Hobeika et al. BMC Pharmacology and Toxicology
https://doi.org/10.1186/s40360-020-0390-y
(2020) 21:15
RESEARCH ARTICLE
Open Access
Are antibiotics substandard in Lebanon?
Quantification of active pharmaceutical
ingredients between brand and generics of
selected antibiotics
Eva Hobeika1*†, Joanna Farhat1†, Joseph Saab1, Walid Hleihel1, Samar Azzi-Achkouty2, Georges Sili3,
Souheil Hallit4,5*† and Pascale Salameh5,6,7†
Abstract
Background: In developing countries, brand-generic substitution is not based on validated scientific evidence that
confirm the therapeutic equivalence of the generic to the originator. Rather, decisions are made based on the
availability of generic medications. Substitution by inappropriate preparations applies to antibiotics, which may
increase the risk of resistance in case of underdosing. This analytical study aims to dose and assess for the accuracy
of labeling three oral antibiotic preparations, namely ciprofloxacin hydrochloride, amoxicillin trihydrate and
amoxicillin trihydrate-clavulanate potassium, the active pharmaceutical ingredients (APIs) found in brand and
generic tablets available on the Lebanese market.
Methods: One brand and 4 generics of ciprofloxacin tablets, 3 generic amoxicillin tablets, and 1 brand and 4
generics of amoxicillin-clavulanic acid medications, were quantified, taking 2 batches of each. According to the
United States Pharmacopeia (USP) guidelines, ultra-high pressure liquid chromatography was used to measure the
APIs content within tablets. The USP required assay limit of the API was taken as the main comparison criteria.
Results: Out of the 5 ciprofloxacin medications tested, all 5 were out of the 2% required range, thus being substandard.
For amoxicillin, all 3 medications were within the 20% range. As for amoxicillin-clavulanic acid medications, 4 out of 5
medications met the 30% required range of clavulanic acid and one exceeded the claimed amount of clavulanic acid,
while all 5 met the assay limit for amoxicillin.
Conclusion: These findings raise safety and efficacy concerns, providing solid grounds for potential correlations of
antibiotic resistance/substandard antibiotics.
Keywords: Generic, Brand, Brand-generic substitution, Substandard, Ciprofloxacin hydrochloride, Amoxicillin trihydrate,
Clavulanic acid, Antibiotic resistance, Therapeutic equivalence
* Correspondence: eva.hobeika@hotmail.com; souheilhallit@hotmail.com
†
Eva Hobeika and Joanna Farhat are First authors and Souheil Hallit and
Pascale Salameh are Last co-authors.
1
Faculty of Arts and Sciences, Holy Spirit University of Kaslik (USEK), Jounieh,
Lebanon
4
Faculty of Medicine and Medical Sciences, Holy Spirit University of Kaslik
(USEK), Jounieh, Lebanon
Full list of author information is available at the end of the article
© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Hobeika et al. BMC Pharmacology and Toxicology
(2020) 21:15
Background
The high cost of medications is one of the main barriers
towards medicine access [1]. The unstable financial and
economic situation in developing countries, along with
increased costs of healthcare services have pushed for
the selling of generic medications as alternatives to the
2.6 times more expensive brand medications (in the private sector) [1, 2]. A study conducted in 2012 by Cameron et al. showed that the switch from brand
medications to generics in the private sector could result
in an average of 60% cost savings [3].
To decrease pharmaceutical expenditures and guarantee
a safe substitution, many developed countries introduced
policies for generic drug substitution [2, 4]. In the United
States of America, the Food & Drug Administration
(FDA) defined the characteristics the generic drug must
have compared to the brand: 1) contains the same active
ingredient of the brand drug, 2) be similar in terms of
strength, pharmaceutical form and way of administration,
3) has the same indications, 4) fulfills the same bioequivalence conditions, 5) fulfills the requirement of the batch in
terms of identity, purity and quality, 6) is being manufactured in accordance with the standards of Good Manufacturing Practice, 7) is being compared to the brand in
accordance with the standards of Good Laboratory Practices. When the generic drug fulfills the above-mentioned
conditions, it can be considered as therapeutically equivalent to the brand, i.e. has the same efficacy and safety.
The WHO (World Health Organization) defines generic drugs as “pharmaceutical product, intended to be
interchangeable with an innovator product manufactured without a license from the innovator company and
marketed after the expiry date of the patent or other exclusive rights” [5]. Whereas brand medications are trade
names that can only be manufactured and distributed by
the company owning the patent, both of these drug categories are subject to quality assurance standards set by
regulatory authorities. The United States FDA defines in
its Orange Book pharmaceutical equivalents as “drug
products in identical dosage forms and route of administration that contain identical amounts of the same active
drug ingredient” [6]. Consequently, for generic and
brand medications to be interchangeable, they must contain identical amounts of active pharmaceutical ingredients of similar quality.
At the international level, the International Council on
Harmonization (ICH) sets clearly defined guidelines
intended for the registration protocol of pharmaceutical
products, in particular the third module entitled “quality” that details the quality assurance process [7]. In case
generic or brand medications are inappropriately manufactured, they are termed substandard, substandard
medications being defined by the WHO as “genuine
medicines produced by manufacturer, not meeting
Page 2 of 12
quality specifications set to them by national standards”.
