-degradation. Such findings support the ability of the applied HPLC
method to separate the degradation products (if any) from the parent drugs without
overlapping or broadening of the peaks (specificity).
(a)
Figure 2. Chromatograms for Co-amoxiclav solutions that subjected to (a) HCl
hydrolysis for 2 hrs and (b) thermal degradation for 1 hr on a water bath.
On another hand, the demonstrated high values of % recovery of spiked drug components and
the low values of relative standard deviation (RSD) associated with determination of that %
recovery has guaranteed the accuracy of the method (99.2- 100.2% with RSD of 0.33% and
97.9-100.3% with RSD of 0.96% for Clavulanic acid and Amoxicillin, respectively).
Moreover, values of RSD associated with five determinations of both drugs within and
between days were found to be < 2% and <5% respectively, authenticating precision,
repeatability and reproducibility of the applied method of analysis.[12, 14]
Furthermore, values for resolution between drugs' peaks (7.34), the average theoretical plates
of the two drugs (6262 and 6773 for Clavulanic acid and Amoxicillin, respectively) and the
tailing factors determined from 7 runs for both drugs (<1.5) appear within the required limit
of the official USP method for system suitability.[13]
Content of drug components in reconstituted Co-amoxiclav injectable solutions
Degradation profiles of drug components in reconstituted Co-amoxiclav injectable solutions
that stored under different storage conditions are depicted in Figures 3 and 4.
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(a)
(b)
Figure 3. Degradation profiles of Clavulanic acid and Amoxicillin from reconstituted
co-amoxiclav injection that stored at 4-8˚C in (a) dark and (b) indoor room light for 4
hours
(a)
(b)
Figure 4. Degradation profiles of Clavulanic acid and Amoxicillin from reconstituted
co-amoxiclav injection that stored at 30˚C in (a) dark and (b) indoor room light for 4
hours
From the two figures, it might be apparent that none of the drug components remained within
the official limits (loss 10%) after one hour in all studied conditions. Moreover, it appears
that the loss in Clavulanic acid is higher than that of Amoxicillin.
In fact, both components share the β-lactam ring which is believed to degrade by cleavage as
a consequence of hydrolysis.[15] However, in contrast to Clavulanic acid, presence of large
group side chain [2-amino-2-(p-hydroxyphenyl) acetamido] attached to a fused ring system
β-lactam thiazolidine of Amoxicillin at the 6-position renders it more resistant to be attacked
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by either water or hydroxide ion by virtue of steric hindrance. This observation agreed with
previous works which consider Clavulanic acid as the determinant component for stability of
this combination.[9, 10, 16, 17]
It is worth mentioning that Amoxicillin in injectable Co-amoxiclav is found in form of
sodium salt which is very soluble in water while in oral form is found as trihydrate which is
slightly soluble in water.[4] This makes Amoxicillin more available and liable to hydrolysis in
injectable form and explains the marked difference in its stability between injectable solution
and oral suspension form.[16, 17] In addition to that, pH of Amoxicillin sodium salt solution is
basic, in contrast to that of trihydrate which is acidic, causing the hydrolysis to be more
aggressive.
Evidenced by Figs. 3 and 4 that Clavulanic acid is more affected by changing in storage
temperature rather than light condition where upon storage at 30˚C for 4 hours, Clavulanic
acid measured a content of 20-22% (Fig. 4a and b) which is almost half that displayed with
storage at 4-8˚C (refrigerated) (38-39%) for the same time period regardless the light
conditions (Fig. 3a and b). This, in turn, is confirmed by statistical analysis of effect estimate
for factors in the 22 factorial design which support that storage temperature has a more
profound influence (p= 0.0232) on % content of Clavulanic acid than that of light condition
(p= 0.0452), though both factors are influential whereas the effect of the pooled linear
influence of both factors appears of no consequence (p= 0.6336).
