Received: 20 January 2012 Revised: 5 March 2012 Accepted: 6 March 2012 Published online in Wiley Online Library

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1 Research Article Received: 20 January 2012 Revised: 5 March 2012 Accepted: 6 March 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: /rcm.6224 A ultra-pressure liquid chromatography/triple quadrupole tandem mass spectrometry method for the analysis of 13 eicosanoids in human urine and quantitative 24 hour values in healthy volunteers in a controlled constant diet S. Medina 1, R. Domínguez-Perles 1, J. I. Gil 2, F. Ferreres 1, C. García-Viguera 1, J. M. Martínez-Sanz 3 and A. Gil-Izquierdo 1 * 1 Department of Food Science and Technology, CEBAS-CSIC, Campus de Espinardo, P.O. Box 164, Espinardo (Murcia), Spain 2 Mammary Pathology Department, Hospital José María Morales Meseguer, Avda Marqués de los Vélez, s/n. Murcia, Spain 3 Department of Physical Education and Sports, University of Alicante, Spain RATIONALE: Isoprostanes (IsoPs) are a series of prostaglandin (PG)-like compounds formed non-enzymatically through free-radical-induced peroxidation of arachidonic acid. They are considered as gold-standard biomarkers for oxidative stress, in general, and lipid peroxidation, in particular. METHODS: A new qualitative and quantitative analytical method for the determination of 13 eicosanoids in human urine using solid-phase extraction (SPE) and ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) has been developed. The SPE was optimized by comparison of the extraction efficiency and recoveries of three distinct cartridges: Strata X-AW, C18 Sep-Pak, and Oasis HLB. The UPLC/MS/MS approach in the multiple reaction monitoring (MRM) mode was developed using negative electrospray ionization (ESI). RESULTS: The validated method provides a high-throughput assay with an adequate linearity from 0.16 to 330 ng ml 1. The limit of detection (LOD) and limit of quantification (LOQ) for each analyte showed low intervals ( ng ml 1 and ng ml 1, respectively). Urinary IsoPs were determined in 24 healthy volunteers and ranged from 685 to 3480 ng 24 h 1 and from 864 to 7511 ng 24 h 1 in urine from women and men, respectively. CONCLUSIONS: This analytical method could constitute a useful tool for the determination of oxidative stress biomarkers in clinical studies in which IsoPs may evidence early pathological conditions, as suggested by the determination of the baseline IsoPs content in human urine, since it improves upon the detection capacity of previously described methods. The quantity of IsoPs excreted in urine was higher than that found in previous reports due to the total hydrolysis of the conjugated forms. Copyright 2012 John Wiley & Sons, Ltd. Oxidative stress (OS) leads to generation of reactive oxygen species (ROS) due to the imbalance between pro- and antioxidant states, [1] lipid peroxidation being the most-important phenomenon associated with OS. [2] Quantification of OS is of great importance for the understanding of the physiological and pathophysiological mechanisms associated with aging and degenerative conditions. One of the greatest challenges in the field of redox biology has been the identification of biomarkers in samples obtained by non-invasive methods, in order to assess ROS in vivo in humans. [3] It was in 1990 when a novel family of prostaglandin (PG)-like compounds named isoprostanes (IsoPs) was first reported. These compounds are formed non-enzymatically through free-radicalinduced peroxidation of arachidonic acid in vivo. [4] Then, they are cleaved by phospholipases and free IsoPs are released * Correspondence to: A. Gil-Izquierdo, Department of Food Science and Technology, CEBAS-CSIC, Campus de Espinardo, P.O. Box 164, Espinardo (Murcia), Spain. angelgil@cebas.csic.es from tissues into the circulatory system, where they are partially metabolized. Both metabolized and unmetabolized IsoPs are mainly excreted in the urine. [5] The IsoPs are considered gold-standard biomarkers of OS. Previously published data show them to be prognostic markers in certain pathologies such as cardiovascular disorders, diabetes, atherosclerosis, cancer, and neurological diseases. [2] Indeed, the detection of IsoPs could provide valuable information for the definition of the clinical pharmacology, helping to prevent the development of human degenerative diseases through the early identification of their onset. [6] Several studies have shown that levels of IsoPs augment in certain disorders or experimental conditions. These levels can be influenced by endogenous (gender, age, ethnicity, and pre- or post-menopausal state) and exogenous factors (smoking, alcohol intake, exercise, dietary habits, and drugs intake) that may alter the qualitative and quantitative occurrence of OS phenomena and, consequently, the synthesis of IsoPs. [7] For this reason, IsoPs can be considered as compounds with great clinical importance as useful, early biomarkers for disease diagnosis. Copyright 2012 John Wiley & Sons, Ltd. 1249

