Stability Study of 0.5 g/mL Urea Oral Solution in InOrpha^®

Introduction

Hyponatremia is defined by urea serum concentration below 135 mmol/L and become severe hyponatremia under 130 mmol/L. The etiologies of hyponatremia can be iatrogene (diuretics, antidepressors.), cardiac or adrenal insufficiency, or syndrome of inappropriate secretion of antidiuretic hormone, which is a paraneoplasic syndrome. Urea is the main nitrogen-containing substance in the human urine. It is synthesized by the liver and corresponds to the catabolism of amino-acids in excess in the organism. It represents about half of the daily osmotic load eliminated in the urine. The antinatriuretic and osmotic properties of urea can be used to normalize the serum sodium level [1] and it takes place in the European clinical practice guideline on diagnosis and treatment of hyponatraemia [2]. It is recommended in the 2nd line treatment in moderate to severe hyponatraemia induced by syndrome of inappropriate antidiuretic hormone secretion (SIADH) when water restriction is insufficient. A posology of 0.25–0.5 g/kg daily is suggested. In the literature, a classical delivery of 10 g urea powder to be dissolved in 100 mL water before use is described [2]. Urea is highly soluble in water [3] and is highly polar because of the electronegativity of its nitrogen and oxygen atoms. It will be able to engage three hydrogen bonds: two as donors and one as an acceptor. Its high melting point (132 to 135 °C) suggests that the forces binding the urea molecules to each other in the solid state are relatively important (hydrogen bond type) [3]. Urea is more stable at pH between 4 and 8 and the lowest degradation is found with a buffered solution at pH 6 [4].

The pharmacy has developed a 0.5 g/mL Urea oral liquid solution in InOrpha^®, a common commercial aromatized and suspending vehicle, with better organoleptic characteristics to improve treatment adherence and reduce the preparation time [5]. InOrpha^® was selected because this excipient has many benefits: ready to used, sugar-free and paraben free, buffered at pH 4,7, within urea stability interval. The organoleptic property allows reducing the poor palatability of urea. The microbiological stability is 3 months after opening according to manufacturer recommendations [6, 7].

However, there were no data about urea stability in the suspending vehicle, InOrpha^® [7]. A stability study was carried out according to the recommendations of the International Conference on Harmonization (ICH) to set a shelf life in order to deliver the drug to patients with quality and safety [8]. The purpose of this study was to determine physicochemical and microbiological stability of the oral liquid solution. Many analytical methods for urea in biological, environmental and food sciences were reported in the scientific literature [9, 10, 11, 12] but not for assay in hospital preparation. Even if the high-performance liquid chromatographic (HPLC) with mass spectrometry detection appears to be the most accurate method [12], a simpler solution, developed by Koebel M., with UV-detection provides a detection limit of 0.2 mg/mL in accordance with expectations of the values from raw material tested and especially for this solution [9]. For these reasons, we decided to adapt the Koebel’s method [10] and to validate it for the stability assay of urea.

Materials and methods

Results

Analytical validation

Matrix effect

Chromatographic patterns from InOrpha^® diluted in phosphate buffer and Chromatograms of phosphate buffer alone were identical. Three smallest peaks not exceeding 500 uAU were observed at 1.63 min, 2.05 min and 2.51 min (Figure 1(a) and 1(b)).

Figure 1:

Representative chromatograms for matrix effect tests.

Chromatogram of urea solution diluted at 50 mg/L in phosphate buffer and chromatogram of a 1:10000 dilution in phosphate buffer of the 0.5 g/mL urea oral liquid solution in InOrpha^® gave the same peak at 2.2 min of an intensity of 65 000 uAU . As found during the tests with InOrpha^® and phosphate buffer alone, another peak not exceeding 500 uAU was observed at 2.51 min (Figure 1(c) and 1(d)).

Linearity

Correlation coefficients of the standard triplicate curves tested were respectively 0.9996, 0.9996 and 0.9998. Furthermore, the comparison tests of the variances, the slopes and the ordinates at the origin of the three curves do not show a significant difference to the relative risks α of 5 % (Figure 2).