It is important to note that this term is inclusive of both
branded and generic medications [8]. In oral dosage
forms, quantifying active pharmaceutical ingredient
(API) assay is achieved through the analytical instrumentation of High Pressure Liquid Chromatography (HPLC).
The potency of tablets – subsequently safety – is evaluated, and the tablets are considered substandard if the
measures prove to be outside the accepted range of
quality assurance [9].
Substandard medications can potentially affect clinical
response for all diseases, and have been implicated in
antibiotic resistance worldwide, raising alarming concerns among healthcare professionals. It is a type of drug
resistance, where survival of usually sensitive bacteria, is
observed after exposure to the antibiotic. This change in
definitive survival is accelerated on the basis of factors
related to human practices, with subsequent resistance
selectivity in animals, humans and the environment [10].
Many theories have been put forward to explain the reason for this substantial increase in multiple pathogenic
bacterial strains and their inevitable resistance to antibiotics worldwide, such as antibiotic misuse, antibiotic resistance and related treatment failures, in addition to
poor quality of available medicines in developing countries (substandard drugs) with inadequate compliance
with manufacturing practices, and lack of proper quality
control [11]. Among the various classes of antibiotics,
fluoroquinolones are worthy of attention as they contribute to the rapid emergence of resistance [12], along with
another category that is the penicillin class, falling under
the broad-spectrum antibiotics category, with its extensive use, low-cost and popularity in developing countries
such as Lebanon [13].
In Lebanon, the general population has access to medicines through the private sector, and outpatients buy
their medication mainly from community pharmacies. In
August 2015, the Lebanese Ministry of Public Health
(MOPH) issued ministerial decision no. One thousand,
two hundred ninety-five that implemented the use of the
unified medical prescription form among physicians,
pharmacists and patients. This unified prescription gave
the community pharmacist the right to substitute the
brand medication by a lower-priced generic, unless
otherwise specified by the physician. On the other hand,
the Technical Committee within the MOPH is the authority that licenses medicines in Lebanon, and generics
are considered as such, based on the dosage, dosage
form, and active ingredient(s). The MOPH guidelines for
the drug technical file submission are based on ICH’s
module 3. The ministry’s online website has a section
termed “Quality Assurance of Pharmaceutical Products”,
under which fall these guidelines. Part 3.2.S.4 labeled
“control of drug substance” is divided into the following
Hobeika et al. BMC Pharmacology and Toxicology
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(2020) 21:15
guidelines: specification, analytical procedures, validation
of analytical procedures, batch analyses and justification
of specification [14]. However, in the absence of a central laboratory for assessing medications content, generics are registered in Lebanon based on files
composed by the manufacturer and subsequent brandgeneric substitution is performed based on the availability of medicines rather than on scientific evidence that
accurately report that generic and brand are therapeutically equivalent. The MOPH defines pharmaceutical
“equivalent” as referring to: “drug products, which contain the same active ingredient in the same strength
(concentration) and dosage form, and is intended for the
same route of administration.” The definition carries on
by specifying conditions as: “In general, it has the same
labeling and meets compendial and other standards of
strength, quality, purity and identity.”
The aim of this study is the analytical control of 3 different antibiotic active pharmaceutical ingredients,
allowing the quantitative assessment of their dosage as
compared to the dosage stated on the label, hence being
able to pinpoint substandard medications. This analytical
evaluation of generic medications would set the pace to
a broader objective, by taking a step further towards establishing a Lebanese equivalent to the US Approved
Drug Products with Therapeutic Equivalence Evaluations – commonly known as the Orange Book. Therefore, this step would provide safer and more efficacious
substitution of medications for patients seeking less expensive alternatives.
What is already known about this subject?
In the United States, several studies were conducted
comparing generic to brand medications and labeled
dosage claim to actual experimental dosage, to evaluate
therapeutic equivalence and potency of the medication
under study. On a regional level, in the Middle East, a
single study in Yemen evaluated the accuracy of dosage
labeling of brand and generic antibiotic tablets, hence
the need for the present study since there is no scientific
evidence proving the therapeutic equivalence between
brand and generic medications in Lebanon, particularly
in the case of antibiotics and the increased risk of
resistance.
What this study adds?
The results of this study provided evidence that all
tested brand and generic ciprofloxacin tablets, and one
amoxicillin-clavulanic acid generic tablet, available on
the Lebanese market, had a dosage deviation from what
is mentioned on the label, exceeding the required range
set by the United States Pharmacopeia (USP), and raising concerns of safety, effectiveness and therapeutic
equivalence. Moreover, this study sheds the light on a
new plausible cause underlying the increased occurrence
of antibiotic resistance, through potential correlations
between substandard medications and antibiotic resistance. Finally, it could pave the way for implementing a
Lebanese equivalent to the US Orange Book.
Methods
Material
The following part details the materials used in the experimental and quantitative analysis of the ciprofloxacin,
amoxicillin and amoxicillin/clavulanic acid APIs from
the antibiotic tablets.
Chemical compounds
Table 1 enumerates the chemical compounds used
throughout this study.
