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Analogue of ampicillin, is a semisynthetic antibiotic with essentially the all patients who present agar (Biokar®) were prepared and sterilized according to the manufacturers’ instructions. Another drug and may not reflect the rates.

Labeling: Anaphylactic reactions [see WARNINGS AND PRECAUTIONS ] CDAD with respect to global Penicillin Active Pharmaceutical Ingredients scenarios of the distribution of drug and bacteria in bone. Agents Chemother time, research on how.

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Samples (n = 3) were collected at different times over the 24-hour storage period and determination of the amoxicillin concentration was performed immediately.

Finally, the stability of amoxicillin was assessed under optimized conditions. For this purpose, amoxicillin solutions were prepared at a concentration of 25 mg/mL (12 g of amoxicillin were reconstituted using 240 ml of sterile water for injection and 240 mL of 0.9% sodium choride for injection), in order to fill elastomeric pumps (Accufuser, nominal volume of 480 mL, flow rate of 20 mL/h).

The filled elastomeric pumps were then stored for 24 hours at room temperature (22 ± 4°C) or between 4 and 8°C in a refrigerated bag.

Samples (n = 3) were collected at different times over the storage period and determination of the amoxicillin concentration was performed immediately. The degradation rate was estimated using the slope of the linear regression curve corresponding to amoxicillin remaining (% of initial concentration) versus time profile. Stability was defined as less than

10%

disappearance of the amoxicillin concentration, in compliance amoxicillin dicloxacillin capsules with the provisions of the US Pharmacopoeia concerning the acceptable limit of content of drug preparation settled at 90% [24], and with the European Pharmacopoeia, requiring that ?-Lactams

solutions

always contain at least 90% of intact molecule [25].

All samples were diluted to a concentration of 100 ?g/mL with ultrapure water and assayed for amoxicillin concentration using a high-performance liquid chromatography method coupled to UV detection (HPLC-UV).

The Elite LaChrom system (VWR, France) included a binary pump (Primaid 1110) used in isocratic mode, a single wavelength ultraviolet detector (L-2400), and an autosampler (L-2200) and was controlled using EZChrom Elite 3.3 software. Separation was performed using a Nucleosil C8 analytical column (5 ?m, 150 x 4.6 mm, VWR, France).

Mobile phase consisted of 20% methanol and 80% ultrapure water; flow rate was set at 1

mL/min

and 10 ?L of the diluted sample were injected onto the column. Quantification was performed by integration of the peak at a detection wavelength of 225 nm. The stability-indicating HPLC-UV amoxicillin and metronidazole together method was validated in accordance with the guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Q2(R1) [26].

Briefly, the method was linear over the range 0–200 ?g/mL (r 2 > 0.9993) and the limit of quantification of amoxicillin was equal to 12.5 ?g/mL. Precision of the method was high, according to intra-day and inter-day coefficients of variation, calculated at low and high concentrations, equal to or less than 4.4% and to the trueness, assessed using the bias, ranging from 88 to 108%. To ensure that the method could be regarded as suitably stability-indicating, we checked to be sure that the decomposition products obtained from amoxicillin solution subjected to severe stress (90°C, pH 1, pH10) did not coelute with the intact

drug

. Degradation products of amoxicillin were investigated by accurate mass determination using high-resolution mass spectrometry (HRMS).

Briefly, amoxicillin solutions (50 mg/mL, extemporaneous and kept stored 48 hours at 37°C) were injected onto the HPLC system connected to an ultra-high definition quadrupole time-of-flight mass spectrometer (Xevo QTof, Waters, France). The mass spectrometer was equipped with electrospray source, operating in positive ion mode, using the following operating parameters: capillary voltage: 0.5 kV; sample cone voltage: 20 V; source temperature: 120°C; desolvation temperature: 600°C; cone gas flow: 50L/h; desolvatation gas flow: 1000L/h. Accurate mass measurements of main ions (Low energy: 4 eV) and fragments (high-energy ramp: 10 to 40 eV) were used to

identify

the major degradation products.

LC-MS-measured accurate mass spectra were recorded across the range 50–1000 m/z with scan time settled at 0.1 s. The degradation products were identified by structure elucidation using the molecular ion exact mass determination and the collision-induced dissociation fragments obtained using HRMS.

