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BY 4.0 license Open Access Published by De Gruyter October 9, 2020

Measurement, analysis and prediction of amoxicillin oral dose stability from integrated molecular description approach and accelerated predictive stability (APS)

Camille Merienne ORCID logo, Chloe Marchand, Samira Filali, Damien Salmon, Christine Pivot and Fabrice Pirot

Abstract

Background

Stability of low amoxicillin oral dosage form (5 mg) used in reintroduction drug test was not fully documented. Furthermore, the impact of (1) salt moiety of amoxicillin and (2) amoxicillin – excipient interactions upon the antibiotic formulation stability during the storage was not characterized so that the estimation of the pharmaceutical expiration date from shelf-life was uncertain. Thus, the main goal of this study was to estimate the shelf-life of two formulations of amoxicillin, using a semi-predictive methodology.

Methods

Amoxicillin sodium (AS) and amoxicillin trihydrate (ATH), corresponding to 5-mg amoxicillin, were compounded with microcrystalline cellulose (MCC) in oral hard capsules which were, then, submitted to four environmental conditions (25 °C / 60% or 80% relative humidity (RH); 40 °C / 75% RH; 60 °C / 5% RH) in climatic chambers for 45 and 84 days. Therefore, the characterization of amoxicillin-MCC mixture was assessed by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) The profiles of amoxicillin content (determined by stability indicating chromatographic method) as a function of storage time, temperature and RH were fitted to pre-defined kinetic models performed by accelerated predictive stability (APS).

Results

ATR-FTIR analysis of AS, ATH, MCC and bulk specimens stored in heated and humid atmosphere confirmed water sorption to cellulose described by a broad and unresolved 3600 to 3000 cm−1 band associated with (1) general intramolecular and intermolecular hydrogen bonding between water and hydroxyl groups of the cellulose, and with (2) free hydroxyl in cellulose. Moreover, a dramatic decrease of absorption at 1776 and 1687 cm−1 respectively characteristic of the β-lactam ring (νC=O) and amide group (νC=O), was revealed as a consequence of AS and ATH degradation caused by moisturization of bulk. Amoxicillin degradation was established by chromatographic analysis showing faster AS degradation than ATH throughout time exposure. The combined effects of temperature – RH were successfully modeled by APS, where AS and ATH showed accelerated (auto-catalysis degradation mechanism) and linear degradation, respectively. The faster AS degradation was assumed to be linked to lower hydrogen donor to hydrogen acceptor count ratio and polar surface than ATH, increasing the probability of AS hydrolysis by water adsorption to AS-MCC solid dispersion (e.g., by reduction of protective intramolecular hydrogen bonds between AS molecules). Furthermore, the compounding which involved a drastic homogenization of solids may have affected the crystalline degree of MCC with an increase of amorphous phase more sensitive to water adsorption.

Conclusions

The improvement of amoxicillin compounding for oral dose forms might be rationalized by taking into account the molecular descriptors of salt moiety and excipients, improved by the choice of an appropriate process of production, characterized from infrared vibrational spectroscopy and chromatographic analysis and finally predicted from accelerated stability assays.