Instrumentation
The high pressure liquid chromatographic U-HPLCDAD RS system model used (Thermo Ultimate 3000) is
equipped with quaternary pump, auto sampler, and
thermo stated column compartment with a variable
wavelength detector controlled by the Chem station software. The HPLC system has a photodiode array detector
(DAD) used for quantification studies. A C18 Thermo
Scientific™ Hypersil GOLD™ HPLC column, small and
meant for high pressure (100 × 2.1 mm), packed with
particles of silica, spherical fully porous ultrapure
(1.9 μm) was used as a stationary phase. For amoxicillin
trihydrate and amoxicillin trihydrate-clavulanate potassium, the analysis was carried out on binary system
using the C18 column (250 × 4.0 mm, 4 μm).
Standard and solvents
The standard used was Ciprofloxacin, pharmaceutical
secondary standard; certified reference material purchased from the supplier Sigma-Aldrich, with CAS number: 855721–33-1. Amoxicillin and clavulanate
potassium buffer standards were also provided from
Sigma-Aldrich.
The solvents used were methanol, acetonitrile, sodium
dihydrogen phosphate, triethylamine and phosphoric
acid, all being of analytical grade. Ultra-pure water was
prepared on a milli-Q purification system from Millipore
(18 MΩ.cm). The uncertainty of the weighing balance
was ±1 mg.
Mobile phase
As mobile phase, 87% of phosphoric acid buffer was
added to 13% acetonitrile (ratio 87:13) in a graduated cylinder. The buffer was prepared as follows: 0.025 M
phosphoric acid (equivalent to 2.85 mL of phosphoric
acid in 2 L of ultrapure water), adjusted to a pH 3.0
using triethylamine solution.
Hobeika et al. BMC Pharmacology and Toxicology
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Table 1 Chemical compounds used during this study
Chemical Compounds
Structurea
Formula
CAS number Purity
Supplier
Ciprofloxacin hydrochloride
(reference standard)
C17H19ClFN3O3
85,721–33-1
Pharmaceutical secondary
standard; certified reference
material
Sigma-Aldrich
Methanol
CH4O
67–56-1
≥99.9% (Chromasolv,
for HPLC)
Honeywell
(Riedel-de Haën)
Acetonitrile
C2H3N
75–05-8
≥99.9% (Chromasolv gradient,
for HPLC, gradient grade)
Honeywell
(Riedel-de Haën)
Phosphoric acid
H3O4P
7664-38-2
98% (ACS reagent, reagent ISO) Sigma-Aldrich
Triethylamine
C6H15N
121–44-8
99%
Scharlau
Amoxicillin trihydrate
C16H19N3O5S .3 H2O 61,336–70-7
Pharmaceutical Secondary
Standard; Certified Reference
Material
Sigma-Aldrich
Potassim clavulanate
C8H8NO5K
VETRANAL™
Analytical standard
Sigma-Aldrich
61,177–45-5
a
Source for structures: American Chemical Society. Source for Ciprofloxacin structure: US FDA
Name and address of the companies that provided the compounds:
1) Name: Ibra HadadAddress: Jdeideh – Nahr El Mott, UniLeb Building, 2nd Floor.ibra@ibrahadad.comCompounds provided: ciprofloxacin hydrochloride (reference
standard), acetonitrile, methanol, phosphoric acid, amoxicillin trihydrate, potassium clavulanate. Compounds purchased from: the United States of America
2) Name: BioDiagnosticAddress: El Bouchrieh Industrial City, Saint Jean Center, 2nd Floor.www.biodiagnostic-lb.comCompound provided: triethylamine.Compound
purchased from: Spain
For amoxicillin and amoxicillin trihydrate-clavulanate
analysis, a degassed mixture of 5 volumes of methanol
and 95 volumes of sodium dihydrogen phosphate (7.8 g
L− 1) was used, adjusted to pH 4.5 using dilute orthophosphoric acid solution.
Methods
The following part details the methods followed in the
experimental and analytical protocol of the ciprofloxacin
API from the 1 brand and 4 generic antibiotics (tablets),
as well as the API from amoxicillin and amoxicillinclavulanic acid medications.
Analysis
Analysis of ciprofloxacin was carried out on binary system
using the high pressure C18 column (100 × 2.1 mm, 1.9 μm)
with UV-detection of 278 nm. The column’s temperature
was set at 30 °C using 1 μL injection volume. A C18 HPLC
column (250 × 4.6 mm) packed with particles of silica gel,
the surface of which has been modified with chemicallybonded octadecylsilyl groups (5 μm) was used as a stationary
phase for amoxicillin and clavulanic acid analysis. The UVdetection is of 220 nm, with ambient temperature column
and using 60 μL injection volume. The analysis was conducted using isocratic conditions; with the mobile phase
mixture being consistent over the entire run time.
Calibration standards
Calibration standards have spanned the range from 5 to
200% of the expected API concentration in the experimental samples and 6 standards have been used to construct the calibration curve.
Normal unknown sample
The “normal” unknown sample was prepared in the 95–
105% range for the analysis all APIs.
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Standard stock solution
Standard stock solution was prepared by dissolving Ciprofloxacin hydrochloride standard equivalent to 40 mg of
Ciprofloxacin in 100 mg of mobile phase (87:13 phosphoric acid buffer:acetonitrile). This standard solution
was further diluted with mobile phase to get the standard solution concentration of Ciprofloxacin ranging
from 1.0 mg.mL− 1 to 38 mg.mL− 1.