2 S. Medina et al Urine is considered as an ideal biological material for the measurement of IsoPs because, unlike plasma, it has low lipid concentrations; this minimizes artefacts generated by lipid autooxidation during sampling. [8] An additional problem lies in the selection of the most-appropriate methodological procedure to be used for analysis, since there are a wide variety of analytical methods, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) detection as well as immunological approaches (radio and enzymatic). Nevertheless, recently, the analysis of contents of IsoPs in biological samples has been carried out by ultra-pressure liquid chromatography (UPLC) coupled to triple quadrupole mass spectrometry (UPLC/MS/MS), because of its higher speed of analysis, selectivity, and sensitivity. [2] To the best of our knowledge, so far, only two methods have been developed for detection of IsoPs by UPLC/MS/MS [9,10] and the existing methods have room for improvement. Regarding the purification procedure, solidphase extraction (SPE) seems to be the most efficient technique. However, for IsoPs, a wide variety of cartridges with different compounds have been used and no comparative studies have been carried out to determine which among them are the most efficient and selective. The aim of this work was to develop a robust and reliable method for the identification and quantification of 13 eicosanoids in human urine, using SPE as the extraction procedure and UPLC/MS/MS, operating in multiple reaction monitoring (MRM) mode, as the acquisition and detection system. A comparative study of different SPE cartridges was carried out in order to establish the most efficient one, while the UPLC/MS/MS technology provides a more selective and sensitive method for quantification of IsoPs in urine. To finish, detection and quantification of the eicosanoids were carried out in healthy and sedentary volunteers with a similar controlled diet. EXPERIMENTAL Chemicals and reagents Synthetic PGs, including 8-iso-prostaglandin F 2a, 8-isoprostaglandin F 2a -d4 (contains 4 deuterium atoms at positions 3, 3, 4, and 4 ), 8-iso-15(R)-prostaglandin F 2a, 11b-prostaglandin F 2a, 2,3-dinor-8-iso-prostaglandin F 2a, 9,11-dideoxy-9a,11a-methanoepoxy-prostaglandin F 2a (U-46619), 9,11-dideoxy-9a,11a-epoxymethano-prostaglandin F 2a (U-44069), 2,3-dinor-6-keto-prostaglandin F 1a (sodium salt), 2,3-dinor-11b-prostaglandin F 2a, 6-keto-prostaglandin F 1a, 8-iso-15-keto-prostaglandin F 2a, tetranor-pgfm (tetranorprostaglandin F metabolite), tetranor-pgem (tetranorprostaglandin E metabolite), and authentic markers of 11-dehydro-thromboxane B 2 and 11-dehydro-thromboxane B 2 -d4 (contains 4 deuterium atoms at positions 3, 3, 4,and 4 ), were purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). Hydrolytic b-glucuronidase, type H2 from Helix pomatia, was purchased from Sigma-Aldrich (St. Louis, MO, USA). All LC-MS grade solvents were obtained from J.T. Baker (Phillipsburg, New Jersey, USA) and Bis-Tris (bis (2-hydroxyethyl)amino-tris(hydroxymethyl)methane) from Sigma-Aldrich. Reagents such as formic acid, chlorhydric acid, hexane, ethyl acetate, and potassium hydroxide were purchased from Panreac (Castellar del Vallés, Barcelona, Spain). Three types of SPE cartridges were used in this study: Strata cartridge (Strata X-AW, 100 mg 3 ml 1, Phenomenex, Torrance, CA, USA), C18 Sep-Pak classic cartridge, and Oasis HLB (both 200 mg 6 cc 1 ), both from Waters (Milford, MA, USA). Selection of volunteers Urine was collected from 24 volunteers (12 males and 12 females) aged 31 to 45 years, selected among healthy subjects based on a clinical examination (Table 1). The volunteers were non-smokers, had stable food habits, and did not receive any medication or drug intake. The dietary surveys showed that all volunteers followed a recommended isocaloric diet and the daily energy and nutrient intakes estimated from the previous 16-days food record did not differ from the intervention period (24 h) (Table 1). The nutritional variables analyzed included energy intake, total proteins, carbohydrates, dietary fiber, sugars, total lipids, saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), vitamin C, vitamin A, vitamin E, calcium, iron, and selenium (Table 1). In addition, the subjects were asked to maintain their lifestyle habits and to report any illness or abnormality during the study period. The participants were