Similarly, effect estimate for the two factors encourages the assumption that storage
temperature has a considerable influence on % content of Amoxicillin (p= 0.0116) followed
by the linear interactive influence of both temperature and light (p= 0.0341) with light
condition, as an individual factor, receiving the least influential effect (p= 0.0484). Therefore,
refrigerated Amoxicillin solution stored in light exhibited 70% content (Fig. 3b) which
account for 1.4 times that measured by drug solution stored in light at 30˚C (Fig. 4b) and 1.1
times that showed by drug solution stored refrigerated (4-8˚C) but in dark (Fig. 3a).
Degradation kinetics of Clavulanic acid and Amoxicillin in reconstituted injectable
solutions
Data of % content-time profile for both drugs were fitted to zero (linear) and first order
(exponential) decay models in a search for degradation kinetics characterizing the two drugs
(Table 1). Based on the associated high values of correlation coefficient (r ranged 0.984-
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0.999), it might be assumed that both drug components follow the first order decay model
(Table 1). For both drug components, refrigerated samples (4-8˚C) that stored in dark show
the lowest degradation rate constant (k= 0.136 and 0.029/hr for Clavulanic acid and
Amoxicillin, respectively) as compared to those stored at 30˚C in light which measure highest
degradation rates (k= 0.336 and 0.129/hr for Clavulanic acid and Amoxicillin,
correspondingly).
Table 1: Estimated parameters of the degradation kinetics of drug components in
injectable solutions stored under different storage conditions.
Drug
component
Storage
Conditions
Zero order
model
First order model
Estimated T90%
K (%/hr)
R
K (hr-1)
R
Clavulanic acid
In dark at 30˚C
13.01
0.962
0.304
0.995
12 min
In light at 30˚C
14.24
0.978
0.336
0.999
10 min
In dark at 4-8˚C
6.76
0.985
0.136
0.993
52 min
In light at 4-8˚C
10.02
0.985
0.179
0.997
29 min
Amoxicillin
In dark at 30˚C
6.46
0.988
0.101
0.990
49 min
In light at 30˚C
8.36
0.998
0.129
0.999
47 min
In dark at 4-8˚C
1.90
0.986
0.029
0.988
217 min
In light at 4-8˚C
3.47
0.975
0.046
0.984
116 min
K and r stands for order rate constant and correlation coefficient of fitting to the decay
model, respectively; T90% stands for time to reach 90% of the initial drug content
In contrast to many reported works held previously to assess stability of components in Co-
amoxiclav drug combination, the investigated source of light in this study provides light that
is similar in brightness and energy to the ambient indoor room lighting. This might be
important because any substance that is susceptible to photolysis is affected by certain
wavelengths that confer specific activation energy required for that photolysis reaction.[18, 19]
Within each storage condition, degradation rate constant demonstrated by Clavulanic acid is
greater than that of Amoxicillin at least by 2-folds, supporting the assumption made before
that Clavulanic acid is the least stable component of this drug combination.
In another occasion, time for decay to reach 90% of initial drug content (T90%) for both drug
components under different storage conditions was estimated from k values associated with
best fitted first-order equations (Table 1). As degradation rate of Clavulanic acid appears
more higher than that of Amoxicillin, stability of the combination is, therefore, expected to be
determined mainly by the degradation profile of the former. Calculated T90% (Shelf-life) of
Clavulanic acid, and consequently of Co-amoxiclav injectable solution is about one hour in
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the most stable storage condition (4-8˚C in dark) and just 10 minutes in the least stable one
(30˚C in indoor room light).
Whilst values of k and T90% shown in Table 1 clearly ensure the role of temperature changes
on the degradation rates of both drugs, the effect of light condition become more profound
with the refrigerated samples.
Colour and pH changes in reconstituted Co-amoxiclav injectable solutions
With time, all reconstituted Co-amoxiclav solutions developed physical instability which is
manifested in form of colour change in the sequence pale yellow, dark yellow, orange and
dark brown without precipitation being observed in any of the stored samples (Table 2).
