Determination of urea content in urea cream by centrifugal partition chromatography

摘要

Abstract The objective of this study is to establish a centrifugal partition chromatography (CPC) method for determination of the urea ingredient in urea cream. The mechanism of this method is that urea is determined by UV detector at 430?nm after being extracted from the cream and derivatized on line via Ehrlich reaction in rotor of CPC, where the reaction products dissolve in the mobile phase and the cream matrix retains in the stationary phase. The mixed solvent consisting of n -hexane, methanol, hydrochloric acid and p -dimethylaminobenzaldehyde with a ratio of 1000?mL:1000?mL:18?mL:2.0?g is used for solvent system of CPC. The CPC method proposed offers good precision and convenience without complex sample pretreatment processes. Graphical abstract Figure options Download full-size image Download as PowerPoint slide Keywords Centrifugal partition chromatography ; Content determination ; Urea cream prs.rt("abs_end"); 1. Introduction Cream is one of the common dosage forms for topical applications. The assay of active pharmaceutical ingredients in cream preparations usually requires tedious processes for sample pretreatment, including heating, dissolving, freezing, extracting, and diluting to a constant volume. These procedures may cause systematic errors or the degradation of some heat-labile ingredients during sample pretreatment. Additionally, other factors, such as extraction methods, pH value, time of heating and freezing, or mode and intensity of flask shaking, may also influence final analysis results. Cream or ointment containing urea is usually used to treat hand and feet chapping, and the urea-content determination in pharmaceutical preparations is mainly carried out by UV-Vis spectrophotometry, infrared spectroscopy, and urease methods [1] . According to Chinese Pharmacopoeia, Japanese Pharmacopoeia Fifteenth Edition , European Pharmacopoeia v7 and US Pharmacopoeia 35 , the current methods for urea-content determination in raw material is titrimetry, which is not suitable for urea cream, because the cream matrix usually interferes with the color change of the indicator, causing unstable results with a relative standard deviation (RSD) 2% [2] and [3] . According to British Phamacopoeia 2009, Volume III, the determination of urea cream is fulfilled by the urease method [4] and [5] . In our study, a centrifugal partition chromatography (CPC) method for the determination of urea cream was proposed for the first time. In the CPC method, p -dimethylamino- benzaldehyde (PDAB) is a derivatization agent that reacts with the urea in the cream inside the CPC partition cells. The derivatization product is separated by CPC and detected by UV detector, thus achieving the combination of chromatography and spectrophotometry. Counter-current chromatographic (CCC) methods are a form of liquid-liquid partition chromatography invented in the 1940s [6] , [7] , [8] and [9] . In modern era of CCC, CPC is equipped with a horizontal rotor consisting of the superposition of disks engraved with small cells that are connected by head/tail ducts on the basis of hydrostatic equilibrium systems. Currently, CPC is widely applied to analyze natural products, pharmaceuticals, and other synthetic organic and inorganic chemicals due to its high stationary phase retention, rapid separation and convenient operation [10] , [11] and [12] . Since CPC does not need any solid support [13] , the stable and uniform emulsion samples can be injected into the apparatus without pretreatment, which simplifies sample preparation, minimizes systematical error, and eliminates the influence of cream matrix on assay results. Therefore, developing a simple, rapid, and precise CPC method for the determination of the urea ingredient in urea cream is advantageous and practical. 2. Methods 2.1. Apparatus CPC 240 (Sanki Engineering, Kyoto, Japan) is equipped with a Waters 2707 autosampler, a Waters 2489 UV detector, and a Waters 1515 pump (Waters; Milford, MA, USA). The rotor of the CPC 240 is made up of 12 disks, with each disk consisting of 178 partition cells. Each partition cell consists of a channel and a duct, and the total volume for 12 disks is ～240?mL, 85% of which is for the channels and 15% for the ducts. 2.2. Materials and reagents The urea reference substance and PDAB were provided by National Institutes for Food and Drug Control. The urea cream sample (containing urea 0.1?g·g?1) was purchased from Shanghai General Pharmaceutical Co., Ltd., Shanghai. n -Hexane (analytical grade) and hydrochloric acid (analytical reagent: ～36% to 38%) were obtained from Fuyu Fine Chemical Co., Ltd, Tianjin, China. Methanol (high-performance liquid chromatography grade) was purchased from Concord Technology Co., Ltd, Tianjin, China. Ultrapure water was made by the Merck Millipore MILLI-Q system (EMD Millipore; Billerica, MA, USA). 2.3. Preparation of standard solutions and test solutions The standard stock solution was prepared as follows: 50?mg of urea r