pharmaceutics
`Article
`Stability of Ophthalmic Atropine Solutions for Child
`Myopia Control
`Baptiste Berton 1, Philip Chennell 2, *
` , Mouloud Yessaad 1, Yassine Bouattour 2
` ,
`Mireille Jouannet 1, Mathieu Wasiak 1 and Valérie Sautou 2
`1 CHU Clermont-Ferrand, Pôle Pharmacie, F-63003 Clermont-Ferrand, France;
`baptiste-berton@hotmail.fr (B.B.); myessaad@chu-clermontferrand.fr (M.Y.);
`mjouannet@chu-clermontferrand.fr (M.J.); mwasiak@chu-clermontferrand.fr (M.W.)
`2 CNRS, SIGMA, ICCF, CHU Clermont-Ferrand, Université Clermont Auvergne,
`63000 Clermont-Ferrand, France; ybouattour@chu-clermontferrand.fr (Y.B.);
`vsautou@chu-clermontferrand.fr (V .S.)
`* Correspondence: pchennell@chu-clermontferrand.fr
`Received: 25 June 2020; Accepted: 7 August 2020; Published: 17 August 2020
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`/gid00048/gid00043/gid00031/gid00028/gid00047/gid00032/gid00046
`Abstract: Myopia is an ophthalmic condition a ffecting more than 1 /5th of the world population,
`especially children. Low-dose atropine eyedrops have been shown to limit myopia evolution during
`treatment. However, there are currently no commercial industrial forms available and there is
`little data published concerning the stability of medications prepared by compounding pharmacies.
`The objective of this study was to evaluate the stability of two 0.1 mg /mL atropine formulations
`(with and without antimicrobiobial preservatives) for 6 months in two di fferent low-density
`polyethylene (LDPE) multidose eyedroppers. Analyses used were the following: visual inspection,
`turbidity, chromaticity measurements, osmolality and pH measurements, atropine quantification by a
`stability-indicating liquid chromatography method, breakdown product research, and sterility assay.
`In an in-use study, atropine quantification was also performed on the drops emitted from the multidose
`eyedroppers. All tested parameters remained stable during the 6 months period, with atropine
`concentrations above 94.7% of initial concentration. A breakdown product (tropic acid) did increase
`slowly over time but remained well below usually admitted concentrations. Atropine concentrations
`remained stable during the in-use study. Both formulations of 0.1 mg /mL of atropine (with and
`without antimicrobial preservative) were proved to be physicochemically stable for 6 months at 25◦C
`when stored in LDPE bottles, with an identical microbial shelf-life.
`Keywords: atropine; ophthalmic solution; stability; myopia
`1. Introduction
`Myopia (or short-sightedness) is an ophthalmic condition that leads to blurred long-distance
`vision, generally characterized by a refractive error of −0.5 or −1 diopters. Overall, it has been
`estimated that currently 1.4 billion people in the world are myopic (22.9% of the population), and crude
`estimations suggest that, by 2050, there will be 4.7 billion people a ffected (nearly 50% of world
`population) [ 1]. The prevalence of myopia is variable between countries, a ffecting for example
`about 30% of young adults in Europe [ 2], 59% in North America [ 3], to more than 95% in some
`student populations of Asian countries [4,5], with onset of the disease occurring during childhood
`and adolescence. Many risk factors have been suggested or clearly identified, such as time spent
`performing close-vision activities, such as reading or looking at smart-device screens [ 6], lack of
`physical exercise, or exposure to sunlight [3,7], often all linked to higher education rates [ 8]. If left
`uncorrected, myopia has been shown to have major consequences on children’s level of education,
`Pharmaceutics 2020, 12, 781; doi:10.3390/pharmaceutics12080781 www.mdpi.com /journal/pharmaceutics
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`quality of life, and personal and psychological well-being [9], and its economic impact on society has
`been estimated at US$244 billion from global potential productivity loss [10]. Several recommendations
`have been proposed to limit the onset of myopia, like encouraging children spending time outside,
`limiting close-vision activities, and adapting light conditions [11], but whilst fundamental, they might
`not be sufficient or easily implemented. Current therapeutic options all have limitations; for example,
`orthokeratology (reshaping of the cornea by a hard, hydrophilic, gas-permeable contact lens worn
`during sleep and removed during the day) does not stop disease progression to returning to its
`previous rate after treatment discontinuation and requires high levels of patient compliance [ 12].