To each volumetric flask containing the diluted stock
solution, 0.2 mL of 7% phosphoric acid was added. Finally, the 6 standard solutions and 1 normal unknown
sample were emptied into small vials, to run through the
U-HPLC. Regarding amoxicillin and clavulanic acid, the
standard stock solution was prepared by dissolving
amoxicillin trihydrate standard equivalent to 20 mg of
clavulanic acid in 60 mL of ultra-pure water and then diluted to 100 mL with the same solvent. This stock solution was further diluted with water to get standard
solution concentrations of amoxicillin ranging from
7.5 μg/mL to 150 μg/mL and those for clavulanic acid
ranging from 1.5 μg/mL to 30 μg/mL.
were withdrawn using a sterile syringe, and filtered using
a 0.45 μm filter tip.
From this filtered solution, 5 mL were withdrawn
using a pipette and emptied into a 25 mL volumetric
flask. Mobile phase solution (87:13) was added until the
meniscus trait. The flask was put for about 10 s on the
sonicator for homogenization. These steps were repeated
for all 3 replicates. From this 25 mL volumetric flask, a
small volume was withdrawn using a dropper and emptied to fill the U-HPLC vial.
The same process was applied for the sample preparation of amoxicillin and clavulanic acid, but with the following modifications: 10 tablets of each medicine was
weighed and powdered, then a quantity of this powder,
containing the equivalent of about 0.875 g of amoxicillin
and 0.125 g of clavulanic acid, accurately weighed, was
transferred into a 1000 mL volumetric flask, and 600 mL
of ultrapure water was added and the flask was sonicated
and shaken for 10 min. The solution was made up to
volume with ultrapure water and filtered.
Repetition of samples
Identity test
The retention times of the principal ciprofloxacin API
peak in the chromatogram obtained with standard
solutions.
Unknown sample preparation
Equivalence of amount
The amount of ciprofloxacin, amoxicillin and clavulanic
acid in solution for each sample of the generic and
brand antibiotic should not be less than 85% of the
amount declared on the label (USP Monograph). In this
analysis, the weight of the unknown sample was taken as
equivalent to the mass of one tablet, including approximately the total amount of APIs mentioned on the label.
Preparation of samples
Two batches of ciprofloxacin generic and brand antibiotic were picked. From each batch, 5 tablets were
weighed and powdered. Therefore, 10 tablets in total
were mixed and their average was accurately weighed
using the diagonal sampling method: a systematic random picking technique (each 100 mg was taken from a
side of the spread total powdered mass). This average
mass, equal to the mass of 1 tablet, was transferred to a
500 mL volumetric flask. Mobile phase (87:13) was
added until the meniscus trait, along with 0.1 mL of 7%
phosphoric acid.
The flask was shaken and sonicated for 20 min to ensure proper homogenization. This was followed by a
settlement time of 10 min. A small volume of the drug
solution was then emptied into a beaker, and 15 mL
The same process mentioned above was repeated 3
times for every 2 batches of the same generic or brand
antibiotic. Therefore, for each drug, 3 pills were typically
analyzed. The same protocol was repeated for the 2
brand and 11 generic antibiotic drugs. All vials were put
consecutively in the tray, and the U-HPLC instrument
was run.
Peak detection and chromatograms
The chromatogram peak corresponding to ciprofloxacin
detection is in the range of 2–3.5 min. The reason why
there is a shift in the retention time as compared to the
Merck-Millipore ciprofloxacin assay of 10–12 min, is because of the use of the ultra-high pressure column. It is
a ThermoFischer Scientific supplied Hypersil Gold C18
column, having a 100 × 2.1 mm length, with a 1.9 μm
particle size, therefore subjecting the column to a much
higher pressure, thus minimizing the time needed to detect the peak, and leading to a better optimization
(Fig. 1).
As for the second set of results, two chromatogram
peaks were observed corresponding to the amoxicillinclavulanic acid tablets. Although both APIs are concurrent, the detection of the peaks is characteristic of each
API, with the potassium clavulanate peak retention time
at 5.8 min, and the amoxicillin trihydrate peak retention
time at 10.3 min (Fig. 2).
Results
The results section will tackle the precision of the standard solutions measurements, the accuracy of measurements of ciprofloxacin normal unknown and finally the
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Fig. 1 Chromatogram representing ciprofloxacin peak
results of the ciprofloxacin assay. The following part details the results of the liquid chromatographic analysis of
ciprofloxacin hydrochloride API molecule.
results obtained for the normal unknown samples and
the unknown samples will be accepted since this preliminary control check is valid.
Identity test and precision of measurements
This part will focus on the precision of the U-HPLC
DAD RS and subsequently the results. It shows how
close the measurements are to each other, by injecting
the standard after every three unknown sample runs, as
a quality check and assaying within 2% RSD of the 3 initial injections and be within the 0.3-min time range. The
following measurements are targeted mainly to the precision of the instrument, showing that the results from
the instrumentation are valid and deemed acceptable.
