Pharm Technol Hosp Pharm 2018; 3(4): 199–206

Nelly Lonca*, Fabienne Maillard, Géraldine Leguelinel, Tahmer Sharkawi and Ian Soulairol

Validation of an HPLC Assay Method for Routine

QC Testing and Stability Study of Compounded
Low-Dose Capsules of Acetylsalicylic Acid
https://doi.org/10.1515/pthp-2018-0022 Introduction
Received June 01, 2018; revised September 11, 2018; accepted
September 12, 2018
Acetylsalicylic Acid (ASA) is used in medicine since the
Abstract early twentieth century. It is an anti-inflammatory non-
steroidal drug used at high doses mainly in the sympto-
Background: The intolerance to Acetylsalicylic Acid
matic treatment of slight to moderate pain and inflamma-
(ASA) can be detected by conducting oral provocation
tion and/or febrile states and at low doses in the
testing (OPT), which is to gradually introduce low doses
prevention of secondary thromboembolic complications
of ASA. To perform this test, hospital pharmacies com-
in patients with ischemic or artheromateous disease and
pound small batches of different low-dosage ASA cap-
prevention of thromboembolic events after surgery or
sules. This work aims to validate a method for fast HPLC-
vascular intervention [1].
UV assay that allows routine quality control and physi-
Ferdinand Widal syndrome was described for the first
cochemical stability studies of capsules.
time in 1922. It consists of a triad of symptoms composed
Methods: The chromatographic separation is performed
of chronic rhinosinusitis with or without nasal polyposis
using a C18 column Kinetex (100 A, 50 × 4.6 mm, 2.6 µm)
and intolerance to aspirin and/or to other non-steroidal
equipped with a precolumn C18. Separation is achieved
anti-inflammatory drugs [2]. The intolerance to aspirin
using a mobile phase composed of water-acetonitrile-
appears after several years of evolution. It is character-
orthophosphoric acid (68:32:0.2 v/v/v) at a flow rate of
ized by the appearance of acute rhinoconjunctivitis with
0.8 mL/min and UV detection at 237 nm.
urticaria, associated with significant asthma and poten-
Results: Validation shows that the method was suitable
tially leading to angioedema and/or anaphylactic shock.
for routine analysis and could be used to perform stabi-
There are no biological test for diagnosing aspirin intol-
lity studies.
erance [3]. The clinical diagnosis is made following
Conclusions: The 5, 25, 100 and 250 mg dosed capsules
patient questioning and thorough clinical examination,
show acceptable stability over 12 months, while the 1 mg
but must always be confirmed by conducting oral provo-
dosed capsule show an unacceptable degradation of more
cation testing (OPT). The OPT remains an essential tool to
than 15 % after 3 months. Therefore, hospital pharmacy
make this diagnosis. OPT aims to gradually introduce
can plan the manufacture of capsules and anticipate the
very low doses of ASA in a patient suspected of intoler-
requests of doctors.
ance. Because of its danger, it must be performed within
an intensive care unit of a hospital [4, 5].
Keywords: acetylsalicylic acid, stability-indicating,
Compounding specific low-dosage capsules of ASA
HPLC-UV, hard capsule, allergology, stability study
has several advantages over diluting existing industrially
prepared dosage forms for performing the OPT. First of
all, by compounding capsules, there is more control of
the administered dose, which allows securing the medi-
*Corresponding author: Nelly Lonca, Department of Pharmacy, CHU
cal management of the patient. Secondly, it simplifies the
Caremeau, Nimes, France, E-mail: nelly.lonca@chu-nimes.fr
Fabienne Maillard, Géraldine Leguelinel, Department of Pharmacy, CHU preparation and administration of the ASA dose by the
Caremeau, Nimes, France, E-mail: fabienne.maillard@chu-nimes.fr; medical staff in the service.
geraldine.leguelinel@chu-nimes.fr However, batch compounding requires qualitative
Tahmer Sharkawi, UMR 5253, Equipe MACS, ICGM, University of and quantitative controls on routine batch production
Montpellier, Montpellier, France
[6]. Moreover, it is essential to perform stability studies
Ian Soulairol, Department of Pharmacy, CHU Caremeau, Nimes, France;
UMR 5253, Equipe MACS, ICGM, University of Montpellier, Montpellier, to ensure sufficient expiry dates since these preparations
France, E-mail: ian.soulairol@chu-nimes.fr are not used very frequently [7]. Also, as part of the

