Please cite this article in press as: P. Araujo, et al., J. Chromatogr. A (2012), http://dx.doi.org/10.1016/j.chroma.2012.08.056 ARTICLE IN PRESS G Model CHROMA-353559; No. of Pages 9 Journal of Chromatography A, xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Journal of Chromatography A jou rn al h om epage: www.elsevier.com/locat e/chroma Application of Doehlert uniform shell designs for selecting optimal amounts of internal standards in the analysis of prostaglandins and leukotrienes by liquid chromatography–tandem mass spectrometry Pedro Araujo a,∗ , Steve Janagap a,b , Elisabeth Holen a a National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029, Nordnes, N-5817 Bergen, Norway b Department of Chemistry, College of Arts and Sciences, University of the Philippines, Visayas, 5023 Miagao, Iloilo, Philippines a r t i c l e i n f o Article history: Received 8 May 2012 Received in revised form 13 August 2012 Accepted 16 August 2012 Available online xxx Keywords: Response factor Doehlert uniform shell designs Cell culture Eicosanoids Multiple internal standards a b s t r a c t A protocol for the analysis of multiple prostaglandins and leukotrienes in cell culture media by using mul- tiple internal standards was validated. A two-factor Doehlert design was used to determine the behaviour of the relationship analyte/internal standard (namely: PGE 2 /PGE 2 -d 4 , PGE 3 /PGE 2 -d 4 , LTB 4 /LTB 4 -d 4 and LTB 5 /LTB 4 -d 4 ) and to select the optimal amounts of deuterated internal standards for quantifying simulta- neously the prostaglandins and leukotrienes in cell culture media by LC–MS/MS. The selection of optimal amounts of internal standards was based on mathematical models that allow visualizing concentration regions where the response factors remain constant over a wide range of analytical concentrations. The linearity of the calibration curves for each analyte at the optimal levels suggested by the mathematical models was statistically confirmed by means of the ratio lack-of-fit to pure error. The validated protocol was successfully applied in the simultaneous quantification of pro- and anti-inflammatory eicosanoids in stimulated cod head kidney cell culture media. The two-factor Doehlert design has permitted to estimate the experimental response as a function of six variables (PGE 2 , PGE 3 , LTB 4 , LTB 5 , PGE 2 -d 4 and LTB 4 -d 4 ) which represents a substantial reduction of resources, time and experiments of approximately 84% (7 × 3 experiments) when compared with the full six-factor Doehlert design (43 × 3 experiments). © 2012 Elsevier B.V. All rights reserved. 1. Introduction Inflammatory diseases affect over 100 million people world- wide and create a burden in health care costs, lost productivity and reduced participation in family life [1,2]. High incidences of inflammatory bowel diseases (IBD) in Europe are found in Scan- dinavian countries while the Mediterranean countries report the lower rates [3,4]. Changes in dietary habits have been proposed as one of the possible causes of inflammatory diseases, particu- larly in the type and amount of essential fatty acids [5–7]. The relative abundance of omega 6 (-6) and omega 3 (-3) polyun- saturated fatty acids (PUFA) has been suggested to be of major significance in human health and immune system [6–8]. It is well- documented that -6 arachidonic acid (20:4-6, ARA) through the action of cyclooxygenase (COX) and lipooxygenase (LOX) enzymes is converted into biologically active eicosanoids known as the 2- series prostaglandins and 4-series leukotrienes. Studies performed on fish and mammals have suggested that eicosanoids such as prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) play a primordial ∗ Corresponding author. Tel.: +47 95285039; fax: +47 55905299. E-mail address: pedro.araujo@nifes.no (P. Araujo). role in several pathological conditions such as inflammation [9] and pain [10]. The importance of incorporating dietary -3 PUFA in patients with rheumatoid arthritis due to the presence of eicosapentaenoic acid (20:5-3, EPA), which can alleviate inflammatory diseases by reducing the contribution of arachidonate-derived eicosanoids such as PGE 2 and LTB 4 and promoting anti-inflammatory metabo- lites such as PGE 3 and LTB 5 has also been demonstrated [11]. Dietary fish oils rich in EPA and docosahexaenoic acid (22:6-3, DHA) are known to replace ARA and to improve the immunopathol- ogy in New Zealand mice [12]. These observations suggest that the quantitative assessment of eicosanoid metabolites from the COX and LOX pathways is vital for evaluating the behaviour of the precursor molecules in the cell membrane and studying how these substrate molecules affect the synthesis of leukotrienes and prostaglandins. Cell culture experiments are ideal approaches for the examina- tion of the causal relationship between PUFA and eicosanoids. The manipulation of the levels of ARA and EPA in cell culture experi- ments allows the quantitative estimation of their related COX- and LOX-related metabolites using different analytical techniques such as enzyme-linked immunosorbent assay (ELISA), gas chromatogra- phy mass spectrometry (GC–MS) and liquid chromatography–mass 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.08.056