Organic acid anhydrides (OAAs) are low molecular weight, reactive compounds extensively used in industry. Exposure to these compounds may lead to allergic symptoms such as rhinitis and asthma. It is important to develop better and more informative methods for assessment of exposure to OAAs. The aim of this study was to develop a method for analysis of specific hexahydrophthalic anhydride (HHPA)-adducted tryptic peptides of human serum albumin (HSA) in nasal lavage (NAL). Furthermore, these peptides were evaluated as biomarkers of exposure. The proteins in the NAL samples were reduced, alkylated and digested with trypsin and the obtained peptides were analyzed using liquid chromatography/tandem mass spectrometry. The total amount of hydrolyzable HHPA in an HHPA-HSA conjugate was used for calibration. A deuterium-labeled HHPA-HSA conjugate was used as internal standard. Five volunteers were exposed to 10, 40 and 80 microg/m3 of HHPA in an exposure chamber and NAL samples were collected before and after exposure. Acceptable precisions of the assay at 13-14% were found for three adducted peptides. The mean levels of these three peptides for the five subjects ranged between 5-22, 15-75 and 33-125 pmol/mL NAL for the exposures at 10, 40 and 80 microg/m3, respectively. High correlations between air levels and the measured peptides were found on an individual basis but there were large inter-individual differences ranging between 63 and 110% for the three peptides. The large differences remained after protein adjustments. It was possible to detect exposures below 10 microg/m3 with the method. Thus, these adducted peptides may be used as biomarkers of exposure, which may better estimate the risk than previous biomarkers developed for OAAs.