The results demonstrated that colour change was aggressive and faster in reconstituted drug
samples that stored at 30˚C in either dark or light condition. It should be noted, however, that
drug samples showed enhanced and profound colour changes are those which characterized
by enhanced chemical degradation rates (samples stored at 30˚C in either dark or light
condition, Table 2). Accordingly, the observed physical instability (colour change) in these
samples could possibly be related to the chemical instability.
Table 2: Colour and pH changes of the reconstituted co-amoxiclav injectable solutions
under different storage conditions.
Storage Conditions
Time (hrs)
Colour
pH
In dark at 4-8˚C
0
Pale Yellow
8.60 ± 0.02
2
Pale Yellow
4
Dark Yellow
8.54±0.01
In light at 30˚C
0
Pale Yellow
8.56±0.02
2
Pale Yellow
4
Dark Brown
8.33±0.05
In light at 4-8˚C
0
Pale Yellow
8.51±0.03
2
Pale Yellow
4
Dark Yellow
8.34±0.03
In dark at 30˚C
0
Pale Yellow
8.60±0.01
2
Dark Yellow
4
Orange
8.24±0.01
The average starting pH values for all reconstituted solutions were almost similar and ranged
8.5-8.6 (Table 2). Moreover, under all storage conditions the pH decreases with time but
remaining within the acceptable range for intravenous injectable solutions (pH 3-9). These
results contradict findings of relevant works on stability of oral Co-amoxiclav suspensions
in which an increase in pH with time was reported.[17, 20] In general, the pH of the drug
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combination in aqueous solution is influenced by generation of pH-influencing degradants of
Amoxicillin and Clavulanic acid in addition to contained degradants of both drug components
when initially prepared as a result of the fermentation process by which they are prepared.[20]
In the light of such scenario, the dissimilarity in pH changing profile between the present
study and other reported ones could possibly be attributed to the unlike chemical form and
source of the contained active ingredients of the investigated dosage forms.
It is observed that the least change in pH is associated with samples which stored refrigerated
in dark condition (Table 2) and, therefore, change in pH could also be related to chemical and
physical instability.
Sterility test
Sterility of parenteral products is important issue of their quality and it might not be
surprising that a product could possibly fails the requirement of sterility though there is no
evidence of chemical or physical instability.
In this study, no turbidity referring to microbiological instability has been observed in all the
bottles of liquid culture media for both fungi and bacteria (Thioglycollate Medium, Tryptone
Soya Broth). Therefore, all tested samples under different storage conditions have complied
with the official test for sterility.
CONCLUSION
Storage of co-amoxiclav injectable solutions after reconstitution may be hazardous. It
undergoes first order rapid hydrolysis especially Clavulanic acid, which is the least stable
component, and might lose its stability in no more than one hour even if kept protected from
light under refrigeration. The influence of storage temperature on degradation of the two drug
components is evidenced whereas that of lighting condition appear to be associated mainly
with degradation of Clavulanic acid. Both colour and pH of reconstituted solutions change
with time under all storage conditions, especially in solutions stored at 30˚C in indoor room
lighting. All reconstituted solutions complied with the test for sterility and best storage
conditions for reconstituted co-amoxiclav injectable solutions was determined as refrigerated
at 4-8˚C in dark with associated shelf life of 1 hour.
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ACKNOWLEDGEMENTS
Authors would like to acknowledge G.M.C., Ltd. (Sudan) and Gulf Pharmaceutical Industries
(UAE) for the materials donation. Technical assistance provided by Tahir M. Tahir and
Ibrahem M. Ismael (Faculty of Pharmacy, University of Khartoum) during the experimental
part of this study is highly admitted. Quality control department of Central Medical Supply
(Sudan) is highly indebted for their kind instruments providing.
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International Conference on Harmonization (ICH): 2003.
