Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 23–32 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis jou rn al h om epage: www.elsevier.com/locate/jpba Application of design of experiments and design space methodology for the HPLC-UV separation optimization of aporphine alkaloids from leaves of Spirospermum penduliflorum Thouars Mamy H. Rafamantanana a,b,∗ , Benjamin Debrus c , Guy E. Raoelison b , Eric Rozet c,1 , Pierre Lebrun c , Suzanne Uverg-Ratsimamanga b , Philippe Hubert c , Joëlle Quetin-Leclercq a a Groupe de Recherche en Pharmacognosie, Louvain Drug Research Institute (LDRI), Université catholique de Louvain, Av. Mounier 7230, 1200 Bruxelles, Belgium b Institut Malgache de Recherches Appliquées (IMRA), BP 3833, Itaosy, Antananarivo 102, Madagascar c Laboratoire de Chimie Analytique, Département de Pharmacie, CIRM, Université de Liège, CHU, Av. de l’hôpital 1, B36, B-4000 Liège, Belgium a r t i c l e i n f o Article history: Received 14 October 2011 Received in revised form 22 December 2011 Accepted 23 December 2011 Available online 25 January 2012 Keywords: Design of experiments Spirospermum penduliflorum Quantitative determination Validation Dicentrine a b s t r a c t Spirospermum penduliflorum Thouars (Menispermaceae) is an endemic species of Madagascar tradition- ally used as vasorelaxant. Recently, two aporphine alkaloids known to possess antihypertensive activity (dicentrine and neolitsine) were isolated and identified from the leaves of this plant. In the present study, a HPLC-UV method allowing the separation of all alkaloids and the quantification of dicentrine in the alka- loidic extract of leaves was developed using design of experiments and design space methodology. Three common chromatographic parameters (i.e. the mobile phase pH, the initial proportion of methanol and the gradient slope) were selected to construct a full factorial design of 36 experimental conditions. The times at the beginning, the apex (i.e. the retention time) and the end of each peak were recorded and modelled by multiple linear equations. The corresponding residuals were normally distributed which confirmed that the models can be used for the prediction of the retention times and to optimize the separation. The optimal separation was predicted at pH 3, with a gradient starting at 32% of methanol and a gradient slope of 0.42%/min. Good agreement was obtained between predicted and experimental chromatograms. The method was also validated using total error concept. Using the accuracy profile approach, validation results gave a LOD and LOQ for dicentrine of 3 g/ml and 10 g/ml, respectively. A relative standard deviation for intermediate precision lower than 10% was obtained. This method was found to provide accurate results in the concentration range of 10–75 g/ml of dicentrine and is suitable for routine analysis. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Nowadays, the traditional medicine still holds an important place to cure diseases in developing countries. It is mainly due to the non accessibility of modern medicine by the local population. The safety, effectiveness and quality of finished herbal medicinal products depend on the quality of their source materials, which can include hundreds of natural constituents, and how elements are handled through production processes. Spirospermum penduliflorum Thouars (Menispermaceae) was chosen among a list of medicinal plants used by Malagasy pop- ulation because it is an endemic species of Madagascar. This ∗ Corresponding author at: Groupe de Recherche en Pharmacognosie, Louvain Drug Research Institute (LDRI), Université catholique de Louvain, Av. Mounier 7230, 1200 Bruxelles, Belgium. E-mail address: mamy.rafamantanana@uclouvain.be (M.H. Rafamantanana). 1 F.R.S.-FNRS Postdoctoral Researcher (Belgium). plant is rich in alkaloids. The decoction of all parts is tradi- tionally used as anticholinergic and vasorelaxant [1] and the decoction of leaves is also used for the treatment of malaria and as a chloroquine adjuvant. The dried leaves are also smoked for pulmonary tuberculosis treatment. The decoction of roots was taken as cholagogue, tonic and for hepatic disorders [2]. Dif- ferent studies on root extracts of S. penduliflorum allowed the isolation of some active molecules: columbine (clerodane-type diterpenoid), palmitine (protoberberine-type quaternary alkaloid) and limacine (bisbenzylisoquinoline alkaloid) [3]. Recently, Raoeli- son et al. found a vasorelaxant activity on isolated rat aorta using a methanol/dichloromethane leaves extract (under publication). Two aporphine alkaloids were then isolated by bioguided frac- tionation: dicentrine (Fig. 1) and neolitsine both known to possess antihypertensive activities. In their experimental model, dicentrine gave an EC50 value of 0.15 ± 0.04 g/ml on rat aorta relaxation. In the previous studies, Teng et al. also demonstrated that dicentrine is a strong vascular -1 adrenoceptor antagonist 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.12.028