Chemical structures of (A) gliotoxin, (B) quercetin (IS). The structures were draw in the ChemSketch 11.0 software [16]. doi:10.1371/journal.pone.0092851.g001 

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... of aspergillosis is complex, particularly in immunocompromised patients. Signs and symptoms are non- specific, colonization is difficult to distinguish from invasive disease, blood cultures are commonly negative and patients are often unable to undergo invasive diagnostic procedures. This situation has led to the strategy of initiating empirical therapy in high-risk patients [3]. Typically, the diagnosis is based on histopathology or culture [4]. However, because the symptoms are similar to those of other diseases, the start of the correct treatment is delayed, reducing its effectiveness and, in some cases, leading to death [5]. Many studies have been published reporting conventional techniques of diagnosing A. fumigatus , including microbiological, histological and radiological analyses. However, these techniques present disadvantages, such as low sensitivity and specificity, high rates of false-positive results and a long time required to confirm the diagnosis [6]. Biopsies are also commonly used to diagnose A. fumigatus ; however, invasive procedures are not encouraged because of the weakened state of the patient [7–9]. The diagnosis of A. fumigatus has been accomplished using enzyme immunoassay tests against the galactomannan antigen [4]. Although this immunoassay looks promising, studies have demonstrated that the quantification of galactomannans by this technique can promote false-positive results because of the administration of antibiotics and can result from infections by fungi other than Aspergillus . Other tests used to diagnose invasive aspergillosis include (1 R 3)- b -D-glucan and polymerase chain reaction (PCR); however, these methods have not yet been evaluated for their validity in diagnosing this condition [4]. Several reports suggest that the suppression of the immune function of the host by mycotoxins (secondary metabolites released by fungi) is one of the possible mechanisms underlying why the fungus is not counteracted by the immune system [2,11]. Gliotoxin is one of the most toxic metabolites produced during the growth of several species of fungi, including Aspergillus fumigatus , Eurotium chevalieri , Gliocladium fimbriatum as well as Penicillium and Trichoderma spp. This toxicity is the result of the intramolecular disulfide bridge, which is the subject of many investigations of its structure and activity [12]. Sutton and coworkers (1996) demonstrated that the immune suppression produced by gliotoxin may promote the establishment of invasive aspergillosis [13]. Studies conducted in the lungs of rats with induced invasive aspergillosis showed that the presence of gliotoxin diminished the recognition of the fungus by the cells of the immune system [14]. Gliotoxin was also found in mice and humans with aspergillosis [14]. Based on these findings, it is evident that detection of gliotoxin by a sensitive, precise and accurate method may be an option for diagnosing invasive aspergillosis. Among the existing methods for the detection and quantification of gliotoxin, a semi-quantitative bioassay based on an automated microplate-reader operated at 630 nm was developed by Grovel and coworkers in 2006. This bioassay allowed for the rapid screening of samples for gliotoxin concentration in extracts with limits of detection of approximately 18 to 20 ng [15]. Gliotoxin was also measured by LC-MS-MS in the sera of mice (36.5 6 30.28 ng mL 2 1 ) with experimentally induced invasive aspergillosis (IA) and in the sera of cancer patients with documented (proven or probable) IA (65–785 ng mL 2 1 ) [14]. None of the described methods have precise materials and methods, making them difficult to reproduce. Additionally, none have been fully validated according to the modern worldwide regulations; thus, the reliability of their results cannot be ensured. The aim of this work is the development and validation of a new method based on high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) to detect gliotoxin in human serum for the early diagnosis of invasive aspergillosis. A pool of blank, hemolyzed and lipemic serum was obtained from healthy volunteers and were used in validation experiments (Section 6). Serum samples from patients hospitalized after bone marrow transplantation and patients undergoing chemotherapy were provided by the Hospital de Clinicas – UFPR and used for method application (Section 8). Methanol and acetonitrile (HPLC grade) were purchased from Tedia (Fairfield, Ohio, USA), and formic acid (88%) was obtained from J. T. Baker Chemicals B.V. (Deventer, The Netherlands). Ammonium formate (97%) was purchased from Acros Organics (New Jersey, USA). Ultrapure water was obtained using a Milli-Q TM purification system from the Millipore Corporation (Bedford, USA). Ether (99%), ethyl acetate (99.8%), a gliotoxin standard (99.0%) and the internal standard (IS) quercetin (99.0%) were purchased from Sigma Aldrich (St. Louis, USA). The structure of each metabolite is shown in Figure 1. Stock solutions of gliotoxin and IS were prepared separately in methanol at concentrations of 1 mg mL 2 1 . All stock solutions were stored at 4 u C. Working standard solutions were freshly prepared from these stock solutions as needed for each experiment via appropriate dilution with acetonitrile/water (98:2 v/v ). HPLC-MS/MS analyses were performed using an Agilent 1200 HPLC System (Wilmington, USA) consisting of a G1312B binary pump, a G1379B degasser and a G1316B column oven. The HPLC was connected to a CTC Sample Manager (Model 2777, Waters Corporation, Milford, USA). The HPLC system was coupled to an Applied Biosystems MDS Sciex API 3200 Triple Quadrupole Mass Spectrometer (Toronto, Canada) equipped with a Harvard 22 Dual Model syringe pump (Harvard Apparatus, South Natick, USA) and an electrospray ionization (ESI) source. The mobile phase consisted of a gradient of water (A) and acetonitrile/water 95:5 v/v (B), both containing 1 mM ammonium formate, eluted using the following gradient program: t 0 to 0.10 min : A = 65%; t 0.11 to 0.70 min : A = 0%; t 0.71 to 4.0 min : A = 65%. The flow rate was 0.45 mL min 2 1 . The analytical separations were achieved on an XBridge Shield C18 150 6 2.1 mm (5 m m particle size) column coupled with an XBridge C18 10 6 2.1 mm (5 m m particle size) guard column (Waters Corporation, Milford, USA). The injection volume was 20 m L, and the column temperature was maintained at 45 u C. Data acquisition was performed with the MS Workstation using Analyst 1.4 software (ABI/Sciex). The ESI source was operated in the negative ion mode for monitoring gliotoxin and IS. Quantification was performed in the Multiple Reaction Monitoring (MRM) mode while maintaining the dwell time at 450 ms. The ion source parameters were as follows: curtain gas (CUR), 10 psi; collision gas (CAD), 6 psi; ion spray voltage (ISV), 2 4500 V; nebulizer gas (GS1), 45 psi; turbo gas (GS2), 45 psi and temperature, 450 u C. The ion transitions and individual compound parameters, including the declustering potential (DP), entrance potential (EP), collision cell entrance potential (CEP), collision energy (CE) and cell exit potential (CXP), are shown in Table 1. The high-purity nitrogen and zero- grade air used as the CUR, GS1, GS2 and CAD gases were produced using a high-purity nitrogen generator from PEAK Scientific Instruments (Chicago, USA). To determine the best extraction procedures for gliotoxin and IS in human serum, several methods were tested, including protein precipitation, liquid-liquid extraction and solid phase extraction. The analyses were performed using six replicates. For each compound and each extraction procedure, the sensitivity and accuracy of the method were evaluated using the peak area, and the reproducibility was determined by the relative standard deviation (RSD%) of the peak area. 4.1. Serum fortification. A 200 m L aliquot of blank serum (serum free of analytes and IS) was transferred into a 2 mL plastic centrifuge tube. The serum samples were each spiked with 50 m L of the working standard solution and 50 m L of the working IS solution to obtain final concentrations of 250 ng mL 2 1 of gliotoxin and 5.0 ng mL 2 1 of IS. The samples were vortexed for 3 min and then subjected to different extraction procedures. 4.2. Extraction procedures. 4.2.1 Protein precipitation: A 1.2 mL aliquot of acetonitrile or methanol was added to the centrifuge tubes containing the homogeneous spiked serum (Section 4.1). The samples were vortexed for 3 min and then centrifuged at 14000 rpm for 10 min at 25 C (Eppendorf 5810R, Hamburg, Germany). The total volume of the supernatant was poured into a new plastic centrifuge tube and evaporated until dry in a sample concentrator (25 u C) (CentriVap Labconco, Kansas City, USA). The samples were resuspended in 200 m L of acetonitrile/water (98:2 v/v ) containing 0.1% formic acid and vortexed for 3 min. 4.2.2 Solid phase extraction: The total volume of the homogeneous spiked serum (Section 4.1) was transferred to an Oasis HLB Sample Extraction Cartridge TM (1cc, 30 mg) previously condi- tioned with methanol and ultrapure water. The extraction cartridge was flushed twice with water (1 mL each), and the retained analytes were recovered by washing the cartridges with 1 mL of pure acetonitrile. The eluate was evaporated until dry in a sample concentrator (25 u C) and redissolved in 200 m L of acetonitrile/water (98:2 v/v ) containing 0.1% formic acid by vortexing for 3 min. 4.2.3 Liquid-liquid extraction: A 1.2 mL aliquot of one of the various extraction solvents (toluene, diethyl ether, ethyl acetate, dichloromethane, dichloroethane, diisopropyl ether, acetone and mixtures of these solvents in the proportions given in Figure 2) was added to each individual centrifuge tube, each containing the homogeneous spiked serum (Section 4.1). The samples were vortexed for 3 min and then centrifuged at 14000 rpm for 10 min at 25 u C. A 1.2 mL aliquot of each sample ...