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200 N. Lonca et al.: Validation of an HPLC Assay Method
production of capsules content uniformity as it is
described in the European Pharmacopeia must be carried
out on 10 capsules [6].
The objective of this work is to validate a method for
fast HPLC assay of ASA, for routine quality control of ASA
batches produced at different dosages and which can
also be used to check the stability of such preparations.
Materials and methods
Chemicals
Chemical reference substances of ASA and salicylic acid
(SA) were provided by the European Directorate for the
Quality of Medicines and Healthcare (EDQM, France). SA
is well known as the degradation product of ASA [8].
European Pharmacopeia (EP) conforming ASA was
provided by Cooper (COOPER, France). HPLC grade pur-
ified water was obtained by reverse osmosis and filtration
on a Millipore simplicity system (Mercks Millipore, USA)
and used to prepare all solutions. Acetonitrile and ortho-
phosphoric acid (≥85 %) were respectively purchased by
Sigma Aldrich (Sigma Aldrich, USA) and international
VWR (VWR International, France). All chemicals used
were of analytical grade.
For the capsule compounding, carmine and lactose
were provided by COOPER (Cooper, France) and comply
with the requirements of the European Pharmacopoeia.
Gelatin capsules provided by Fagron (Fagron, France)
were used.
HPLC instrumentation and conditions
A Prominence HPLC system (Shimadzu, Japan) composed of
two LC-20AD pumps, an injector Sil-20AC, a CTO-20A oven
and a diode array detector SPD-M20A was used and run with
the LC-Solution software (Shimadzu, Japan). The chromato-
graphic separation was performed using a C18 column
Kinetex (100 A, 50 × 4.6 mm, 2.6 μm) (Phenomenex, USA)
equipped with a precolumn C18 (4.6 mm) (Phenomenex,
USA) and an in-line filter for UHPLC (Phenomenex, USA).
Separation was achieved using a mobile phase (MP) com-
posed of: water-acetonitrile-orthophosphoric acid (68: 32:
0.2, v/v/v) at a flow rate of 0.8 mL/min and UV detection at
237 nm. Column oven temperature was set to 30 ± 5 °C, injec-
tor at 23 ± 5 °C and detector cell at 40 °C ± 1 °C.
Preparation of stock and standard solutions
Acetylsalicylic acid stock solution (12.5 mg/mL) was pre-
pared in MP and used to prepare 5 standard solutions
(0.025, 0.05, 0.075, 0.1 and 0.25 mg/mL). A salicylic acid
stock solution (8.15 mg/mL) was also prepared with the
MP, and diluted 20 times prior to its injection.
Forced degradation
Forced degradation studies [9] of AAS and capsule of
AAS were performed for validate the stability indicating
power of the analytical procedure used. A mass of
125 mg AAS or capsule containing 5 mg of AAS were
diluted in 10 mL 0.1 HCl or 0.1 NaOH and heat at 60 °C
for 5 hours. Acid and base treated sample were neutra-
lized and were then diluted to a concentration of
0.125 mg/mL with the mobile phase. The samples were
analyzed using HPLC method. The chromatogram
obtained from each degradation samples (Figure 2(a),
2(b) for AAS and Figure 2(c), 2(d) for AAS capsules) was
compared with chromatograms obtained with AAS and
AS solutions (0.075 mg/mL) (Figure 1(a), 1(b)).
Preparation of capsules
Batches of 100 capsules were prepared using a semi-
automatic capsule filler with lever lock and stainless
steel plate (LGA®, France). Five different dosages were
prepared, 1, 5, 25, 100 and 250 mg. Table 1 summarizes
the composition of the different dosed capsules. Lactose
monohydrate was used as filler and carmine was used as
visual control agent of homogeneity of the mixture. The
capsules were packaged in individual opaque blisters
ensuring UV protection (Practidose®France). The blister
used ensures an UV protection and is composed of cells
that can be detached individually after the labels have
been glued. The labels are made of a special paper with
an aluminium insulation and an adhesive.
HPLC validation
The analytical validation was performed according to the
recommendations of the commission report of SFSTP [10]
and NF T90-210 guide lines [11].
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Specificity – degradation products
Specificity was evaluated by injecting a solution of exci-
pients (lactose and carmine) at 5.5 mg/mL in MP.
Forced degradation of 4 solutions presented in § 2.4.
was also conducted. These solutions were diluted to 1/3
in the MP before injection.
N. Lonca et al.: Validation of an HPLC Assay Method 201
Figure 1: Representative chromatogram ASA chemi-
cal reference substance and chemical structure of
ASA (a), chromatogram SA chemical reference sub-
stance and chemical structure of SA (b) and solu-
tion of ASA capsule 0.075 mg/mL (c).
Linearity
Linearity was checked with MP solutions of ASA alone or
ASA mixed with the excipients and repeated five times to
ensure reproducibility. ASA concentration ranged
between 0.025 and 0.25 mg/mL. Five ASA solutions of differ-
ent concentrations were prepared from a stock solution to
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 202 N. Lonca et al.: Validation of an HPLC Assay Method

395 445
SA SA
345 a 395 b
295 345
Intensity (mAU)

Intensity (mAU)
295
245
245
195
195
145
145
95 95
45 ASA 45 ASA
-5 -5
0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5
Time (min) Time (min)

495 495
c d
SA SA
395 395
Intensity (mAU)

Intensity (mAU)
295 295

195 195

95 95
ASA ASA
-5 -5
0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5
Time (min) Time (min)

Figure 2: Representative chromatogram ASA Acid hydrolysis (a), ASA hydrolysis alkaline (b), ASA capsule acid hydrolysis (c) and ASA
capsule alkaline hydrolysis (d).

