Fully automated analytical procedure for propofol determination by sequential injection technique with spectrophotometric and fluorimetric detections Ivana Šrámková a , Célia G. Amorim b , Hana Sklenářová a,n , Maria C.B.M. Montenegro b , Burkhard Horstkotte a , Alberto N. Araújo b , Petr Solich a a Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic b REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal article info Article history: Received 5 June 2013 Received in revised form 26 September 2013 Accepted 30 September 2013 Available online 10 October 2013 Keywords: Propofol Sequential Injection Analysis Emulsions Horseradish peroxidase 4-Aminoantipyrine abstract In this work, an application of an enzymatic reaction for the determination of the highly hydrophobic drug propofol in emulsion dosage form is presented. Emulsions represent a complex and therefore challenging matrix for analysis. Ethanol was used for breakage of a lipid emulsion, which enabled optical detection. A fully automated method based on Sequential Injection Analysis was developed, allowing propofol determination without the requirement of tedious sample pre-treatment. The method was based on spectrophotometric detection after the enzymatic oxidation catalysed by horseradish peroxidase and subsequent coupling with 4-aminoantipyrine leading to a coloured product with an absorbance maximum at 485 nm. This procedure was compared with a simple fluorimetric method, which was based on the direct selective fluorescence emission of propofol in ethanol at 347 nm. Both methods provide comparable validation parameters with linear working ranges of 0.005– 0.100 mg mL À1 and 0.004–0.243 mg mL À1 for the spectrophotometric and fluorimetric methods, respectively. The detection and quantitation limits achieved with the spectrophotometric method were 0.0016 and 0.0053 mg mL À1 , respectively. The fluorimetric method provided the detection limit of 0.0013 mg mL À1 and limit of quantitation of 0.0043 mg mL À1 . The RSD did not exceed 5% and 2% (n ¼10), correspondingly. A sample throughput of approx. 14 h À1 for the spectrophotometric and 68 h À1 for the fluorimetric detection was achieved. Both methods proved to be suitable for the determination of propofol in pharmaceutical formulation with average recovery values of 98.1 and 98.5%. & 2013 Elsevier B.V. All rights reserved. 1. Introduction Sequential Injection Analysis (SIA) is a technique which is valued for its simplicity, easy control, versatility, repeatability, easy manipulation with solutions in a closed system, and the possibility of automation of complex analytical protocols. All these features make this technique suitable for the analysis of samples with complex matrices, such as pharmaceutical formulations or food [1], where a fully automated sample pre-treatment would bring a significant benefit. Several methods using SIA system as a tool for analysis of different pharmaceutical formulations have been suggested, measuring the drug content or evaluating various pharmaco- technological parameters stated in Pharmacopoeias such as dissolution [2] or liberation; even applications to semi-solid formulations such as ointments have been reported [3]. Emulsions are often used as pharmaceutical dosage form due to their capacity to dissolve and stabilize lipophilic compounds, while achieving a high applicability and bioavailability of the active substance. They are a heterogeneous mixtures of two immiscible liquids, one forming microdroplets within the other liquid. Emulsions can be administered either by the oral, topic, or parenteral route. If a pharmaceutically active substance possesses lipophilic properties and has to be administered intravenously, oil- in-water emulsion is generally prepared as a dosage form. Usually, a pharmaceutical emulsion represents a complex matrix for analysis, as it contains, apart from the active substance, two different solvents, generally water and vegetable oil as main constituents and further additives such as antimicrobials, anti- oxidants and surfactants which are necessary to maintain the required stability of the active substance and the matrix over the declared time. Both, the main emulsion constituents as well as the additives can significantly affect the analysis and therefore, Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/talanta Talanta 0039-9140/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.talanta.2013.09.059 n Corresponding author. Tel.: þ420 495067453; fax: þ420 495067164. E-mail address: Hana.Sklenarova@faf.cuni.cz (H. Sklenářová). Talanta 118 (2014) 104–110