Introduction

The molecular, crystal or amorphous structure of one or some active pharmaceutical ingredients (API) mixed with several excipients affects the overall properties of pharmaceutical products in an intricate network of physicochemical interplay and influences. Therefore, the prediction of pharmaceutical product stability is made difficult and need the measurement and the individual chemical analysis of each API and excipients in essential long-term stability studies. The moisture absorbability of excipients and the salt moiety of API are well-known to impact e.g., the solubility and the hydrolysis of API. This property can modify with unexpected extent the forthcoming stability of initial dried solid-solid dispersion in oral dosage formulation. Amoxicillin (i.e. beta-lactam antibiotic with broad spectrum bactericidal action), API sensitive to hydrolysis, shows high efficacy and good tolerance. However, after oral administration of high amoxicillin dose, allergenicity was reported in 0.5–2.0 % of cases, including mainly cutaneous reaction (rash, hives, and itching), and also some severe adverse reactions such as angioedema and potentially fatal anaphylaxis [1]. To assess the potential hypersensitivity reaction to amoxicillin, often overestimated [2], [3], the reintroduction drug test, exploring the allergy to amoxicillin treatments, is achieved by oral administration of low doses of antibiotic in patients. In absence of commercial oral dose forms, the reintroduction of amoxicillin test needs a hospital pharmaceutical compounding which implies an analysis of feasibility including technical and safety concerns. The choice of amoxicillin chemical moiety (e.g., amorphous amoxicillin sodium salt [AS] or amoxicillin trihydrate crystalline form [ATH]) as well as excipients is of crucial importance for a reasonable rationalization of formulation and proper estimation of the stability of oral dose form and shelf-life. The choice of amoxicillin moiety may be dictated by the availability of raw materials or the existence of commercial pharmaceutical specialties on the market. The latter, as a raw material, has the advantage of avoiding possible identification or purity checking prior to its use in a formulation. Furthermore, the choice of the diluent excipient is often made by using the main ingredient of the commercial amoxicillin specialties. However, although the process of preparation is straightforward, the chemical reactions between low doses of API dispersed in large amounts of excipients may be different from those predicted with commercial amoxicillin specialties having served as ingredients. Thus, it may complicate an estimation of API stability in new formulation entity and the prediction of medication shelf-life. The first objective of this study was to compare the chemical stability of AS and ATH loaded formulations in climatic chambers (i.e., temperature and relative humidity (RH) controlled) during accelerated stability test in order to discriminate the most stable oral dose form. The second objective was to predict the highest pharmaceutical shelf-life from degradation models in Accelerated Predictive Stability (APS) [4–7]. The specific objectives of the study was to characterize the mechanisms of drug instability from molecular descriptor analysis, vibrational spectroscopy, chromatographic assays and kinetic modeling which allow to rationalize the choice of API, ingredients and process of preparation for reintroduction amoxicillin test.

Material and methods

Molecular descriptors and physicochemical properties

AS and ATH molecular descriptors and physicochemical properties, collected from Drugbank® and Chemaxon® on-line databases, are shown in Table 1.

Table 1:

Molecular descriptors and physicochemical properties of AS and ATH.

Physicochemical parametersASATH
Formulas
CAS numbera34642-77-861336-70-7
Molecular mass (g/mol)a387.39419.45
Hydrogen acceptor counta66
Hydrogen donor counta34
Polar surface (Å)a135.8132.96
Heavy atom counta2628
Rotatable bounda44
Number of ringsa33
Water solubility (mg/mL)a1.990.96
LogPb−2.3−2.3

  1. aEstimated from DrugBank®.

  2. bEstimated from Chemaxon®.

AS and ATH oral dosage forms

Commercial pharmaceutical specialties of amoxicillin were used as API sourcing for the preparation of amoxicillin hard capsules (AS: Amoxicillin 2G IV, Panpharma®, Fougères, France; ATH: Clamoxyl® 500 mg oral hard capsules, GSK®, Brentford, United Kingdom). Microcrystalline cellulose (MCC) was used as inactive ingredient and API diluent (Cooper® Melun, France). The main excipient of Clamoxyl® was the magnesium stearate. Once a technical and regulatory analysis of feasibility and risk was done, AS and ATH oral hard capsules were compounded according to qualitative and quantitative composition given in Table 2, in the respect of good preparation practices of medicinal products in healthcare establishments [8]. After a manual homogenization of AS, ATH, MCC powders in a mortar, a 5-mg amoxicillin mixture was loaded by a hand-operated capsule filling machine (LGA®, La Seyne-sur-Mer, France) in gelatin hard capsules (0.2 cm3, Cooper®, Melun, France). Then, they were packaged (per 25 unit doses) in 50-mL white polypropylene jars (Gravis, Neuville-sur-Saône, France).