`The use of correction glasses or lenses does not address the root-cause, refractive surgery is only
`curative, and its cost-effectiveness is still uncertain [13]. Pharmacological treatments have however
`shown some very promising potential. Of these, mydriatic agents such as atropine or tropicamide
`gained interest early on [ 14], with atropine being the most studied. Its biological action has been
`surmised as involving a complex interplay with receptors on di fferent ocular tissues at multiple
`levels, leading to a decrease in change in the cycloplegic refraction and axial length elongation [15].
`Initially tested at 1% concentration during the ATOM1 trial, atropine eyedrops were proved to be
`effective in controlling myopic progression but caused important visual side e ffects resulting from
`cycloplegia and mydriasis [ 16]. Since then, several clinical trials have evaluated the safety and
`efficiency of atropine eye drops at lower concentrations. The ATOM2 trial studied myopia progression
`in 400 children 2 years after treatment with 0.01%, 0.1%, and 0.5% and found the 0.01% concentration
`to be the concentration causing the least side e ffects for comparable efficacy in controlling myopia
`progression [17,18]. Very recently, the LAMP phase 2 trial report confirmed that concentrations ranging
`from 0.01% to 0.05% were well tolerated in 383 children after two years of treatment, with patients
`experiencing rare and mild side effects [19], even if the need for photochomratic glasses was higher
`than 30% for treated patients. The ATOM1, ATOM2, and LAMP trials were all conducted on Asian
`patients, and some have highlighted the need for high-quality evidence from European populations on
`atropine effectiveness in controlling myopia progression [20]. However, a smaller study on European
`paediatric patients also concluded that 0.01% atropine eye drops slowed the rate of myopia progression
`whilst retaining a favourable safety profile [21].
`Despite all this recent and very favourable clinical data, there is still currently no commercially
`available low dose formulation of atropine eyedrops. In order to treat patients, hospital and
`compounding pharmacies could produce the desired ophthalmic solution, but the lack of long-term
`validated stability data severely limits their conservation period by imposing short expiration dates
`after preparation [22,23]. Indeed, few studies have been published concerning atropine eyedrops
`stability, but the analyses were either lacking several tests or suffered from shortcomings concerning
`breakdown product research [24–26]. As single-dose container technology is not readily available to
`compounding pharmacies, multidose eyedroppers (often in low dose polypropylene) are the most
`used container and therefore the most studied [27–30]. However, not all of those devices possess a
`system allowing their content to be preservative-free, and their contents must therefore be preserved.
`Because 0.1 mg/mL is the concentration with currently the most data concerning safety and efficacy,
`the aim of this study was therefore to assess the physicochemical stability and to control the sterility of
`two 0.1 mg/mL atropine ophthalmic solutions (with and without an antimicrobial conservative) in
`two different low-density polyethylene (LDPE) multidose eyedroppers (one with a sterility-preserving
`technology allowing the absence of an antimicrobial preservative in the formulation and the other
`without such a system in order to allow choice of container depending on stability data) at 25◦C for six
`months in unopened eyedroppers.
`2. Materials and Methods
`2.1. Preparation and Storage of Atropine Solution Formulations
`Two different formulations of 0.1 mg/mL (0.01%) atropine ophthalmic solutions were prepared:
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`- atropine solution with an antimicrobial preservative (cetrimide) for use with ethylene oxide
`sterilized white opaque LDPE squeezable multidose eyedroppers (reference VPLA25B10;
`Laboratoire CAT®, Lorris, France)
`- atropine solution without the preservative for use within gamma-sterilized white opaque LDPE
`squeezable multidose eyedropper (reference 10002134) equipped with sterility preserving Novelia®
`caps (reference 20050772; Nemera, La Verpillère, Cedex France).