2. Draft guidance for industry: Stability testing for drug substances and drug products.
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3. Chambers HF, Petri WA. Chemotherapy of Microbial Disease. In: Brunton LL, Parker
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... 12 Literature indicates that the main constraints of coamoxiclav stability include infusion diluent and storage temperature. Co-amoxiclav has been found to be less stable at higher temperatures, with data suggesting that shelf-life ranges between 1 and 5.5 hrs at room temperature in water for injection (WFI) and up to 8 hrs at 4°C. [13][14][15][16] To expand the breadth of current knowledge, this study utilises the bench-to-bedside approach, where challenges experienced in practice are addressed in the laboratory. Coamoxiclav stability is a crucial parameter that needs to be determined to assess the feasibility of administration via continuous/prolonged infusions. ...
... Greatest stability of amoxicillin was achieved in WFI and saline solutions at 4°C, where shelf-lives of 9.6 and 10.0 hrs, respectively, were determined (Table 2). Data obtained indicated co-amoxiclav stability superior to that previously proposed [13][14][15][16] making it suitable for extended infusion therapy. Prolonged co-amoxiclav infusion would improve the effectiveness of therapy without altering the dose or dosing schedule, giving no increase in toxicity. ...
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Rationale: Previously, we have been able to outpace bacterial mutation by replacing increasingly ineffective antibiotics with new agents. However, with the discovery of new antibiotics diminishing, optimising the administration of existing broad-spectrum antibiotics such as co-amoxiclav has become a necessity. Methods: A stability indicating HPLC method was developed and validated in compliance with International Council for Harmonisation (ICH) guidelines. Stability of co-amoxiclav at clinical concentration was evaluated at three temperatures (4°C, ambient (23-25°C) and 37°C) in three diluents (water for injection (WFI), 0.9% w/v NaCl and Ringer's solution). To establish whether there were significant differences at the level of both diluent and temperature, results were analysed using analysis of covariance (ANCOVA) to assess differences between the attained slopes of regression. Results: Data obtained indicated co-amoxiclav stability superior to that previously proposed making it suitable for extended infusion therapy. The degradation of amoxicillin appeared to follow a linear trend, with the rate of degradation elevated at higher temperatures, demonstrated by the magnitude of the regression slopes in these conditions. Analysis of regression slopes via ANCOVA demonstrated that diluent and temperature both significantly affected co-amoxiclav stability. Amoxicillin retained 90% of its initial concentration for 7.8 to 10 hrs when stored at 4°C, 5.9 to 8.8 hrs at ambient and 3.5 to 4.5 hrs when incubated at 37°C. Conclusion: Co-amoxiclav is suitable for administration via prolonged infusion. Findings from this study aid in ameliorating current dosing regimens to optimise antibiotic efficacy. Other valuable applications conferred from these findings include the ability to pre-prepare solutions for use in bolus administration, minimising preparation time and workload.
... The results of the analysis of variance show that the temperature effect is much more significant than CAo and the combined interaction effect, influencing the final CA concentration negatively in the period between 0 and 42 h. The trends of CA decomposition in fermentation broth seem to deviate from first and pseudofirst-order kinetics previously reported for CA solutions prepared with standard reactant or commercial formulations [10,19,23,[25][26][27]. Similar behaviors to those observed in this work can be observed in the data presented by other authors for CA from fermentation broths in the range of 10 to 40 °C [10,17]. ...
... The results of the observed rate constants kobs,1 for t < 5.5 h and kobs,2 for t > 5.5 h are presented in Table 1. The trends of CA decomposition in fermentation broth seem to deviate from first and pseudo-firstorder kinetics previously reported for CA solutions prepared with standard reactant or commercial formulations [10,19,23,[25][26][27]. Similar behaviors to those observed in this work can be observed in the data presented by other authors for CA from fermentation broths in the range of 10 to 40 • C [10,17]. ...