asked to keep a menstrual log; during this study, the women were not in their menstrual days. Written, informed consent was obtained from all the subjects and the study was approved by the Bioethics Committee of the University Hospital. Urine samples were collected after 24 h, aliquoted immediately, and stored at 80 C until posterior analysis. Collection and preparation of urine samples The urine samples were thawed at room temperature and centrifuged (11000 g, 5 min). The supernatant (400 ml) was mixed with the internal standards (8-iso-PGF 2a -d4 and 11-dehydro-thromboxane B 2 -d4). The SPE protocol was different and specific for each type of cartridge used (Fig. 1). The use of the Strata X-AW, C18 Sep-Pak, and Oasis-HLB cartridges followed the protocols described by Taylor et al., [11] Cavalca et al., [12] and Zhang et al., [10] respectively. The eluted compounds were dried completely using a SpeedVac concentrator (Savant SPD121P, Thermo Scientific, MA, USA). The extracts were reconstituted with the mobile phase A/B (90:10, v/v), solvent A being H 2 O/0.01 % acetic acid and solvent B MeOH/0.01 % acetic acid, to avoid the influence of the different solvents used in the different methods on the ionization of the sample at the electrospray interface and to make comparable the extraction results. The samples were injected and analyzed by UPLC/MS/MS. In order to determine the concentration of IsoPs in the baseline urine of the healthy volunteers selected, the 24-h urine of each subject was collected and the total volume determined. This volume was used for the absolute determination of the IsoPs excreted in 24 h. Given that IsoPs are excreted both as the free form and as glucuronide and sulfate derivatives, [13] 1 ml of baseline urine was hydrolyzed by b-glucuronidase, type H2 from Helix pomatia, for 2 h at 37 C. The samples were clarified further with MeOH/HCl (200 mm) and centrifuged at g, for 5 min. [13,14] The supernatant underwent an SPE using the Strata X-AW cartridge, selected according to the conclusions obtained in wileyonlinelibrary.com/journal/rcm Copyright 2012 John Wiley & Sons, Ltd.

3 Simultaneous analysis of 13 eicosanoids in human urine by UPLC/MS/MS Table 1. Body weight, height, BMI, age and daily energy and dietary intake of the 24 subjects at baseline Women (n = 12) Men (n = 12) Weight (kg) Height (m) BMI (kg m 2 ) Age (years) Estimated means Energy intake (kcal day 1 ) Total proteins (g day 1 ) Carbohydrates (g day 1 ) Dietary fiber (g day 1 ) Sugars (g day 1 ) Total lipids (g day 1 ) SFA Z (g day 1 ) MUFA Y (g day 1 ) PUFA X (g day 1 ) Vitamin C (mg day 1 ) Vitamin A (mg day 1 ) Vitamin E (mg day 1 ) Calcium (mg day 1 ) Iron (mg day 1 ) Selenium (mg day 1 ) Data are presented as mean SD. The data represent the daily energy and dietary intake. Z SFA: Saturated fatty acid, Y MUFA: Monounsaturated fatty acid, X PUFA: Polyunsaturated fatty acid. the comparative study of SPE techniques (see Results section), using the specific protocol described in Fig. 1. The eluted compounds were dried completely and the dried extract was reconstituted, as described above. Twenty microliters of each sample were injected and analyzed by UPLC/MS/MS, using the optimized method. Ion trap-ms/ms and UPLC/MS/MS analyses Previous to the development and creation of the UPLC/MS/ MS method for qualitative and quantitative detection of eicosanoids, all the IsoPs and thromboxane (TX), at 1 mm, were infused in methanol/water (50:50, v/v) into the ESI source of a Bruker ion trap mass spectrometer (model ultra- HCT) operating with nitrogen as flow injection and drying gas, electrospray ionization (ESI) source temperature, and capillary voltage of 5 psi, 3 L min 1, 350 C, and 4000 kv, respectively. The objective of this preliminary step was to know the exact pool of fragment ions generated at the MS/MS event of the corresponding deprotonated molecular ions of the whole IsoPs and TX detailed in this study. These data facilitated the UPLC/MS/MS optimization, as well as the elucidation of the most-sensitive MRM transition and the array of transitions with a satisfactory signal-to-noise (S/N) ratio, to confirm qualitatively the occurrence of the corresponding compound in urine. Separation of IsoPs was performed using a UPLC system coupled with a 6460 tandem mass spectrometer (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was carried out on a ZORBAX Eclipse Plus C18 column ( mm, 1.8 mm; Agilent Technologies). The column temperatures were 5 C (left) and 5 C (right). The MRM was performed using negative ESI mode and the dwell time was 25 ms for all MRM transitions. The MS analysis was applied in the MRM mode, assigning preferential MRM transition of the corresponding analytes (see