`The details of each formulation are presented in Table 1. All compounds that were used were of
`pharmaceutical grade.
`Table 1. Composition of the tested atropine ophthalmic formulations. q.s: quantity su fficient.
`Chemical Components Formulation (mg)
`Without Preservative With Preservative
`Atropine sulphate (batch 18276508, exp. 31/01/2021, Inresa, France) 100 100
`Natrium dihydrogenophosphate dihydrate (NaH2PO4)
`(batch 190298040, exp. 30/11/2021, Inresa, France) 7800 7800
`Dinatrium monohydrogenophosphate dodecahydrate (Na2HPO4)
`(batch 18129611, exp. 30/04/2023, Inresa, France) 4480 4480
`Cetrimide (batch 16F08-B01-334049, exp. 05/2020,
`Fagron, Netherlands) 100
`Sodium chloride (NaCl) 0.9% (Versylene®; Fresenius Kabi France,
`Louviers, France) q.s 1000 mL q.s 1000 mL
`The formulations were prepared by dissolving atropine into the 0.9% sodium chloride solution
`at room temperature under gentle agitation before adding the hydrogenophosphate bu ffer and,
`finally, if needed, the preservative (cetrimide). For the purpose of the study, batch size was 1 L for
`both formulations. Cetrimide at a concentration of 0.01% was chosen because it is a preservative
`commonly used for the antimicrobial preservation of ophthalmic solutions, with an efficacy similar to
`benzalkonium chloride [31,32].
`The obtained atropine solutions were filtered through a 0.22-µm filter (Stericup® Sterile Vacuum
`Filtration Systems, Merck Millipore, MC2, Clermont-Ferrand, France) and then sterilely distributed
`(6 mL per unit, for a maximum filling capacity of 8 mL for both multidose eyedroppers) into the
`eyedroppers under the laminar airflow of an ISO 4.8 microbiological safety cabinet using a conditioning
`pump (Repeater pump, Baxter, Guyancourt, France). The solution was distributed into the two different
`low-density polyethylene (LDPE) eyedroppers.
`2.2. Study Design
`The stability of the 0.1 mg/mL atropine solutions was studied for 180 days at 25 ◦C in unopened
`eyedroppers and in simulated use conditions for 6 days.
`2.2.1. Stability of 0.1 mg/mL Atropine in Unopened Multidose Eyedroppers
`The eyedroppers containing atropine were stored upwards in an ICH Q1B compliant climate
`chamber (BINDER GmbH, Tuttlingen, Germany) at 25 ◦C ± 2 ◦C and 60 ± 5% residual humidity
`until analysis.
`Immediately after preparation (day 0) and at days 8, 15, 30, 90, and 180, five units per kind
`of eyedropper were submitted to the following analyses: visual inspection, chromaticity analysis,
`atropine quantification, breakdown products (BPs) research (i.e., looking specifically for products
`resulting from the degradation of atropine), osmolality, pH, and turbidity. Sterility was also assessed
`using five units for each kind of eyedropper and storage temperature immediately after preparation and
`after 60 and 180 days of storage. Initial day 0 analyses were performed immediately after conditioning
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`within 4 h after the end of the preparation of the solutions to have results as representative as possible
`of initial conditions (least degradation or modification of parameters).
`2.2.2. Evaluation of Atropine Concentrations in Eye Drops during Simulated Use
`Thirty eyedroppers were subjected to simulated patient use: every day for 6 days, one drop
`from each eyedroppers was manually emitted (i.e., the drop was squeezed out of the bottle as if to
`be administered to the eye, but instead of being administered, it was collected for analysis) at room
`temperature. Atropine quantification was then realized in triplicate from 10 collected and pooled
`drops. In between use, the bottles were stored vertically at 25 ◦C.
`2.3. Analyses Performed on the Atropine Solutions
`2.3.1. Visual Inspection
`The multidose eyedroppers were emptied into glass test tubes, and the atropine solutions were
`visually inspected under day light and under polarized white light from an inspection station (LV28,
`Allen and Co., Liverpool, UK). Aspect and colour of the solutions were noted, and a screening for
`visible macroparticles, haziness, or gas development was performed.