Table 2:

Composition of amoxicillin sodium (AS) and amoxicillin trihydrate (ATH) based formulations compounded in the present study.

Oral dose formsAmount (mg)
AS formulation
 AS5.31
 Equivalent to amoxicillin per capsule5.00
 MCC65
 Ratio AS/MCC (%)8.2
ATH formulation
 ATH5.74
 Equivalent to amoxicillin per capsule5.00
 MCC60
 Ratio ATH/MCC (%)9.6

Accelerated predictive stability study conditions

The hard capsules were maintained in uncapped jars stored in climatic chambers regulated in temperature and RH allowing to test API and oral dosage form stability to standards of the International Conference on Harmonization (ICH, 25 °C / 60% RH and 40 °C / 75% RH) [9] as well as in custom environmental conditions (25 °C / 80% RH and 60 °C / 5 % RH). Identification assay of oral dosage form and the assessment of amoxicillin content (see next sections) were carried out (n = 5 dose units) immediately after compounding and after different time exposure in high, medium or low humidity conditions as reported in Table 3.

Table 3:

Experimental design of the ASAP study for AS and ATH formulations. Storage in ICH and non ICH conditions and time of storage.

Storage conditionsTime of storage (days)
AS formulationATH formulation
25 ± 2 °C, 60 ± 5% RH (ICH)0 ; 25 ; 56 ; 840 ; 15 ; 30 ; 45
60 ± 2 °C, 5 ± 2% RH (non ICH)0 ; 25 ; 56 ; 840 ; 15 ; 30 ; 45
25 ± 2 °C, 80 ± 5% RH (non ICH)0 ; 1 ; 2 ; 250 ; 15 ; 30 ; 45
40 ± 2 °C, 75 ± 5% RH (ICH)0 ; 1 ; 2 ; 250 ; 15 ; 30 ; 45

  1. RH, relative humidity.

Multicomponent infrared analysis of amoxicillin dosage forms

A multicomponent analysis of amoxicillin mixture contained in hard capsules exposed to controlled temperature and RH was realized by attenuated total reflectance Fourier-transformed infrared spectroscopy (ATR-FTIR, 3800–400 cm−1, Nicolet®iS 50 FT-IR Spectrometer, ThermoScientific®, Thermo Nicolet Corp.®, Waltham, MA), immediately after compounding and after a regular interval of time exposure. ATR-FTIR spectrum of individual components (AS, ATH and MCC) was recorded by a diamond crystal containing a standard deuterated triglycine sulfate detector (10-cm optical path, 32 scans with spectral resolution of 4 cm−1). The spectra were compared to amoxicillin mixture and amoxicillin pharmacopeia standard [10]. Omnic Spectra® software was used to analyze individual and multi-component spectra.

Amoxicillin content and degradation analysis by stability-indicating HPLC-UV method

Amoxicillin content of oral dosage forms exposed to ICH and custom environmental conditions was quantified by high performance liquid chromatography and ultra-violet detection (HPLC-UV). HPLC system (Agilent 1290 Infinity Quaternary LC System, Les Ulis, France) was composed of a binary pump, a vacuum degasser, a thermostated column compartment (25 °C), an autosampler and a diode array detector. The stationary phase was a Kinetex® C18 150 × 4.6; 2.6 µm column (Phenomenex®, Torrance, CA). Mobile phase was an isocratic mixture of water/acetonitrile (90/10, v/v) filtered and degassed through nylon membranes (0.20 μm) under vacuum before use. The flow rate was 1 mL/min. The wavelength of quantification was 273 nm while the degradation products were searched between 190 and 400 nm. The injection volume was 10 µL. The analytical method was validated according to ICH and French Society of Pharmaceutical Science and Technology recommendations [11]: linearity (R2>0.99), repeatability (2.6%), reproducibility (5.8%), accuracy (3.26%), and accuracy profile (β=80%, α<10%). A sufficient resolution was searched between amoxicillin and the degradation products generated according to the « Methodological guidelines for stability of hospital pharmaceutical preparations » [12].