Three injections of the external standard (and the
unknown samples) have shown a peak area of APIs
within the required 2.0% relative standard deviation
(RSD), and the range of retention time is within 0.3 min
(Additional file 1: Table S1, Additional file 2: Table S2
and Additional file 3: Table S3). Therefore, the following
Accuracy of measurements
This part focuses on the accuracy of the method applied
and subsequently the accuracy of the results. A normal unknown solution is a solution that is prepared with a similar
expected concentration of the unknown sample issued
from the unknown sample preparation tablets. The relative
deviation computes the ratio of difference between the experimental value obtained and the true expected value
(Additional file 4: Tables S4, Additional file 5: Table S5,
Additional file 6: Table S6 and Additional file 7: Table S7).
It allows showing how much the experimental value has deviated from the true value. The results for the APIs show a
good agreement between the true expected concentration
and the experimental one with a relative deviation lower
than the required range of deviation ±5%.
Fig. 2 Chromatogram representing potassium clavulanate and amoxicillin trihydrate peaks
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Active pharmaceutical ingredient assay
The objective of this assay is the analytical control of the
antibiotic API: ciprofloxacin (1 brand and 4 generic
medications), amoxicillin (3 generic medications), and
amoxicillin + clavulanic acid (1 brand and 4 generic
medications), thus allowing the quantitative assessment
of their dosage as compared to the dosage stated on the
label. Included in the supplemental materials part are
the calibration curves (Additional file 8: Figure S1, Additional file 9: Figure S2 and Additional file 10: Figure S3)
corresponding to each run of sample sequence. From
this curve and with the corresponding detection peak,
the mass of API per tablet was calculated. The mean
amount of API per tablet was then calculated based on
the average of the 3 sample repetitions of each
medication.
Tables 2, 3, 4, and 5 focus on the results of the API
assay. The strategy of sampling used is the diagonal
method, whereby the crushed antibiotic tablets or capsules were dispersed on a paper in a rectangular form
and each 100 mg was taken in a diagonal mode (at opposite sides). The purpose behind this choice of sampling is to minimize the error coming from the sampling
technique. Typically, an analysis of 3 tablets from each
brand and generic antibiotic drug was performed (each
weighed mass has an equivalent mass of 1 tablet).
For each of the weighed masses tested, a calculation of
the total amount of ciprofloxacin in the medium using the
declared amount of ciprofloxacin was computed. A relative deviation of ±2% was observed for all drugs (Table 2),
thus being non-compliant with the declared contents of
the unknown samples based on the USP requirement that
Table 2 Amount in mg of ciprofloxacin in the tested weighed mass (runs 1, 2 and 3)
Relative
deviation© (%)
14.45
3.05
−5.27
98–102%
474.38
7.25
1.53
−5.12
98–102%
466.70
6.33
1.35
−6.66
98–102%
543.78
13.89
2.55
8.76
98–102%
525.33
11.15
2.12
5.07
98–102%
529.45
17.12
3.23
5.89
98–102%
483.55
2.18
0.45
−3.29
98–102%
495.70
3.42
0.69
−0.86
98–102%
487.38
5.94
1.22
−2.52
98–102%
Batch number
Mass of unknown
sample (mg)
Mass of ciprofloxacin
(CIP) per tablet (mg)
Mean amount of
ciprofloxacin (CIP)
per tablet (mg)
SD
Drug 1.1 (run 1)
BXJ1KK2 BXJ22L1
762.7
464.8028
473.66
Drug 1.2
762.8
465.8469
Drug 1.3
762.9
490.3392
Drug 4.1
0802138 0802139
762.2
482.6215
762.4
468.9408
762.4
471.5772
763.5
461.0619
Drug 5.2
763.6
465.4737
Drug 5.3
763.6
473.5532
Drug 4.2
Drug 4.3
Drug 5.1
M101 M102
Drug 3.1 (run 2)
NOB51 N2KX1
824.2
537.1647
824.4
534.4266
824.2
559.7423
824.7
536.2957
Drug 3 (2).2
824.8
514.0106
Drug 3 (2).3
824.9
525.6963
Drug 3.2
Drug 3.3
Drug 3 (2).1
M12A1 M3R01
Drug 3 (3).1
N2 L01 N2KX1
822.8
524.1745
822.9
515.5925
822.9
548.5816
773.9
485.4494
Drug 2.2
773.8
481.1726
Drug 2.3
773.7
484.0210
Drug 3 (3).2
Drug 3 (3).3
Drug 2.1
4710 2966
Drug2 [1].1(run 3)
5006 5006
774.2
493.3421
774.1
499.6229
774.1
494.1410
772.9
491.9032
Drug 2 (2).2
772.8
480.6563
Drug 2 (2).3
772.8
489.5944
Drug 2 (1).2
Drug 2 (1).3
Drug 2 (2).1
a: SD =
4710 5006
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
rffiP
ðx
xÞ
n
b: RSD = SD
100 c: RD =
X
experimental expected
expected
(a)
RSD
(b)
(%)
Medicine
USP
requirement
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Table 3 Amount in mg of amoxicillin in the tested weighed mass
Medicine
Mass of unknown
sample
Mass of amoxicillinper
pill
Mean amount
of amox (mg)
SD (a)
RSD (b) (%)
Relative
deviation (%)©
USP
requirement
Drug1.1
0.5886
544.29
547.14
2.60
0.47
9.42
90–120%
Drug1.2
0.581
549.42
Drug1.3
0.5832
547.705
Drug2.1
0.6058
554.11
556.90
4.55
0.81
11.38
90–120%
Drug2.2
0.6022
554.44
Drug2.3
0.6068
562.16
Drug3.1
0.6058
551.62
549.67
5.41
0.98
9.93
90–120%
Drug3.2
0.6022
553.83
Drug3.3
0.6068
543.55
a: SD =
rP
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðx
xÞ
n
b: RSD = SD
100 c: RD =
X
experimental expected
expected
clearly states that the sample fails analysis if the assay is
under < 98% or > 102% of the stated API content.