`2.3.2. Chromaticity Analysis
`Chromaticity and luminance were measured with a UV-visible spectrophotometer (V670,
`Jasco®, Lisses, France) using the mode Color Diagnosis of the built-in software (Spectra Manager®,
`version 2.12.00). The xyY CIE colorimetric system was used. Chromaticity was presented as a
`two-dimensionl diagram (x and y axes) representing the whole the colour system independently of
`luminance. uminance is defined as the visual sensation of luminosity of a surface measured by the
`ratio of the colour’s luminosity (in cd·cm−2) over the luminosity of pure white (reference colour) times
`100; its value Y ranges therefore between 0 (no luminosity) and 100 (maximum luminosity).
`2.3.3. Atropine Quantification and BPs Research
`Chemicals and Instrumentation
`For each unit, atropine was quantified and BPs were detected using the liquid chromatography
`(LC) method described by the European Pharmacopeia, Atropine monography [33]. The LC system
`that was used was a Prominence-I LC2030C 3D with diode array detection (Shimadzu France SAS,
`Marne La Vallée, France), and the associated software used to record and interpret chromatograms
`was LabSolutions® version 5.82. The LC separation column used was a C18 Synergi® Fusion-RP 80
`(150 × 4.6 mm, 4 µm) with an associated guard column (Phenomenex, Le Pecq, France).
`The mobile phase was a gradient mixture of phases A and B. Phase A consisted of an aqueous
`solution of 3.5 g of sodium dodecyl sulphate (SDS) (CAS 1561-21-3, purity > 99%, Sigma-Aldrich,
`St. Louis, MO, USA) in 606 mL of a 7 g/L solution of potassium dihydrogen phosphate (CAS 10049-21-5,
`purity > 99%, Sigma-Aldrich, St. Louis, MO, USA) previously adjusted to pH 3.3 with orthophosphoric
`acid (20,624.295, purity > 85%, Normapur, Prolabo, Paris, France) and mixed with 320 mL of acetonitrile
`(34851-2, purity > 99.9%, Honeywell, Charlotte, NC, USA). The final pH of phase A was of 3.9.
`Phase B consisted of 100% acetonitrile. The gradient used is presented in Table 2. All solvents were of
`analytical grade.
`The flow rate through the column for the analysis was set at 1 mL /min, with column
`thermo-regulation set to a temperature of 30 ◦C. The injection volume was of 20 µL. The quantification
`wavelength was set up at 210 nm. BP detection was realized by screening with a diode array detector
`(DAD) detector from 190 nm to 800 nm.
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`Table 2. Gradient used for the liquid chromatography (LC) mobile phase.
`Time (min) Mobile Phase (%)
`A B
`0 95 5
`2 95 5
`20 70 30
`21 95 5
`25 95 5
`Method Validation
`Linearity was initially verified by preparing one calibration curve daily for three days using five
`concentrations of atropine (European Pharmacopoeia reference standard Y0000878 (Sigma-Aldrich,
`MC2, Clermont-Ferrand, France) at 10, 20, 60, 100, and 140 µg/mL, diluted in deionized water.
`Each calibration curve should have a determination coe fficient R 2 equal or higher than 0.999.
`Homogeneity of the curves was verified using a Cochran test. ANOVA tests were applied to determine
`applicability. Each day for three days, six solutions of atropine 0.1 mg/mL were prepared, analysed,
`and quantified using a calibration curve prepared the same day. To verify the method precision,
`repeatability was estimated by calculating relative standard deviation (RSD) of intraday analysis and
`intermediate precision was evaluated using an RSD of inter-days analysis. Both RSDs should be less
`than 5%. Specificity was assessed by comparing the UV spectra DAD detector. Method accuracy was
`verified by evaluating the recovery of five theoretical concentrations to experimental values found
`using mean curve equation, and results should be found within the range of 95–105%. The overall
`accuracy profile was constructed according to Hubert et al. [34–36].
