Journal of Chromatography B, 877 (2009) 719–724 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Application of liquid chromatography–mass spectrometry to measure short chain fatty acids in blood Hans M.H. van Eijk ∗ , Johanne G. Bloemen, Cornelis H.C. Dejong Maastricht University Medical Centre, Department of Surgery, NUTRIM School for Nutrition, Toxicology & Metabolism, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands article info Article history: Received 30 September 2008 Accepted 28 January 2009 Available online 5 February 2009 Keywords: Short chain fatty acids Mass spectrometry Liquid chromatography abstract A new liquid chromatography–mass spectrometry method is described to determine concentrations of the short chain fatty acids acetic acid, propionic acid and butyric acid (SCFAs) in human blood plasma. The method is based on reversed phase chromatography followed by post-column neutralization of the mobile phase with ammonia and a consecutive measurement of the SCFAs ammonia adducts using negative electro spray ionization. Sample preparation involved simple organic acid deproteinization, resulting in 100% recovery. SCFAs eluted baseline separated within a 25 min run cycle. A linear response was obtained in the range between 0 and 250 mol/l (R 2 ranged from 0.997 to 0.9999). The limit of detection ranged from 0.05 mol/l for propionic and butyric acid and 0.1 mol/l for acetic acid. The method was tested by analyzing plasma of arterial blood, from portal vein and hepatic vein blood from patients undergoing a pylorus-preserving pancreaticoduodenectomy. As expected, the highest SCFA concentrations were found in portal plasma, hepatic vein levels were in between, while arterial concentrations were lowest. This newly developed method is suitable to determine SCFA concentrations in human plasma samples. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Bacterial fermentation in the colon of complex carbohydrates that escape digestion in the small intestine results in produc- tion of acetic acid, propionic acid and butyric acid, also known as short chain fatty acids (SCFAs) [1]. SCFA in general exert a vari- ety of effects important for intestinal health and function and in particular butyric acid serves as principal metabolic fuel for colonic epithelial cells [1,2]. Under normal conditions the liver takes up most of the surplus of intestinal butyric and propionic acid, converting it mainly into acetoacetic acid, resulting in low sys- temic propionic and butyric acid concentrations, while acetic acid levels, which escapes liver metabolism, are usually much higher [3]. Presently, numerous studies are undertaken in an effort to increase intestinal SCFA concentrations by providing human sub- jects with pre-biotics. However, it is still unclear to what extent the liver is capable of metabolizing an increased SCFA inflow, both under normal and pathological conditions. This is important in pre- venting too high systemic concentrations, because such elevated levels are considered toxic [4,5]. To expand our knowledge about interorgan exchange of SCFA in humans and especially the role of gut and liver in SCFA metabolism in vivo, we aimed to measure SCFA ∗ Corresponding author. Tel.: +31 433881496; fax: +31 433884154. E-mail address: hmh.vaneijk@ah.unimaas.nl (H.M.H. van Eijk). concentrations in portal venous, hepatic venous and arterial blood, collected during upper abdominal surgery. Although several methods have been reported to measure SCFAs in blood [6–8]. Most of these methods however, require laborious sample preparation and/or apply acid deproteinization. Recently, we reported a liquid chromatography–mass spectrometry method to measure SCFA concentrations in blood [9], in which we also showed acid deproteinization does not provide a 100% recovery for SCFAs. We now aim at developing a new method with reduced run times, enhance sensitivity and a reduce need of analytical col- umn and mass spectrometer (MS)—system regeneration to allow analyses of large numbers of samples collected in patient studies. Therefore, a new approach was developed in which plasma sam- ples were deproteinized with organic acid, the supernatant was separated on a reversed phase column with hydrochloric acid acid- ified water and an ethanol gradient. Next, the column effluent was mixed with ammonia, thus increasing the pH to allow SCFA mea- surement through negative ionization electro spray MS. 2. Materials and methods 2.1. Chemicals All chemicals used were of analytical grade, and unless spec- ified otherwise, purchased from Sigma–Aldrich (Zwijndrecht, the Netherlands). Ultra-pure water was generated through a Super-Q water purification system (Millipore, Amsterdam, the Netherlands). 1570-0232/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2009.01.039