Figure 2:

Linear standard calibration of average measured concentrations.

Precision

Precision was tested for both properties: repeatability and intermediate precision (reproducibility). The maximum deviation of the RSD % was 0.33 for the repeatability and 1.78 for the intermediate precision.

Accuracy

The RSD % were estimated to 0.36, 0.31 and 0.20 respectively on the 30, 50 and 60 mg/L concentrations of the targeted control samples. The relative errors were estimated to 2.35 %, 4.69 % and 1.87 % respectively on the same serie of concentrations of quality control samples.

Specificity

After 4 days at 75 °C, a decrease of urea concentration was observed from 47.5 mg/L to 37.6 mg/L which corresponds to about 21 % of loss, without the appearance of degradation peaks, undetectable at 190 nm (Figure 3).

Figure 3:

Representative chromatograms of urea standard solution, urea stressed using warm at 75 °C and phosphate buffer stressed using warm at 75 °C.

Detection and quantification limits

The detection limit was determined to 0.45 mg/L (or 9.10 ng) and the quantification limit was 1.52 mg/L (or 30.34 ng).

Discussion

Regarding to the method validation, all the parameters were acceptable for a stability indicating assays. No matrix effect of the suspending vehicle InOrpha^® was observed. Phosphate buffer used as mobile phase and diluent presents three small peaks in the chromatogram that have no consequence for urea quantification because of their low intensity in comparison with urea peak . The method was considered as linear because the correlation coefficient was over 0.999 for the mean curve. Moreover, accuracy and fidelity were validated because values were inferior to 5 %.

The method is stable and reliable to detect and quantify any degradation in the drug product during stability study. Indeed, the forced degradation study allowed to highlighting the degradation of the API with a loss of 21 % in urea concentration even if no degradation products could be detectable onto chromatograms. This lack of degradation products

Picture 1:

Precipitation and massive crystallization of urea solution in fridge (5 °C+/− 3 °C).

detection could be explained by the urea degradation way in aqueous solution leading to cyanate and ammonium ions, (NH₂)₂CO → CNO^– + NH₄⁺. Cyanate ion further readily undergoes conversion to CO₂ and ammonia [17]. At pH 7, ammonia remains as ammonium ions, so the column will not retain it, in addition to being undetectable at 190 nm.

This proposed UV-HPLC method was found to be simple, rapid, linear, accurate and stability indicating. Thus, it can be used for the assay of urea during the stability study and for routine quality control analysis in syrup, suspension preparation or for raw urea assay.

During 90 days, no significant variation of the urea concentration was observed in the solution. This concentration was always higher than 95 % of the initial concentration while the acceptance threshold was 90 %.

The pH measurements were not realized during this stability study. A previous study has shown that pH remained stable after 32 days for samples stored at fridge whereas a linear increase (r²=0.99) from 5.3 to 7.2 of pH was observed for the samples stored at room temperature. At room temperature, the pH not exceeds 6.3 after fifteen days. In absence of other date, it seems better to not exceed fifteen days for stability at room temperature. Beyond fifteen days, the pH variation exceed 1 unit and can’t be considered negligible [5].

Visual inspections revealed a crystallization of urea when the solution was stored at low temperature between +2 °C and+8 °C. Indeed, the 0.5 g/mL urea solution is highly concentrated and close to the saturation threshold, while urea solubility increases with temperature. Because of macroscopic changes observed, the oral liquid can’t be considered stable between+2 °C and+8 °C. Therefore, storage at room temperature was proposed for better homogenization of the solution and more convenient use.

Conclusion

Although, all the preparations retain more than 95 % of the initial concentration after 90 days in all storage conditions, macroscopic and pH change have to be taken into account. The 0.5 g/mL urea oral liquid solution in InOrpha^® remains stable for 15 days at room temperature (24 °C±1 °C) in amber glass bottles.