Table 1: Formulation for 100 Capsules of ASA. conducted for 3 mixtures corresponding to 1, 5 and
25 mg/capsule.
Dosage  mg  mg  mg  mg  mg For each mixture, 6 samples were prepared and ana-
ASA  mg  mg  mg  mg  mg
lyzed. This operation was performed 4 more times at the
Lactose . g . g . g . g g rate of once a day in reproducible conditions by varying
Carmine  mg  mg  mg  mg  mg one parameter at a time. The varied parameters were:
Capsule N° N° N° N° N° operator, stock solution, column, and day.

12.5 mg/mL. Excipient interference was evaluated by using 3

Stability of standard solutions
identical powder mixtures as those used to produce the 1, 5
and 25 mg dosed capsules (Table 1). For these solutions ASA
concentrations ranged from 0.05, 0.25 and 0.125 mg/mL. Stability of solutions in the mobile phase (ASA alone or
mixed with excipients) was evaluated on the same solu-
tions to the linearity test at different times 0, 4, 8 and
12 hours. They were stored in the automatic injector at
Robustness
23 ± 5 °C. For the stability study, analysis was performed
in triplicate, each time using a different stock solution.
Robustness was evaluated by increase or decrease in flow
rate of mobile phase (0.6 mL/min or 1.0 mL/min). The
evaluation of this parameter was based on the resolution
between the peak of ASA and SA. Stability study of ASA capsules

The study focused on five dosages 1, 5, 25, 100 and

Accuracy (reproducibility-repeatability) 250 mg. Each dosage assay was conducted on 4 batches,
with the exception of the 1 mg dosage for which the study
Accuracy of the assay has been studied with respect to was conducted on 5 batches. The capsules were packaged
both repeatability and reproducibility. Accuracy was in individual blister and stored in normal conditions at a

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 N. Lonca et al.: Validation of an HPLC Assay Method 203

controlled room temperature (20 ± 5 °C). The batches
were analyzed at 0, 3, 6, 9 and 12 months using the
method of HPLC described above.
Content uniformity was based on the European
Pharmacopoeia (2.9.40 Uniformity of content of single-
dose preparations) [12]. For the 1 and 5 mg dosed capsules,
the content determination at t0 requires to individually
titrate 10 capsules. For the 25, 100 and 250 mg dosed cap-
sules, content determination was performed by emptying
and weighing individually the content of ten capsules.
Three samples of 25 mg of powder of the total emptied con-
tent was assayed and used for the content determination.
The average concentration expressed in mg/mL was
calculated and the percentage of degradation relative to
the initial value determined with the following equation:
tx − t0
% of degradation = × 100
t0
tx = content determination at time “x” and t0 = content
determination at the beginning of the study.
Product stability is demonstrated if the percentage
degradation is less than 10 %.
– There is a linear relationship between concentration
and the detected signal (y = 336.47x + 0.67)
– The intercept cannot be equated to 0 (with a risk
of 5 %)
– The slopes and intercepts were not significantly dif-
ferent between the active ingredient alone and phar-
maceutical form reconstituted (with a risk of 5 %,
t = 0.5256 and 0.6127).
Accuracy
The relative standard deviation (RSD) for repeatability and
reproducibility were determined for each concentration
tested (0.05, 0.125 and 0.25 mg/mL). They were all less
than 5 %. All data are presented in Table 2. Any precisions
did not exceed the required RSD value (5 %). The maximum
value (4.14 %) was observed for worst concentrations. The
intermediate precision (repeatability and reproducibility)
test was satisfactory. This assay method can be used routi-
nely to control the quality of compounded acetylsalicylic
acid capsules as well as a tool to perform stability studies.

Results Table 2: Accuracy result.