Degradation kinetic modelling

Amoxicillin dosage forms were expected to degrade faster when they are exposed to elevated stress conditions such as high temperature and / or humidity. In order to characterize the mechanisms underlying the pattern of amoxicillin degradation in solid dispersion (i.e., amoxicillin – MCC as drug–polymer two-component system), oral dosage forms were exposed to several temperature/RH combinations with the purpose of increasing the rate of amoxicillin degradation. The modifications of physicochemical properties of amoxicillin – MCC component system were estimated from vibrational spectroscopy while the amoxicillin content was tracked by a stability indicating chromatographic method. Indeed, in drug–polymer two-component system, two molecules (amoxicillin and MCC) could interact and have a cumulative effect on degradation causing a lower degradation rates at the beginning which increases gradually with time. Profiles of amoxicillin degradation were fitted to different mathematical models combining the effects of temperature and humidity in solid-state kinetics [5], [6]. In the model A, the degradation rate increases exponentially with the humidity. In the model B, the degradation rate raises to the power of a constant [6], [7]. Most pharmaceutical and biological models are based on the exponential pattern [13]. Degradation rate models A and B, detailed in Table 4, were fitted by using the statistical computing and graphic software “R” [14]. Models were evaluated through the Bayesian Information Criterion (BIC), Akaike Information Criterion (AIC) and the Relative Mean Square Error (RMSE) [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Finally, the best fitted model was used to predict the long-term degradation rate of amoxicillin over 24 months in the common ICH environmental conditions (25 °C / 60% RH, 30 °C / 65% RH and 40 °C / 75% RH).

Table 4:

Mathematical Models used for fitting experimental data of amoxicillin degradation in normal and accelerated environmental conditions.

Kinetic modelsFormulas
LinearHumidity as factor (A)α(t)=α(0)+K1.exp(K2T).exp(H.N1).t
Humidity as power (B)α(t)=α(0)+K1.exp(K2T).HN1.t
ShapedHumidity as factor (A)α(t)=α(0)+K1.exp(K2T).exp(H.N1).tN2
Humidity as power (B)α(t)=α(0)+K1.exp(K2T).HN1.tN2
AcceleratingHumidity as factor (A)α(t)=α(0).exp(K1.exp(K2T).exp(H.N1).t)
Humidity as power (B)α(t)=α(0).exp(K1.exp(K2T).HN1.t)
DeceleratingHumidity as factor (A)α(t)=α(0)+K3.(1exp(K1.exp(K2T).exp(H.N1).t))
Humidity as power (B)α(t)=α(0)+K3.(1exp(K1.exp(K2T).HN1.t))

  1. α is the extent of the reaction. t is the time. K1, K2 and K3 are respectively the fitted frequency factor, activation energy divided by R and asymptotic limit of the degradant at t=∞. H is the humidity descriptor and T is the temperature (Kelvin). N1 is the fitted exponent on humidity and N2 is the fitted shape parameter.