`The matrix effect was evaluated by reproducing the previous methodology with the presence of
`all excipients present in the formulation (including the preservative) and by comparing the calibration
`curves and intercepts.
`Atropine impurities described in European Pharmacopeia (atropine impurity B CRS, atropine for
`peak identification CRS (containing impurities A, D, E, F, G, and H) and tropic acid R (impurity C))
`were identified with the same method. Their retention times were collected for potential identification
`and quantification during stability studies.
`In order to exclude potential interference of degradation products with atropine quantification,
`atropine 0.1 mg/mL solutions was subjected to the following forced degradation conditions: 0.1, 0.5,
`and 1 N hydrochloric acid for 150 min at 25 ◦C; 0.1, 0.5, and 1 N chloride acid for 150 min at 90 ◦C; 0.1,
`0.5, and 1 N sodium hydroxide for 30 min at 25◦C; 15% hydrogen peroxide for 60 min at 60◦C and 90◦C;
`and 30% hydrogen peroxide for 60 and 180 min at 90 ◦C. Susceptibility to light was performed 3 times
`after solution preparation for 180 min and 4 and 8 days using an UVA light in climatic chamber (25◦C).
`Tropic acid was quantified at 210 nm using the same method as for atropine quantification in the
`presence of a phosphate buffer, using a calibration curve ranging from 0.1 to 5.0µg/mL validated using
`the same methodology as previously described for the validation of the atropine quantification method.
`2.3.4. Osmolality, pH, and Turbidity Measurements
`For each unit, pH measurements were made using a SevenMultiTM pH-meter with an InLabTM
`Micro Pro glass electrode (Mettler-Toledo, Viroflay, France). Measures were preceded and followed
`by instrument validation using standard buffer solution of pH 4 and pH 7 (HANNAH® Instrument,
`Tannerries, France). Osmolality was measured for each solution using an osmometer Model 2020
`Osmometer® (Advanced instruments Inc., Radiometer, SAS, Neuilly Plaisance, France). Turbidity was
`measured using a 2100Q Portable Turbidimeter (Hach Lange, Marne La Vallée, France), by pooling the
`five samples per analysed experimental condition and assay time to obtain the necessary volume for
`the analysis. The results were expressed in Formazin Nephelometric Units (FNU).
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`2.3.5. Sterility Assay
`Sterility was assessed using the European Pharmacopoeia sterility assay (2.6.1). Multidose eyedroppers
`were opened under the laminar air flow of an ISO 4.8 microbiological safety cabinet, and the contents
`were filtered under vacuum using a Nalgene® analytical test filter funnel onto a 47-mm diameter cellulose
`nitrate membrane with a pore size of 0.45 mm (ref 147-0045, Thermo Scientific, purchased from MC2,
`Clermont-Ferrand CEDEX, France). The membranes were then rinsed with 500 mL deionized water
`(V ersylene®; Fresenius Kabi, France, Louviers, France) and divided into two equal parts. Each individual
`part was transferred to each of a fluid thioglycolate and soya tripcase medium and incubated at 30–35◦C
`or 20–25◦C, respectively , for 14 days. The culture medium was then examined for colonies.
`2.4. Data Analysis—Acceptability Criteria
`The stability of diluted atropine solutions was assessed using the following parameters:
`visual aspect of the solution, turbidity, pH, osmolality, atropine concentration, and presence or
`absence of BPs.
`The study was conducted following methodological guidelines issued by the International
`Conference on Harmonisation for stability studies [37] and recommendations issued by the French
`Society of Clinical Pharmacy (SFPC) and by the Evaluation and Research Group on Protection in
`Controlled Atmosphere [ 38]. A variation of concentration outside the 90–110% range of initial
`concentration (including the limits of a 95% confidence interval of the measures) was considered as
`being a sign of instability. Presence of BPs and the variation of the physicochemical parameters were
`also considered a sign of atropine instability but were interpreted with regards to quantities found in
`commercial ophthalmic atropine solution (see Supplementary data file S1). The observed solutions
`must be limpid, of unchanged colour, and clear of visible signs of haziness or precipitation. Since there
`are no standards that define acceptable pH or osmolality variation, pH measures were considered
`acceptable if they did not vary by more than one pH unit from the initial value [38], and osmolality
`results were interpreted considering clinical tolerance of the preparation.
