Journal of Chromatography B, 814 (2005) 21–28 LC–MS analysis of phospholipids and lysophospholipids in human bronchoalveolar lavage fluid  Begona Barroso ∗ , Rainer Bischoff Center of Pharmacy, Bioanalysis and Toxicology, University of Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands Received 1 June 2004; accepted 15 July 2004 Available online 11 September 2004 Abstract A reversed phase HPLC method was developed for the simultaneous analysis of different phospholipids and lysophospholipids in hu- man bronchoalveolar lavage fluid (BALF). Separation was achieved using a pellicular C8 column at elevated temperatures with an increasing gradient of acetonitrile containing 0.1% formic acid. Detection was carried out by electrospray ionization ion-trap mass spectrometry. Calibra- tion graphs for selected phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and lysophosphatidylcholine) showed linearity up to 50ng allowing quantitative determinations. Identification of the individual species within each class was possible with tandem mass spectrometry. Analysis of BALF phospholipids was performed after liquid/liquid extraction with a mixture of chloro- form/methanol/acetic acid. Recoveries ranged from 69 to 97% with standard deviations of less than 6%. The limit of detection varied slightly between different classes but was in the range 0.05–0.25 ng total injected amount. © 2004 Elsevier B.V. All rights reserved. Keywords: Phospholipids; Lysophospholipids; BALF; HPLC/MS 1. Introduction Pulmonary surfactant is an essential fluid produced by alveolar type II cells, covering the entire surface of the lungs [1]. It consists of a complex mixture of lipids (about 90%) and specific surfactant-associated proteins (10%). Of the lipids 80–90% are phospholipids (PLs) and 10–20% con- sist of cholesterol, triglycerides and free fatty acids. The principal phospholipids are phosphatidylcholine (PC) species (aproximately 85%) containing high amounts of saturated  Presented at the Biomarker Discovery Symposium, Rotterdam, The Netherlands, May 14, 2004. Abbreviations: CID, collision induced dissociation; TIC, total ion chromatogram; EIC, extracted ion chromatogram; LC–MS, liquid chromatography–mass spectrometry; COPD, chronic obstructive pulmonary disease; BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid; PL, phospholipid; LPL, lysophospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; LPC, lysophos- phatidylcholine ∗ Corresponding author. E-mail address: b.barroso@farm.rug.nl (B. Barroso). palmitic acid (C16:0) and phosphatidylglycerol (PG). Other phospholipids usually found in small amounts are phos- phatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) [2]. The most important role of surfactant is to decrease the surface tension at the air–liquid interface and, therefore, to reduce the tendency of alveoli to collapse during expiration. It also contributes to the regulation of airway fluid balance, improves bronchial clearance and sets up a barrier to inhaled agents. Apart from this biophysical function, surfactant is in- volved in immunomodulation [3]. Biochemical and biophys- ical surfactant abnormalities have been found in various lung diseases such as acute respiratory distress syndrome (ARDS), pneumonia and lung edema. In our research we are interested in disease processes in- volving chronic lung inflammation, such as chronic obstruc- tive pulmonary disease (COPD) and Bronchiolitis Obliterans (BO). A decrease in the total amount of phospholipids, which has been observed in patients with chronic lung inflamma- tion [4], is known to enhance injury by elastase secreted from neutrophils and to induce collapse of bronchioles [5]. 1570-0232/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2004.07.044