Results

Multicomponent infrared analysis of amoxicillin dosage forms

ATR-FTIR spectra of (A) AS, (B) ATH USP standard, (C) MCC as individual components, (D, E) AS and (F, G) ATH loaded formulations immediately after compounding, 25 days of storage at 40 °C / 75% HR and 45 days at 25 °C / 60% RH are shown in Figure 1a. An enlargement of ATR-FTIR regions between 1900 and 1600 cm−1 allowed to distinguish typical 1776 and 1687 cm−1 absorption peaks characteristic of the β-lactam ring (νC=O) and amide group (νC=O). Interestingly, MCC presented two distinct domains with strongly enhanced IR absorption between (1) 3700–3000 cm−1 and (2) 1000–500 cm−1. However, water absorption spectrum presented very intense and broad bands over the full mid-infrared region (notably at 3300 cm−1) which was responsible for an increase of IR signal of amoxicillin dosage forms exposed to humidity. Furthermore, the impact of moisture upon AS and ATH – MCC systems was clearly evidenced by the dramatic decrease of 1776 and 1687 cm−1 signals which may be explained by the fast hydrolysis of amoxicillin submitted to humidity and heat. Moreover, the degradation rate of ATH, roughly estimated by measuring the absorbance of the band corresponding to the carbonyl present in the drug molecule [16] was lower than the one of AS. This confirms the importance of amoxicillin moiety in product stability.

Figure 1: (a) ATR-FTIR spectra of (A) AS, (B) ATH USP standard, (C) MCC as individual components, (D, E) AS dosage forms immediately after compounding and after 25 days of storage at 40 °C / 75% HR, (F, G) ATH dosage forms immediately after compounding and after 45 days of storage at 25 °C / 60 % RH. (b): Enlargement of vibrations between 1600 and 1800 cm−1 allowing to distinguish typical 1776 and 1687 cm−1 absorption peaks characteristic of the β-lactam ring (νC=O) and amide group (νC=O).

Figure 1:

(a) ATR-FTIR spectra of (A) AS, (B) ATH USP standard, (C) MCC as individual components, (D, E) AS dosage forms immediately after compounding and after 25 days of storage at 40 °C / 75% HR, (F, G) ATH dosage forms immediately after compounding and after 45 days of storage at 25 °C / 60 % RH. (b): Enlargement of vibrations between 1600 and 1800 cm−1 allowing to distinguish typical 1776 and 1687 cm−1 absorption peaks characteristic of the β-lactam ring (νC=O) and amide group (νC=O).

Amoxicillin content and degradation analysis by stability-indicating HPLC-UV method

Typical chromatograms of AS-MCC and ATH-MCC mixtures obtained immediately after compounding and after exposure to humidity and heat are presented in Figure 2. Different patterns of drug degradation were clearly evidenced as function of environmental conditions and amoxicillin moiety. Thus, as shown in Figure 3, the profiles of AS and ATH degradation showed that AS was relatively stable in normal and low humidity environment (25 °C / 60% RH and 60 °C / 5% RH) but was very unstable in high humidity environments (25 °C / 80% RH and 40 °C / 75% RH) (Figure 3A). The rate of degradation of ATH was lower than 10% and followed zero-order kinetic whatever the environment considered (Figure 3B). Therefore, the humidity is a determining factor in the rate of degradation of AS. It followed a zero-order kinetic at normal and low RH but an exponential decay at high RH (after 25 days at 25 °C / 60% RH and 60 °C / 5% RH, 93 and 98% AS remained, respectively, while after 2 days of storage, at 25 °C /80% RH and 40 °C /75% RH, 68 and 30% AS remained, respectively).

Figure 2: HPLC-chromatograms of (A, B) AS dosage forms immediately after compounding and after 45 days of storage at 25 °C / 60% RH (C, D) ATH loaded formulations immediately after compounding and after 25 days of storage at 40 °C / 75% HR.

Figure 2:

HPLC-chromatograms of (A, B) AS dosage forms immediately after compounding and after 45 days of storage at 25 °C / 60% RH (C, D) ATH loaded formulations immediately after compounding and after 25 days of storage at 40 °C / 75% HR.

Figure 3: Degradation profiles of AS (Figure 3A) and ATH (Figure 3B) from dosage forms stored at (□) 25 °C / 60% RH (○) 60 °C / 5% RH (■) 25 °C / 80% RH; and (▲) 40 °C / 75% RH. Each data is the mean ± standard deviation of five experimental determinations.