In the case of amoxicillin, all 3 medications are within
the 20% range (Table 3). USP requirement clearly states
that sample fails analysis if the assay is under < 90% or >
120% of the stated API content.
In the case of amoxicillin-clavulanic acid medications
(Tables 4 and 5), 4 out of 5 medications met the clavulanic
acid’s 20% range, with one medication (drug 4) exceeding
the declared content amount by 1.31% (21.31%) (Table 4).
Discussion
This study offers insight into the quality of some of the
antimicrobial generic drugs available in Lebanon. The results show a marked difference between the amount of API
found in the generic drug tablets, and the actual dosage
mentioned on the label. The drugs that have shown an
amount less than that mentioned on the label can be contributors to antibiotic resistance since the dose taken by the
patient is relatively less than that thought to be prescribed
by the physician. Thus, not all bacteria are killed, leading to
a consequential resistance of these remainder bacteria [12].
For amoxicillin, deviation from the labeled claim for potency was not found in the case of analysis of 3 different
generic preparations. All 3 generic antibiotics were within
the acceptable potency range of 90–120% set by the USP.
Thus, a positive relative deviation, lower than 20%, is confirmed for all 3 medications. Therefore, these generic
medications qualify as potent and safe, meeting the standard requirements.
Table 4 Amount in mg of clavulanic acid in the tested weighed pill
Medicine
Mass of unknown
sample
Mass of clavulanic
acid (CA) per pill
Mean amount of clavulanic
acid (CA) (in mg)
SD(a)
RSD (b) (%)
Relative deviation
(%)©
USP
requirement
Drug1.1
1478.1
150.452
147.44
2.91
1.97
17.95
90–120%
Drug1.2
1451.1
147.259
Drug1.3
1471.8
144.623
Drug2.1
1477.9
146.529
148.29
1.69
1.14
18.63
90–120%
Drug2.2
1544.8
148.447
Drug2.3
1548.8
149.912
Drug3.1
1463.0
141.884
144.37
2.56
1.77
15.5
90–120%
Drug3.2
1459.1
144.227
Drug3.3
1474.1
147.013
Drug4.1
1501.6
150.575
151.64
1.39
0.92
21.31
90–120%
Drug4.2
1501.3
151.137
Drug4.3
1512.2
153.226
Drug5.1
1513.3
147.668
147.73
0.88
0.6
18.18
90–120%
Drug5.2
1519
146.885
Drug5.3
1532.6
148.665
a: SD =
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
rffiP
ðx
xÞ
n
b: RSD = SD
100 c: RD =
X
experimental expected
expected
Hobeika et al. BMC Pharmacology and Toxicology
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(2020) 21:15
Table 5 Amount in mg of amoxicillin in the tested weighed pill
Medicine
Mass of unknown
sample
Mass of amoxicillin
per pill
Mean amount of
amoxicillin (in mg)
SD(a)
RSD(b) (%)
Relative
deviation© (%)
USP
requirement
Drug1.1
1478.1
986.3631
969.46
15.39
1.58
10.79
90–120%
Drug1.2
1451.1
965.7939
Drug1.3
1471.8
956.2252
Drug2.1
1477.9
969.4269
983.60
12.97
1.31
12.41
90–120%
Drug2.2
1544.8
986.4853
Drug2.3
1548.8
994.8977
Drug3.1
1463.0
926.9293
952.01
28.73
3.01
8.80
90–120%
Drug3.2
1459.1
945.758
Drug3.3
1474.1
983.3707
Drug4.1
1501.6
1001.293
1003.43
6.30
0.62
14.67
90–120%
Drug4.2
1501.3
998.4876
Drug4.3
1512.2
1010.529
Drug5.1
1513.3
978.5518
985.95
6.49
0.65
12.68
90–120%
Drug5.2
1519
990.6797
Drug5.3
1532.6
988.6453
a: SD =
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
rP
ðx
xÞ
n
b: RSD = SD
100 c: RD =
X
experimental expected
expected
For amoxicillin/clavulanate preparations, there is a deviation from the labeled claim for potency. Although a
relative deviation, lower than 20%, is confirmed for the
amoxicillin value in all 5 medications, the relative deviation lower than 20% is confirmed for 4 out of the 5
medications with regards to the clavulanic acid content.
Drug 4 exceeded this range. In fact, based on healthcare
professionals’ observation, patients who have taken drug
4 have reported a high rate of diarrhea. While patients
taking other preparations with the same APIs did not report as much diarrhea; it is believed that the increased
amount of clavulanic acid could be responsible for this
undesired effect. A study by Evans et al. validates this assumption, since it mentions that antibiotic-associated
diarrhea has a significantly higher occurrence rate (10–
25%) when the treatment is amoxicillin-clavulanic acid,
as compared to amoxicillin alone. Increased doses of
amoxicillin-clavulanic acid can cause increased small intestine motility (through harmful effect of the natural
microbiota), therefore leading to diarrhea [15].