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Clavulanic acid (CA) is a β-lactam antibiotic inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. CA is produced in submerged cultures by the filamentous Gram-positive bacterium Streptomyces clavuligerus; yield and downstream process are compromised by a degradation phenomenon, which is not yet completely elucidated. In this contribution, a study of degradation kinetics of CA at low temperatures (−80, −20, 4, and 25 °C) and pH 6.8 in chemically-defined fermentation broths is presented. Samples of CA in the fermentation broths showed a fast decline of concentration during the first 5 h followed by a slower, but stable, reaction rate in the subsequent hours. A reversible-irreversible kinetic model was applied to explain the degradation rate of CA, its dependence on temperature and concentration. Kinetic parameters for the equilibrium and irreversible reactions were calculated and the proposed kinetic model was validated with experimental data of CA degradation ranging 16.3 mg/L to 127.0 mg/L. Degradation of the chromophore CA-imidazole, which is commonly used for quantifications by High Performance Liquid Chromatography, was also studied at 4 °C and 25 °C, showing a rapid rate of degradation according to irreversible first-order kinetics. A hydrolysis reaction mechanism is proposed as the cause of CA-imidazole loss in aqueous solutions.
... 16 Even though clavulanic acid is not available in pharmaceutical formulation and is not licensed to be administered alone, this study paves the way for innovation to overcome stability concerns. Data obtained indicated stability superior to that previously proposed 6,11,13,18,19 rendering it suitable for extended or continuous infusion therapy. Results obtained are in alignment with those recently published, suggesting that storage temperature significantly influences the stability of amoxicillin and clavulanic acid (Tables 1-3). ...
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Rationale: With the discovery of new antibiotics diminishing, optimising the administration of existing antibiotics such as amoxicillin-clavulanic acid has become a necessity. At present, the optimal approach for enhancing the effectiveness of time-dependent antibiotics involves extending the time at which antibiotic concentrations are maintained above the minimal inhibitory concentration by prolonging the infusion time. This pharmacodynamic rationale cannot be applied to co-amoxiclav because of poor stability at room temperature. The aim of this study was to establish the shelf-life of amoxicillin and clavulanic acid prepared in separate containers to determine the feasibility of 24-hr continuous infusion therapy. Methods: A previously developed and validated stability-indicating HPLC method was used to establish the shelf-life of reconstituted amoxicillin and clavulanic acid when prepared in separate containers. Stability at clinical concentration was evaluated at three temperatures. To establish whether there were significant differences at the level of both active ingredients and temperature, results were analysed using analysis of covariance (ANCOVA) to assess differences between the attained slopes of regression. Results: Data obtained indicated amoxicillin and clavulanic acid stability superior to that previously proposed making it suitable for continuous infusion therapy. Analysis of regression slopes via ANCOVA showed that temperature significantly affected amoxicillin and clavulanic acid stability. Amoxicillin retained 90% of its initial concentration for 80.3 hrs when stored at 4°C, 24.8 hrs at 25°C and 9 hrs when incubated at 37°C. Clavulanic acid retained 90% of its initial concentration for 152 hrs when stored at 4°C, 26 hrs at 25°C and 6.4 hrs when incubated at 37°C. Conclusion: Amoxicillin and clavulanic acid are suitable for administration via continuous infusion when prepared, stored, and administered in separate containers. Results obtained from this study aid in ameliorating current dosing regimens to optimise antibiotic efficacy; however, more in-depth amoxicillin and clavulanic acid y-site compatibility studies are warranted.
... In addition to the characteristic low productivity of the bioprocess, the product yield is also compromised by the degradation of CA in aqueous phases and the efficiency of the separation steps required for the obtention of potassium clavulanate (the stable commercial form). Several authors have confirmed the degradation of CA in fermentation broths [67,69,[168][169][170], synthetic buffer [68,171,172], and pure water [173][174][175]. The CA secreted by S. clavuligerus exhibits a high initial reaction rate (~5 h) in aqueous solution (e.g., fermentation broths) followed by a slower degradation rate in the next hours [168,169]. ...