Figure 3:

Degradation profiles of AS (Figure 3A) and ATH (Figure 3B) from dosage forms stored at (□) 25 °C / 60% RH (○) 60 °C / 5% RH (■) 25 °C / 80% RH; and (▲) 40 °C / 75% RH. Each data is the mean ± standard deviation of five experimental determinations.

Degradation kinetic modelling

The profiles of AS and ATH submitted to combined temperature and humidity conditions were fitted to previous models of degradation rate as shown in Table 4. The statistical analysis of experimental data fitted to those models is presented in Table 5. It includes the determination of rate constants, humidity and temperature related exponents and the RMSE, BIC and AIC statistical parameters. Degradation rate of AS was successfully fitted to the “A Accelerated model” characterized by lowest statistical values for RMSE, BIC and AIC (7.4, 368.9 and 360.6, respectively). The experimental data of ATH profile was best fitted to the “A linear model” (i.e., RMSE, BIC and AIC equal to 4.9, 388.1 and 378.9 respectively). Thus, the rate of degradation of amoxicillin in the two-component solid system was clearly influenced by the drug moieties. The determination coefficient was 0.89 for AS and 0.14 for ATH.

Table 5:

Modelling parameters calculated with “R” software.

ModelsFormulationBest fitting parameters
−103• K110−4 • K2K3N1N2RMSEBICAIC
A linearASa8.110.711.411.9386.7378.4
ATH52.65.10.74.9388.1378.6
A shapedASa212.77.67.20.68.0380.0370.4
ATH10.06.61.01.34.9392.1381.0
A deceleratedASaUnfittedUnfittedUnfittedUnfittedUnfittedUnfittedUnfitted
ATH6.05.25.30.84.9392.1381.0
A acceleratedASa0.01211.811.37.4368.9360.6
ATH0.5405.10.74.9388.1378.7
B linearASa238 244.611.37.39.3391.4383.0
ATH93.95.20.24.938.1378.7
B shapedASa143 00010.74.60.58.6388.1378.4
ATH21.66.80.31.44.9392.1381.0
B deceleratedASaUnfittedUnfittedUnfittedUnfittedUnfittedUnfittedUnfitted
ATH12.35.35.20.24.9392.1381.0
B acceleratedASa0.00413.87.18.9380.0371.6
ATH0.965.20.24.9388.1378.7

  1. aData of AS at 25 ± 2 °C, 80 ± 5% RH and 40 ± 2 °C, 75 ± 5% RH after 25 days were excluded from the study taking account the extremely low amoxicillin content.

Shelf-life prediction of AS and ATH dosage form

The degradation models of AS (A accelerated) and ATH (A linear) were used for further shelf-life predictions of amoxicillin dosage forms in three conditions of storage (25 °C / 60% RH, 30 °C / 65% RH and 40 °C / 75% RH) as shown in Figure 4. The shelf-life was defined as the time needed to degrade 10% of the initial AS content, which was the threshold fixed for the study. The shelf life was found equal to 7, 2 days and few hours after storage in 25 °C / 60% RH, 30 °C / 65% RH and 40 °C / 75% RH conditions, respectively. Exponential profiles of AS degradation were clearly predicted (Figure 4A) whatever the storage conditions whereas a linear profile of AS degradation was determined experimentally in 25 °C / 60% RH conditions. Therefore, the predicted shelf-life of AS dosage form was much shorter (7 days) than the one determined experimentally (55 days) as presented in Table 6. Similarly, ATH predicted content of the ATH formulation remained stable (i.e., ATH remained higher than 90%) for 250, 170 and 90 days in respectively 25 °C / 60% RH, 30 °C / 65% RH and 40 °C / 75% RH environments.