Furthermore, deviation from the labeled claim for potency was found during analysis of 1 brand and 4 different generic drugs containing ciprofloxacin as API. USP
requirement regarding ciprofloxacin hydrochloride states
that the sample fails if the assay value is not within the
98–102% range of ciprofloxacin hydrochloride. A relative
deviation exceeding the ±2% required range is observed
for all four generics and even the brand antibiotic.
Therefore, they are considered substandard preparations,
and thus inappropriate for patient access.
An important note is that recent studies show that there
is an increase of ESBL-producing bacteria. Specifically,
fluoroquinolones are among the antibiotics that contribute to the rapid emergence of resistance. Adding to it the
fact that certain generic antibiotics contain less than the
required dose, resistance emergence would be favored
even more. Undesired secondary effects are not reported
at the time, but the main concern arising from these results is antibiotic resistance in case of all of these drugs
since they contain doses less or more than the expected
amount as per label claim. Thus, based on experimental
numerical data, 6 out of the 13 tested brand and generics
antibiotic medications cannot be substituted in place of
their counterparts. These findings would lead to question
the quality of the manufacturing process, affecting safety
and therapeutic equivalence.
Moreover, the interchange cannot be done since the
generic/brand drug does not fulfill the required guidelines set by the ICH and adopted by the MOPH for control of drug substances. More precisely, section 3.2.S.4.2
handling analytical procedures clearly states that “quantitative tests of active moiety in samples of drug substance”, and section 3.2.S.4.4 concerning batch analyses
requires that a “description of batches and results of
batch analyses should be provided” [16]. Therefore, these
6 drugs qualify as substandard and do not meet quality
assurance requirements. No similar studies targeting ciprofloxacin analytical control have been conducted in
Lebanon, but only one study was conducted in the Middle East, specifically in Yemen. It assessed 5 different
Hobeika et al. BMC Pharmacology and Toxicology
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(2020) 21:15
brands of 500 mg ciprofloxacin hydrochloride tablets,
bought from retail pharmacies, and results were however
deemed satisfactory, with all brands meeting British
Pharmacopeia and US Pharmacopeia requirements [17].
Therefore, our results highlight the added risk of having substandard antibiotics, in a community that considers antibiotics as a solution to almost all infectious
cases, and tries to pay less from out of pocket. In a study
conducted in Beirut, 42% out of the 319 participants reported buying antibiotics based on the pharmacist’s advice and not a physician’s prescription (18.8%). The
cause behind this decision was believed that patients
wanted to save time (55.7%) and money i.e. the cost of
physician’s prescription (33.6%) [18]. The misuse of substandard antibiotics available on the market puts patients in a double trouble situation.
Consequently, to adequately address the safety of
medication substitution, a proper guidance of healthcare
professionals towards therapeutic equivalence of brand
versus generic medication is required. The following strategic points aim to serve as guidelines to the making of
clearer choices based on the safety of interchanging brand
and generic medications. Recently, the Lebanese Order of
Pharmacists (OPL), which is the official professional association of pharmacists in Lebanon and the legal partner of
the MOPH, suggested to the MOPH a proposal regarding
the safety of medication substitution. Its aim was to guide
healthcare professionals towards the therapeutic equivalence of generic vs. brand medications.
The proposal included the following strategic points,
to be discussed and agreed upon among all stakeholders:
a) Unified patient information leaflet: The OPL
suggested to the MOPH to use of a unified patient
information leaflet, easily read and understood by
the patient, on all medications that have a common
indication. A template of the leaflet was also
suggested.
b) Reference drugs list: since the generics should be
bioequivalent to the brand, the OPL proposed the
creation of a reference drugs list. The reference
drug is the drug to which all drugs are compared
and that is approved by the MOPH upon the
registration of a new drug; this aims at identifying
discrepancies between multiple bioequivalent
generics when compared to a single standard drug
c) Laboratories following the Good Laboratory
Practices (GLP): the MOPH should have a list of
laboratories that follow the GLP and ensure that
they continue to follow these practices and apply
the terms of the biological match.
d) List of interchangeable medications: the MOPH
should have a list of all generic medications that are
interchangeable with the brand, based on the
therapeutic equivalence. All generics that are not
listed can be prescribed by the doctor but cannot
be considered by the pharmacist as a substitution
option. This list constitutes basic scientific
information for the community pharmacist for
substituting medications in an effective and secure
manner.
e) Rating guidelines: the OPL specified a guideline for
the substitution of generic medications by showing
the results of the therapeutic equivalence between
generics and inserting a rating that allows the
possibility of drug substitution. As for narrow
therapeutic index drugs and sustained-release
medications, they are not to be replaced except
under specific circumstances mentioned in the
booklet according to scientific international
standards.
f) Practical pointers for generic substitution flowchart:
Once all previous points are finalized, the OPL
would develop a chart for the substitution of
generic medications to guide community
pharmacists and alert them upon substituting the
abovementioned medications, taking into
consideration the physiological characteristics of the
patient (age, weight, liver function, kidney function
and other critical conditions).