`3. Results
`3.1. Atropine Quantification and Breakdown Products (BP) Research
`Atropine retention time was of 9.7 ± 0.3 min (Figure 1). The chromatographic method used was
`found linear for concentrations ranging from 0.5 to 140 µg/mL. Average regression equation was y
`= 22429.5x−13.6, where x is the atropine concentration (in µg/mL) and y is the surface area of the
`corresponding chromatogram peak. Interception was not significantly different from zero, and average
`determination coefficient R2 of three calibration curves was 0.99999. No matrix effect was detected.
`The relative mean trueness bias coe fficients were less than 2.75%, except for the 0.5 µg/mL
`calibration point, for which it was of 3.75%. Mean repeatability RSD coefficient and mean intermediate
`precision RSD coefficient were less than 2%. The accuracy profile constructed with the data showed that
`the limits of 95% confidence interval coefficients were all within 3% of the expected value, except for
`the 10 µg/mL calibration point, for which the lower range limit was −8.8%. The limit of detection was
`evaluated at 0.05 µg/mL (signal-to-noise ratio of 3.03 and experimentally confirmed by visual analysis
`of the chromatograms), and the limit of quantification was fixed at 0.5µg/mL, even if the signal-to-noise
`ratio was 46, thus potentially indicating that a lower quantification limit could be reached.
`No impurities were visible in the initial atropine solution on the reference chromatogram at
`210 nm in Figure 1. All the impurities specified by the European Pharmacopeia were detected and
`identified in Figure 2. The chromatograms presented show separately di fferent impurities as they
`come from different European Pharmacopoeia reference solutions and were thus analysed sequentially
`in order to be able to correctly identify each peak using the relative retention times provided in the
`atropine monography. They were all visible at 210 nm, which allowed maximum sensitivity. No other
`impurities were detected at other wavelengths.
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`Figure 1. Reference chromatogram of a 0.1 mg/mL atropine solution at 210 nm and with diode array
`detector screening.
`No impurities were visible in the initial atropine solution on the reference chromatogram at 210
`nm in Figure 1 . All the impurities specified by the European Pharmacopeia were detected and
`identified in Figure 2. The chromatograms presented show separately different impurities as they
`come from different European Pharmacopoeia reference solutions and were thus analysed
`sequentially in order to be able to correctly identify each peak using the relative retention times
`provided in the atropine monography. They were all visible at 210 nm, which allowed maximum
`sensitivity. No other impurities were detected at other wavelengths.
`Figure 1. Reference chromatogram of a 0.1 mg/mL atropine solution at 210 nm and with diode array
`detector screening.
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`Figure 1. Reference chromatogram of a 0.1 mg/mL atropine solution at 210 nm and with diode array
`detector screening.
`No impurities were visible in the initial atropine solution on the reference chromatogram at 210
`nm in Figure 1 . All the impurities specified by the European Pharmacopeia were detected and
`identified in Figure 2. The chromatograms presented show separately different impurities as they
`come from different European Pharmacopoeia reference solutions and were thus analysed
`sequentially in order to be able to correctly identify each peak using the relative retention times
`provided in the atropine monography. They were all visible at 210 nm, which allowed maximum
`sensitivity. No other impurities were detected at other wavelengths.
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`Figure 2. Chromatograms of atropine impurities at 210 nm: (A) reference atropine chromatogram, (B)
`chromatogram of the atropine impurity B CRS solution , (C) chromatogram of atropine for the peak
`identification CRS (containing impurities A, D, E, F, G, and H) solution, and (D) chromatogram of the
`tropic acid R (impurity C) solution. The insets represent a close up of the chromatograms.
`A summary of the impurity retention times and relative retention times (relative to atropine) is
`presented in Table 3.