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Clavulanic acid (CA) is an irreversible β-lactamase enzyme inhibitor with a weak antibacterial activity produced by Streptomyces clavuligerus (S. clavuligerus). CA is typically co-formulated with broad-spectrum β‑lactam antibiotics such as amoxicillin, conferring them high potential to treat diseases caused by bacteria that possess β‑lactam resistance. The clinical importance of CA and the complexity of the production process motivate improvements from an interdisciplinary standpoint by integrating metabolic engineering strategies and knowledge on metabolic and regulatory events through systems biology and multi-omics approaches. In the large-scale bioprocessing, optimization of culture conditions, bioreactor design, agitation regime, as well as advances in CA separation and purification are required to improve the cost structure associated to CA production. This review presents the recent insights in CA production by S. clavuligerus, emphasizing on systems biology approaches, strain engineering, and downstream processing.
... Confirmation of physical degradation of AMX sodium was provided via visible discolouration of AMX sodium DCTs (in the MN patch and individually) when packaged in poly(ester) foil and unpackaged. Reports of physical instability of AMX can be found in the literature [48]. The change of a pharmaceutical products physical appearance can dictate appropriate primary packaging [49]. ...
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As microneedle (MN) patches progress towards commercialisation, there is a need to address issues surrounding their translation from the laboratory to the end-user. One important aspect of MN patches moving forward is appropriate primary packaging. This research focuses on MN patches containing amoxicillin (AMX) sodium for the potential treatment of neonatal sepsis in hot and humid countries. A MN patch consists of a hydrogel-forming MN array and a drug-containing reservoir. Improper primary packaging in hot and humid countries may result in degradation of active pharmaceutical ingredients, with the use of substandard medicines a major health concern. The research presented here, for the first time, seeks to investigate the integrity of MN patches in different primary packaging when stored under accelerated storage conditions, according to international guidelines. At pre-defined intervals, the performance of the MN patch was investigated. Major causes of drug instability are moisture and temperature. To avoid unnecessary degradation, suitable primary packaging was sought. After 168 days, the percentage of AMX sodium recovered from drug-containing reservoirs packaged in Protect™ 470 foil was 103.51 ± 7.03%. However, packaged in poly(ester) foil, the AMX sodium content decreased significantly ( p = 0.0286), which is likely due to the degradation of AMX sodium by the imbibed moisture. Therefore, convincing evidence was provided as to the importance of investigating the stability of MN patches in primary packaging intended for MN-mediated transdermal delivery so that they are ‘fit for purpose’ when it reaches the end-user. Future work will include qualitative studies to assess MN patch usability. Graphical abstract
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Method validation is one of the measures universally recognized as a necessary part of a comprehensive system of quality assurance in analytical chemistry. In the past, ISO, IUPAC, and AOAC International have cooperated to produce agreed protocols or guidelines on the "Design, conduct and interpretation of method performance studies" [1], on the "Proficiency testing of (chemical) analytical laboratories" [2], on "Internal quality control in analytical chemistry laboratories" [3], and on "The use of recovery information in analytical measurement" [4]. The Working Group that produced these protocols/guidelines has now been mandated by IUPAC to prepare guidelines on the single-laboratory validation of methods of analysis. These guidelines provide minimum recommendations on procedures that should be employed to ensure adequate validation of analytical methods. A draft of the guidelines has been discussed at an International Symposium on the Harmonization of Quality Assurance Systems in Chemical Laboratory, the proceedings from which have been published by the UK Royal Society of Chemistry.
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Improving the stability of potassium clavulanate in admixture with amoxicillin Papers C o-amoxiclav is a fixed combination of the sodium salt of amoxicillin (amoxiNa) and the potassium salt of clavulanic acid (clavK). Although clavulanic acid has weak antibacterial activi-ty when used alone, its combination with certain penicillins results in a synergistic effect which expands the spectrum of activi-ty of the penicillin against many strains of beta-lactamase producing bacteria. Preformulation studies indicate that clavulanic acid undergoes acid-base catalysed reactions depending on pH and the presence of buffer salts. 1 Maximal stability of clavulanic acid is reported to be at pH 6.3. Although amoxicillin is subject to similar degradation pathways it appears to be more stable than clavulanic acid. In fact, evaluation of the stability of this combination in intravenous form 2 as well as in oral suspensions 3,4 showed that amoxicillin was markedly more stable than clavulanic acid.Therefore, in this paper, the stability of the admixture is assessed, in terms of clavK stability only. The pH of the reconstituted intravenous mixture of amoxiNa and clavK is in the range 8.5–9.5. The low stability of clavK at this pH range can affect the efficacy and safety of therapy. Since visible degradation (apparent by a colour change) has been observed before its administration, it is considered of interest to improve the stability of the mixture.