These strategic points would serve as guidelines for
physicians and pharmacists in Lebanon, allowing them
to make clearer choices and safely interchange brands
and generics according to patients’ economic status.
This would be the first step towards the establishment
of an equivalent to the US “Orange Book”.
Limitations of the study
This study focused mainly during the experimental part:
on the therapeutic equivalence concept of the brandgeneric substitution, without having tested for bioequivalence that is another main factor to be taken into consideration for substitution evidence. The assay for
quantification of the medications focused on the analytical
extraction and quantification of the API using U-HPLC.
Another test that can be of value is the dissolution test,
which was not performed during this analytical study and
can give insights into the therapeutic equivalence. This
sample size only included 13 medications; a bigger sample
size would definitely make the results and conclusions
more reliable and statistically significant. The standard solution preparation and the unknown sample preparation
protocols are not identically the same, and the filtration of
the solution before the final dilution was only done in the
case of the unknown sample preparation. After filtration
of the solution containing the API, excipients and impurities, an amount of API may have been lost by adhering to
Hobeika et al. BMC Pharmacology and Toxicology
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(2020) 21:15
the filter. Therefore, there might be an error on the recuperated amount of API. Finally, this study mainly focused
on the quantification assay and drew conclusions regarding potential antibiotic resistance. Additional in vitro studies (susceptibility of bacteria to antibiotics, etc...) may add
further evidence towards the role of substandard medications and their effects on the biological and public health
levels.
Conclusions
The results of this study showed that the tested generic
antibiotics belonging to the fluoroquinolones class, containing ciprofloxacin as the active pharmaceutical ingredient, were found to be substandard to the labeled
dosage claim. In addition, one generic antibiotic containing amoxicillin and clavulanic acid was found to be out
of range for its clavulanic acid content. These findings
raise questions regarding the quality of medications
available in Lebanon and open the door towards a better
testing of generic medications, and the establishing of a
database accessible to healthcare professionals for an informed and secure choice of medications.
Authors’ contributions
EH carried out the research and drafted the manuscript; JS designed and
approved the experimental protocols to be followed in the study; JF and EH
were involved in the data analysis; WH, SAA and GS reviewed the
manuscript and corrected it for intellectual content; SH and PS assisted in
drafting; PS designed the study; all authors reviewed the final manuscript
and gave their consent. All authors read and approved the final manuscript.
Funding
This study is an initiative of the Order of Pharmacists of Lebanon and was
carried out at its request. The authors would like to thank the National
Council of Scientific Research CNRSL for supporting this study through the
Doctoral Fellowship Program and the Lebanese Order of Pharmacists for
providing drug substances for this study. Funders had no role in the design
of the study and collection, analysis, and interpretation of data and in
writing the manuscript.
Availability of data and materials
All data generated or analysed during this study are included in this
published article and its supplementary information files.
Ethics approval and consent to participate
Ethical approval was not needed since no participants were involved in this
study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Additional file 2: Table S2. Precision of peak detection of the standard
solutions (amoxicillin 500 mg medications).
Author details
1
Faculty of Arts and Sciences, Holy Spirit University of Kaslik (USEK), Jounieh,
Lebanon. 2School of Engineering, Holy Spirit University of Kaslik (USEK),
Jounieh, Lebanon. 3Drug Information Center, Order of Pharmacists of
Lebanon, Beirut, Lebanon. 4Faculty of Medicine and Medical Sciences, Holy
Spirit University of Kaslik (USEK), Jounieh, Lebanon. 5INSPECT-LB: Institut
National de Sante Publique, Epidemiologie Clinique et Toxicologie, Beirut,
Lebanon. 6Faculty of Pharmacy, Lebanese University, Beirut, Lebanon.
7
Faculty of Medicine, Lebanese University, Beirut, Lebanon.
Additional file 3: Table S3. Precision of peak detection of the standard
solutions (amoxicillin and clavulanic acid medications).
Received: 12 November 2019 Accepted: 31 January 2020
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s40360-020-0390-y.
Additional file 1: Table S1. Precision of peak detection of the standard
solutions (runs 1, 2 and 3).
Additional file 4: Table S4. Accuracy of measurements of ciprofloxacin
solution (runs 1, 2 and 3).
Additional file 5: Table S5. Accuracy of measurements of amoxicillin
solution.
Additional file 6: Table S6. Accuracy of measurements of clavulanic
acid solution.
Additional file 7: Table S7. Accuracy of measurements of amoxicillin
solution.
Additional file 8: Figure S1. Calibration curve for ciprofloxacin run 1.
Additional file 9: Figure S2. Calibration curve for ciprofloxacin run 2.
Additional file 10: Figure S3. Calibration curve for ciprofloxacin run 3.
Abbreviations
API: Active pharmaceutical ingredients; DAD: Photodiode array detector;
ESBL: Extended Spectrum Beta Lactamase; FDA: Food & Drug Administration;
HPLC: High Pressure Liquid Chromatography; ICH: International Council on
Harmonization; MOPH: Lebanese Ministry of Public Health; OPL: Order of
Pharmacists of Lebanon; RSD: Relative standard deviation; U-HPLC: Ultra High
Performance Liquid Chromatography; USP: United States Pharmacopeia;
WHO: World Health Organization
Acknowledgements
None.
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