Figure 4: Predicted degradation profile with the best fitted kinetics models of AS (Figure 4A) and ATH (Figure 4B). Modeling was performed at common ICH climatic conditions: (□) 25 °C / 60% RH (○) 30 °C / 65% RH; and (∆) 40 °C / 75% RH. The shade of grey depends on the intensity of the environmental constraints. If the environmental stocking conditions of the dosage forms are greater than 40 °C / 75% RH, the predicted degradation profile will be in the darkest grey zone. If the constraints are between 40 °C 75% RH and 30 °C / 65% RH, the degradations profiles will be in the middle grey zone. If the constraints are between 30 °C / 65% RH and 25 °C / 60% RH, the degradations profiles will be in the light grey zone.

Figure 4:

Predicted degradation profile with the best fitted kinetics models of AS (Figure 4A) and ATH (Figure 4B). Modeling was performed at common ICH climatic conditions: (□) 25 °C / 60% RH (○) 30 °C / 65% RH; and (∆) 40 °C / 75% RH. The shade of grey depends on the intensity of the environmental constraints. If the environmental stocking conditions of the dosage forms are greater than 40 °C / 75% RH, the predicted degradation profile will be in the darkest grey zone. If the constraints are between 40 °C 75% RH and 30 °C / 65% RH, the degradations profiles will be in the middle grey zone. If the constraints are between 30 °C / 65% RH and 25 °C / 60% RH, the degradations profiles will be in the light grey zone.

Table 6:

Experimental and predicted shelf life of AS (days), experimental and predicted percentage of ATH remaining at the end of the stability study, and predicted ATH shelf life (days) at the four environmental conditions of the study (25 °C / 60 and 80% RH, 40 °C / 75% RH and 60 °C / 5% RH).

Dosage formsStorage conditions
25 °C / 60% RH25 °C / 80 %RH40 °C / 75 %RH60 °C / 5 %RH
AS shelf life (days)
 Experimental550.50.2570
 Predicted70.750.260
ATH shelf life (days)
 Predicted2502459060
ATH remaining at D45 (%)
 Experimental98999693
 Predicted98.498.695.8692