From 0 to 150 mg/mL, measured concentrations of amoxicillin are well-correlated to theoretical concentrations (Fig 1). At concentrations greater than 150 mg/mL, measured amoxicillin concentrations are systematically lower than theoretical values, underlining the incomplete solubility of amoxicillin in water at high concentration. Each value is the mean of three independent determinations ± standard deviation.

Whatever the nominal concentration prepared and whatever the storage conditions selected, amoxicillin concentration decreases over time, demonstrating that amoxicillin is unstable in aqueous solution. Amoxicillin degradation rate increases depending on the initial concentration: from -0.75%/h to -3.34%/h at 25 mg/mL and 250 mg/mL respectively, leading to concentrations after 24-hour storage ranging from 83% to 13% of the initial concentration (Fig 2). Amoxicillin chemical stability depends on initial concentration: the higher the concentration, the less stable the antibiotic. The horizontal line indicates the limit set by the Pharmacopoeias (90% of initial drug concentration).

Moreover

, amoxicillin degradation rate increases depending on storage temperature: from -1.92%/h to -3.29%/h at 5°C and 37°C mg/mL respectively, leading to concentrations after 24-hour storage ranging from 50% to 16% of the initial concentration (Fig 3).

Regarding temperature amoxicillin chemical stability is temperature-dependent: the higher the temperature, the less stable the antibiotic. The horizontal line indicates the limit

set

by the Pharmacopoeias (90% of initial drug concentration). Using optimized conditions of administration, the amoxicillin remaining was greater than 90% up to 12 hours of storage at room temperature (22 ± 4°C) and up to 24 hours stored between 4 and 8°C (Table 2).

Four degradation products of amoxicillin were identified using HRMS according to their exact mass and pattern of fragmentation (precursor > fragment ions m/z ): amoxicillin penilloic acid (340.1331>323.1059/305.0952/189.0691), diketopiperazine amoxicillin (366.1124>160.0426/114.0374), Amoxicillin penicilloic acid (amoxicilloic acid) (384.1223>367.0954/323.1056/305.09520), and hydroxyphenylglycyl amoxicillin (515.1588>498.1361/349.0988/339.0988) (Figs 4 and 5). The high doses of amoxicillin required for treatment of severe infections should be administrated over a long period, and preferably by continuous intravenous administration [1–9]. According to the limited volume of medical devices available for continuous drug infusion,

particularly

the portable devices used for OPAT, highly concentrated solutions of amoxicillin need to be prepared, leading to issues regarding solubility. In this study, we have demonstrated for the first time that amoxicillin has to be reconstituted at concentrations below or equal to 150 mg/mL to achieve complete solubility, which is mandatory in order to ensure accurate drug dosage and efficacy when using intravenous administration mode.

Moreover, incomplete solubility of a drug may lead to particles in solutions, which may induce serious harmful effects such as pulmonary embolism or systemic inflammatory response syndrome [27–29].

Therefore, an appropriate volume of solvent is required for the reconstitution of amoxicillin to ensure safe administration, excluding the use of low-volume devices such as syringes or small volume portable pumps, when high doses of amoxicillin are to be administrated. This work confirms that amoxicillin degradation is time, temperature and concentration-dependent, resulting in short-term stability of amoxicillin solutions for infusion, particularly at high concentrations.

Consequently, continuous infusion of the high-dose amoxicillin recommended to treat serious infectious diseases requires applying rigorous conditions of administration especially for OPAT.

Elastomeric pumps are commonly used in the setting of OPAT and are available in different volumes and flow rates to allow proper administration of a drug [30]. Therefore, we have investigated different volumes and flow rates through the use of different devices in order amoxicillin for stomach infection to determine which one best helps to achieve amoxicillin concentration-dependent stability.

Regarding storage temperature, elastomeric pumps may be used in different conditions, refrigerated or not.

Moreover, it has been demonstrated that the temperature of drug solutions in elastomeric pumps may dramatically increase

over

the day, especially for continuous infusion, reaching a maximum near body temperature [31].

In this context, we have investigated the impact of potentially different storage temperatures on amoxicillin stability in order to determine the optimal conditions for its administration.



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