`Table 3. Atropine impurities retention times and relative retention times.
`Impurity Retention Times
`Experimental Absolute Retention Time (min) Relative Retention Time
`Atropine 9.7 1
`Impurity A 16.2 1.7
`Impurity B 9.3 0.9
`Impurity C 2.5 0.3
`Impurity D 7.6 0.8
`Impurity E 7.1 0.7
`Impurity F 8.0 0.8
`Impurity G 10.8 1.1
`Impurity H 9.3 0.9
`After forced degradation, BPs were detected with a resolution higher than 1.5 of the atropine
`peak to all its BPs and particularly in alkaline forced conditions. No BPs were detected when atropine
`solutions were exposed to UVA light, and atropine concentration did not vary after 8 days of UV-Vis
`exposure. After 1 h at 15% H2O2 exposure at 60 °C, no loss of atropine concentration was detected.
`After 1 h at 90°, a loss of 7.9% of atropine was noticed, without any breakdown products being
`detected. Chromatogram results are showed in Figure 3.
`Figure 2. Chromatograms of atropine impurities at 210 nm: ( A) reference atropine chromatogram,
`(B) chromatogram of the atropine impurity B CRS solution, (C) chromatogram of atropine for the peak
`identification CRS (containing impurities A, D, E, F, G, and H) solution, and (D) chromatogram of the
`tropic acid R (impurity C) solution. The insets represent a close up of the chromatograms.
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`A summary of the impurity retention times and relative retention times (relative to atropine) is
`presented in Table 3.
`Table 3. Atropine impurities retention times and relative retention times.
`Impurity Retention Times
`Experimental Absolute Retention Time (min) Relative Retention Time
`Atropine 9.7 1
`Impurity A 16.2 1.7
`Impurity B 9.3 0.9
`Impurity C 2.5 0.3
`Impurity D 7.6 0.8
`Impurity E 7.1 0.7
`Impurity F 8.0 0.8
`Impurity G 10.8 1.1
`Impurity H 9.3 0.9
`After forced degradation, BPs were detected with a resolution higher than 1.5 of the atropine
`peak to all its BPs and particularly in alkaline forced conditions. No BPs were detected when atropine
`solutions were exposed to UVA light, and atropine concentration did not vary after 8 days of UV-Vis
`exposure. After 1 h at 15% H 2O2 exposure at 60 ◦C, no loss of atropine concentration was detected.
`After 1 h at 90◦, a loss of 7.9% of atropine was noticed, without any breakdown products being detected.
`Chromatogram results are showed in Figure 3.
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`Figure 3. Chromatograms at 210 nm of breakdown products (BPs) obtained after forced degradation:
`(A) alkaline conditions of NaOH 0.5 N for 0.5 h and (B) acid conditions of HCl 0.1 N for 1 h at 90 °C.
`Detailed results of tropic acid quantification are presented in Supplementary data.
`3.2. Stability of Atropine in Unopened Multidose Eyedroppers
`3.2.1. Physical Stability
`All samples stayed limpid and uncoloured; chromaticity and luminance were unchanged during
`the study for both tested kind of eyedroppers; and there was no appearance of any visible particulate
`matter, haziness, or gas development. Initial turbidity was 0.33 and 0.32 FNU respectively for the
`atropine formulation with and without preservatives and did not vary by more than 0.6 FNU for the
`formulation with antimicrobial preservative or 0.32 FNU for the formulation without preservative
`(Table 4).
`Table 4. Evolution of turbidity over time. n = 1 (pooled volume from 5 units). FNU: Formazin
`Nephelometric Units.
` Turbidity (FNU)
`Day 0 Day 8 Day 15 Day 30 Day 60 Day 90 Day 180
`Atropine solution with preservative conditioned in LDPE
`CAT® eyedroppers 0.33 0.31 0.27 0.78 0.78 0.43 0.93
`Atropine solution without preservative conditioned LDPE
`NOVELIA® eyedroppers 0.32 0.31 0.26 0.54 0.44 0.34 0.64
`Figure 3. Chromatograms at 210 nm of breakdown products (BPs) obtained after forced degradation:
`(A) alkaline conditions of NaOH 0.5 N for 0.5 h and (B) acid conditions of HCl 0.1 N for 1 h at 90 ◦C.