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Study objectives: To study the physical compatibility and chemical stability of amoxicillin, a β-lactam antibiotic combined with clavulanic acid, a β-lactamase inhibitor, using a stability-indicating high-performance liquid chromatographic (HPLC) assay. Methods: The study samples were prepared by adding amoxicillin/clavulanic acid to 0.9% sodium chloride solution in polyolefin bags. The contents of the bags were studied after storage under the following conditions: at ambient temperature without protection from light and at 4°C with protection from light for 72 hours. Both compounds were considered stable if they retained ≥90% of the baseline drug concentration. Three dosages (two adult forms: 2 g/200 mg and 1 g/200 mg, and one paediatric form: 0.5 g/50 mg) were examined under laboratory conditions simulating those used routinely in hospitals. Evaluations for physical compatibility and chemical stability were performed initially and during the storage period. The physical compatibility was assessed using visual observation for signs of discoloration and precipitation at each sampling interval. The optical density was measured to give a measurable reading of the colour; pH values of solutions were also measured. The chemical stability of the drugs was evaluated by using a stability-indicating HPLC assay. Results: When compared with ambient or refrigerated storage conditions, amoxicillin, whatever the studied preparation, was stable for a longer duration than clavulanic acid. The mixture was only stable for four hours at ambient temperature and for eight hours at 4°C. However, we noticed a colour change (from light to dark yellow) of the various reconstituted solutions, undoubtedly because of the pH variations. Conclusion: We recommend that these solutions be kept refrigerated whenever possible.
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Clavulanic acid (CA) is a β-lactam antibiotic that alone exhibits only weak antibacterial activity, but is a potent inhibitor of β-lactamases enzymes. For this reason it is used as a therapeutic in conjunction with penicillins and cephalosporins. However, it is a well-known fact that it is unstable not only during its production phase, but also during downstream processing. Therefore, the main objective of this study was the evaluation of CA long-term stability under different conditions of pH and temperature, in the presence of variable levels of different salts, so as to suggest the best conditions to perform its simultaneous production and recovery by two-phase polymer/salt liquid–liquid extractive fermentation. To this purpose, the CA stability was investigated at different values of pH (4.0–8.0) and temperature (20–45 °C), and the best conditions were met at a pH 6.0–7.2 and 20 °C. Its stability was also investigated at 30 °C in the presence of NaCl, Na2SO4, CaCl2 and MgSO4 at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 6.5). All salts led to increased CA instability with respect to the buffer alone, and this effect decreased in following sequence: Na2SO4 > MgSO4 > CaCl2 > NaCl. Kinetic and thermodynamic parameters of CA degradation were calculated adopting a new model that took into consideration the equilibrium between the active and a reversibly inactivated form of CA after long-time degradation.
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SINCE December 2010 the pharmacovigilance team at the Veterinary Medicines Directorate (VMD) has received 16 reports of adverse events in animals following the administration, under the cascade system, of …
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A study was carried out using high performance liquid chromatography (HPLC) to determine the chemical stability of amoxycillin and potassium clavulanate in 250/62 co-amoxyclav oral suspension (Augmenting stored at room temperature. (RT, 20°C) and 8°C over a period of 11 days. The suspension was judged to be acceptable if its components maintained at least 90% of their label concentrations. During the test period, the amoxycillin component was found to be more stable than the clavulanate. Amoxycillin was stable for 7 days at both temperatures. Potassium clavulanate maintained at least 90% of its initial concentration for 7 days at 8°C but showed more than 40% degradation in the same time period at RT. For potassium clavulate the shelf-life, or time taken for the original concentration to drop to 90% of its value (t90) at RT was found to be 2 days.
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