Discussion

In the present study, the determination of AS and ATH stability in a two-component system (i.e., amoxicillin-MCC) was investigated in order to appreciate the shelf-life of API-excipient mixture filled in hard capsules submitted to four environmental conditions (normal and accelerated) with four time points for a fit-for-purpose APS study. The main difference between both formulations was the raw material: AS and ATH. AS was an amorphous crystal presented as a lyophilizate without any other excipients. ATH was an orthorhombic crystalline structure with magnesium stearate as excipient. In this lean approach, APS application was to test and compare the stability of amoxicillin moieties from straightforward formulation in which amoxicillin mixed with MCC was assayed by HPLC-UV [17]. Furthermore, amoxicillin-MCC mixture integrity was concomitantly evaluated by ATR-FTIR to characterize the stability of the formulation. The qualitative and quantitative results from chromatographic and spectrometric methods clearly showed that ATH was less sensitive to chemical degradation than AS. The AS fast degradation was due to hydrolysis caused by the moisture adsorption onto MCC and AS lyophilized form. The use of MCC as inactive ingredient presented several advantages such as an absence of complexation with amoxicillin. This was deduced from vibrational spectrometry analysis of raw ingredients and dosage forms showing no shifts of 1,776 and 1,687 cm−1 signals (i.e., νC=O β-lactam ring and amide group) [18]. However, the potential modification of MCC crystallinity by the grinding during the compounding may have had a crucial effect on the pharmaceutical properties of amoxicillin hard capsules. As reported earlier, a variation of structural properties of MCC would affect the extent of moisture absorbability and further amoxicillin hydrolysis [19]. If this hypothesis was confirmed, the duration of power grinding would affect the stability of the preparation. However, such ATR-FTIR spectrometry offered a rapid and versatile method for controlling (1) the moisture adsorption as a potential cause of API hydrolysis and (2) the decrease of the carbonyl liaison signal as a consequence of molecular alteration of API while HPLC assessed the products of degradation. The disordered amorphous structure of AS had likely favored its intrinsic propensity to bind water molecule. As a lyophilizate, it has a natural tendency to absorb environmental water. The AS dried structure is more thermodynamically stable after water sorption, which increases its hydrolysis rate. On the contrary the orthorhombic crystalline structure of ATH can be protective against water, as the major site of hydrolysis in the lactam core is sterically hindered [20], [21]. The ATH sourcing includes magnesium stearate in its formulation. This excipient is sparingly soluble in water. Even diluted in the capsules, the magnesium stearate can enhance the protection of the API against water, and hydrolysis. Furthermore, an estimation of intramolecular hydrogen bonding from the ratio of hydrogen donor to hydrogen acceptor count showed that ATH molecular structure offered more appropriate opportunity for protective intramolecular bonds than AS molecular structure. By limiting the number of inactive ingredients in the formulation, the mechanisms of amoxicillin degradation were explained by physicochemical parameters as the crystallinity of ATH-MCC compounding and the amorphous-crystal heterogeneous structure of AS-MCC mixture. Thus, capsules were stored in an opened jar bottle. Indeed, the aim of this study was to investigate the stability between the two API in the same formulation. The moisture effect was better highlighted with this layout. In a closed jar, the humidity effect would have been weighted by the water permeability of the plastic bottle used. Whereas uncapped jar environmental condition is more aggressive than normal conditions, it allows an analysis of the stability of both formulations in a worst case. Therefore, although a linear rate of degradation of ATH was claimed whatever the conditions of storage, a zero-ordered process of degradation of AS was experimentally shown in normal conditions (25 °C/60% RH) followed by an exponential decay of AS remained in accelerated conditions. It makes difficult to have an accurate prediction of AS dosage form shelf-life from composite linear-exponential model. It was assumed AS rate of degradation found its origin in auto-catalytic reaction primarily induced by surrounding moisture and MCC hydration. ATH degradation was linearly dependent on time without major contribution of temperature-humidity combined factors. The weak rate of degradation and the use of exponential models explained the low value of determination coefficient R2 for ATH modelling and confirmed the linear kinetic of AS degradation. Therefore, it was assumed that diverse chemical pathways were activated according to the extent of temperature and humidity drastically modifying the pattern of AS degradation kinetics. To refine AS kinetic degradation model deriving from the classical solid-state kinetic models and the Waterman and Clancy equations [5], [6], [7], the lean approach should have been done with more than four storage conditions for an accurate deconvolution of the temperature and humidity importance as regard to degradation.

Conclusion

In the absence of commercial oral dose forms, the reintroduction amoxicillin test needs a hospital pharmaceutical compounding which implies an analysis of feasibility comprising proper estimation of stability of oral dose form and shelf-life. The choice of amoxicillin chemical moiety (e.g., AS or ATH) as well as excipients is of crucial importance for a reasonable rationalization of formulation. So far, in the present study, a rationalization of formulation was elaborated by considering (1) amoxicillin and MCC molecular structures, (2) vibrational spectrometry analysis of amoxicillin dosage forms, (3) the HPLC-UV quantification of amoxicillin degradation products (4) and the fit of experimental data to degradation models enabling to predict the shelf-life of amoxicillin dosage forms in a lean approach of storage conditions.


Corresponding author: Camille Merienne, Pharm.D, Service Pharmaceutique, Groupement Hospitalier Centre Edouard Herriot, Place d’Arsonval, 69437 Lyon Cedex 03, Lyon, France. Phone: + 33 04 72 11 11 20, Fax: + 33 04 72 11 78 76, E-mail: , web address: Fripharm.com.

Acknowledgments

Author would like to thank Dr Patrick Nolain for his help in modelling and Dr Jean Vigneron for his critical assessment of this paper.

  1. Research funding: None declared.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Conflict of interest statement: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

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Received: 2020-06-18
Accepted: 2020-09-01
Published Online: 2020-10-09
Published in Print: 2020-05-26

© 2020 Camille Merienne et al., published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.