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`Detailed results of tropic acid quantification are presented in Supplementary data.
`3.2. Stability of Atropine in Unopened Multidose Eyedroppers
`3.2.1. Physical Stability
`All samples stayed limpid and uncoloured; chromaticity and luminance were unchanged during
`the study for both tested kind of eyedroppers; and there was no appearance of any visible particulate
`matter, haziness, or gas development. Initial turbidity was 0.33 and 0.32 FNU respectively for the atropine
`formulation with and without preservatives and did not vary by more than 0.6 FNU for the formulation
`with antimicrobial preservative or 0.32 FNU for the formulation without preservative (Table 4).
`Table 4. Evolution of turbidity over time.n = 1 (pooled volume from 5 units). FNU: Formazin Nephelometric Units.
`T urbidity (FNU)
`Day 0 Day 8 Day 15 Day 30 Day 60 Day 90 Day 180
`Atropine solution with
`preservative conditioned in LDPE
`CAT® eyedroppers
`0.33 0.31 0.27 0.78 0.78 0.43 0.93
`Atropine solution without
`preservative conditioned LDPE
`NOVELIA® eyedroppers
`0.32 0.31 0.26 0.54 0.44 0.34 0.64
`3.2.2. Chemical Stability
`Evolution of pH and osmolality throughout the study is presented Table 5. Throughout the
`study, osmolality did not vary by more than 3.7% (15 mOsm /kg) of the initial osmolality (412 and
`398 mOsm/kg respectively for the atropine solution with and without preservatives) after 6 months of
`storage at 25 ◦C. Moreover, pH did not vary by more than 1.8% (0.1 pH unity) of the initial pH (6.1 for
`both solutions).
`Table 5. Evolution of pH and osmolality over time (n = 5, mean ± 95% confidence interval).
`Day 0 Day 8 Day 15 Day 30 Day 60 Day 90 Day 180
`Atropine solution with
`preservative conditioned in
`LDPE CAT®eyedroppers
`pH 6.10 ±0.01 6.11 ±0.01 6.13 ±0.02 6.13 ±0.01 6.13 ±0.02 6.21 ±0.04 6.12 ±0.01
`Osmolality
`(mOsm/kg) 412±16 400 ±6 403 ±14 393 ±14 400 ±5 413 ±11 418 ±23
`Atropine solution without
`preservative conditioned
`LDPE NOVELIA®
`eyedroppers
`pH 6.10 ±0.01 6.11 ±0.01 6.13 ±0.01 6.14 ±0.02 6.13 ±0.01 6.21 ±0.01 6.09 ±0.01
`Osmolality
`(mOsm/kg) 399±2 401 ±6 409 ±6 405 ±2 415 ±15 408 ±10 405 ±7
`For all studied conditions, mean atropine concentrations did not vary by more than 5.7% of mean
`initial concentrations (as presented in Figure 4). By extrapolation of the degradation rate using a linear
`regression, it could be estimated that atropine concentrations would remain higher than 90% of the
`original concentration for about 300 days.
`Chromatographs showed no sign of BPs until day 8 for both types of LDPE eyedroppers at 25 ◦C.
`After 15 days of storage, one BP appeared, presenting a retention time of 2.10 min (relative retention
`time of 0.2; Figure 5A) seemingly not detected during forced degradation assays but close to that of
`tropic acid (Figure 5B). However, when diluting the know impurity tropic acid in a phosphate buffer
`of the same nature and concentration of the one used for the atropine formulation, its retention time
`changed to be identical to that of the breakdown product (Figure 5C), thus indicating that it is highly
`likely that the misidentified breakdown product is in fact tropic acid.
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`Pharmaceutics 2020, 12, x FOR PEER REVIEW 10 of 18
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`3.2.2. Chemical Stability
`Evolution of pH and osmolality throughout the study is presented Table 5. Throughout the
`st