Dosage of capsules  mg  mg  mg
Method validation
Concentration of . mg/mL . mg/mL . mg/mL
solutions (diluted
Specificity, forced degradation and robustness
injected (mg/mL) solution of
/th)
In chromatographic conditions, solutions references ASA
and SA retention times were respectively 1.32 and 1.74 Accuracy

minutes (Figure 1(a), 1(b)). Figure 1(c) confirms that the SD repetability . . .
excipients (lactose and carmine) did not interfere with the SD accurancy . . .
detection of ASA and its degradation product at 237 nm RSD repetability . . .
wavelength. For all solutions tested, forced degradation (%RSD)
RSD accurancy . . .
in acid and alkaline media showed a decrease in the ASA
(%RSD)
peak and an increase in the SA peak (Figure 2). Accuracy (%) −. . −.
Between 0.6 mL/min and 1.0 mL/min, retention times
range from 1.8 to 1.09 min for ASA and from 2.53 to 1.52
for SA. The resolution between the two peaks is satisfac-
Stability of standard solutions in the automatic injector
tory (between 5.12 and 4.8).
Figure 3(a) shows evolution of content of ASA standard
Linearity solutions (0.025, 0.050, 0.075, 0.100 and 0.250 mg/mL)
stored in the automatic injector at 23 ± 5 °C for 0, 4, 8 and
The assay method of ASA was linear in the tested con- 12 hours. For all solutions, degradation of ASA was less
centration range (0.025–0.25 mg/mL). The correlation and than 1.4 %. Figure 3(b) shows the stability of solutions
determination coefficient were respectively: 0.9978 and prepared from mixtures 1, 5 and 25 mg at times of 0, 4, 8
0.9957. The results allow the following conclusions and 12 hours. All solutions had a degradation rate less
through different statistical tests: than 0.6 % in the automatic injector at 23 ± 5 °C.

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204 N. Lonca et al.: Validation of an HPLC Assay Method
Figure 3: Degradation study in HPLC automatic injector at 23 ± 5 °C for control solutions (a) and solutions made from mixtures of capsule of
1, 5 and 25 mg (b).
All solutions were therefore considered to be stable for
12 hours, with a degradation rate less than 2 %.
Stability study of ASA capsules
Figure 4 shows the evolution of the content of ASA
capsules over time. When considering relative degrada-
tion, capsules containing 1 mg ASA have a very signifi-
cant degradation rate of approximately 15 % at
3 months and greater than 30 % at 12 months. For the
5 mg capsules, ASA degradation rate was around 6 % at
12 months. For other doses tested (25, 100 and 250 mg),
the degradation rate was less than 5 % at 12 months.
Discussion
For the analytical method, the correlation coefficient was
0.9978, so the method was considered as linear.
Moreover, the accuracy, the repeatability and the inter-
mediate precision were systematically inferior to 5 %.
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Figure 4: Evolution of the content of ACAS capsules over time.
So, the method was considered as accurate, repeatable
and with an acceptable intermediate precision.
About the stability indicating capacity of the method,
for each tested condition (acid hydrolysis and alkaline
hydrolysis), the peak of SA, a degradation product of ASA,
was observed. The method is thus stability indicating.
The degradation tests have highlighted the ability of
the method to detect degradation products of ASA. It can
therefore be used to evaluate the stability of the ASA.
About the stability of ASA capsules, when consider-
ing absolute quantities of ASA degradation the difference
is nearly non-significant suggesting that an equal abso-
lute amount of ASA is degraded due to hydrolysis in
capsules. The higher relative degradation for the 1 mg
dosed capsule can also be explained by the antioxidant
properties of ASA as it is known to act an inhibitor of
radical formation and free radical quenching agent [13,
14]. This property is probably responsible for the stability
obtained for capsules containing 5 mg and over ASA as it
would acts as preservative of itself. But for a content of
less than 5 mg, it is too small in regards of the total
amount of ASA in the capsule.
Conclusion
This dosage method is to perform quality control and
stability studies of compounded ASA capsules containing
lactose and carmine. This work is part of a context of small
batch production manufactured within hospital pharmacy.
N. Lonca et al.: Validation of an HPLC Assay Method 205
The HPLC-UV method proposed is simple, cheap and
quick. It is specific, accurate and precise. It can be used
routinely for uniformity testing described in PE. This
dosage method has the ability to separate the drug from
the degradation product and excipients used in this study.
The stability study conducted on the capsules ASA 1, 5, 50,
100 and 250 mg demonstrate that all dosages, except for
the 1 mg capsules, are suitably stable for 12 months.
Therefore, hospital pharmacy can plan the manufacture
of capsules and anticipate the requests of doctors.
It is then possible to respond immediately to this need
in case of emergency, as is the case for some patients with
imperative cardiologic indications have a history of allergy
to ASA. A rapid challenge-desensitization procedure with
compounded low-dose capsules of ASA may allow intro-
duction of ASA at therapeutic doses within a few hours [15].
Conflict of interest statement: The authors state no con-
flict of interest. The authors have read the journal’s
Publication ethics and publication malpractice statement
available at the journal’s website and hereby confirm that
they comply with all its parts applicable to the present
scientific work.
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