Figs. 2 and 3 for original retention times). The mobile phases employed were solvent A (H 2 O/acetic acid; 99.99:0.01, v/v) and solvent B (MeOH/acetic acid; 99.99:0.01, v/v). The flow rate was 0.3 ml min 1 using the linear gradient scheme (t; %B): (0.00; 60), (7.00; 60), (7.01; 73), (10.00; 73), (10.01; 100), (13.50; 100), and (13.51; 60). The sample volume injected was 20 ml. The operating conditions for the MS parameters were as follows: gas flow: 8 L min 1, nebulizer: 30 psi, capillary voltage: 2750 V, nozzle voltage: 1500 V, gas temperature: 325 C, sheath gas temperature: 350 C, and jetstream gas flow: 12 L min 1. The acquisition time was 13.5 min for each sample, with a post-run of 1.5 min for the column equilibration. The MS parameters fragmentor (ion optics; capillary exit voltage) and collision energy were optimized for each compound, to generate the most-abundant product ions. Data acquisition and processing were performed using MassHunter software version B (Agilent Technologies). The urinary concentrations of the IsoPs and the TX were calculated from the area ratio of the ion peak of each compound to that of the corresponding standard in the urine of healthy sedentary volunteers. Sensitivity, precision, and accuracy The sensitivity of the analytical method was evaluated as the limit of detection (LOD), the lowest concentration of analyte that can be detected but not necessarily quantified. In our method, the LOD was established with a S/N ratio of 3:1. In the same way, the second parameter that informs on sensitivity, the limit of quantification (LOQ), was the lowest 1251 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

4 S. Medina et al. Figure 1. Schematic representation of the three distinct solid-phase extraction procedures performed on three separate classes of extraction cartridges: Strata X-AW (a), C18 Sep-Pak (b), and Oasis HLB (c), corresponding to the references Taylor et al., [11] Cavalca et al., [12] and Zhang et al., [10] respectively amount of analyte which could be determined quantitatively. The typical S/N ratio for LOQ of 10:1, previously established, [15] was also used in the present work. The precision (coefficient of variation (CV%)) and accuracy (% deviation) of the intra- and inter-day assays were also evaluated for the method developed. For intra- and interday determinations, three concentrations of the target eicosanoids within the linear range (1, 10, and 25 ng ml 1 ) were analyzed. The analyte concentrations were determined with daily calibration curves. To assess the precision and accuracy of the method, three replicates per specimen and run, and two runs per day, were carried out on five independent days. The CV% was calculated as follow: CV% ¼ ðstandard deviation=meanþ100: The standard deviations were used to assess the accuracy of the method. [16] RESULTS The validation of our method followed the FDA Guidance for Industry-Bioanalytical Method Validation (May, 2001) [16] and fundamental parameters were determined such as recovery, sensitivity, linearity, and precision, among others. Qualitative and quantitative MRM transitions and analytical selectivity IsoPs are characterized by having a free carboxylic group. Thus, negative ESI mode has been applied in this method because ESI results in an abundant [M H] carboxylated ionic environment, which helps the detection of low concentrations of IsoPs. [14] Furthermore, additional complementary MRM transitions (qualitative transitions), for further confirmation of the identity of compounds, have been used (Table 2). Analytical selectivity is the ability of a method to differentiate the specific analyte of interest from other matrix components, such as degradation products or impurities present in urine. The selectivity of this method was developed by analyzing five urine samples of different volunteers (among the 24 samples) and no chromatographic interferences were observed, either with the analytical method itself or with the solvents of the SPE protocol. Calibration curves The quantification of the eicosanoids was carried out by daily preparation of calibration curves using standard solutions. The linearity was assessed using aqueous/methanol solutions (50:50, v/v) of the standards. Calibration curves were wileyonlinelibrary.com/journal/rcm Copyright 2012 John Wiley & Sons, Ltd.

5 Simultaneous analysis of 13 eicosanoids in human urine by UPLC/MS/MS Figure 3. MRM chromatograms for deuterated standards (1 mm each): 8-iso-PGF 2a -d4 (A) and 11-dehydro-TXB 2 -d4 (B). Figure 2. Representative SPE-UPLC/MS/MS chromatograms of the 13 eicosanoids (1 mm, each compound) at the MRM transitions for quantification (Table 1). tetranor-pgfm (A); tetranor-pgem (B); 2,3-dinor-6-keto-prostaglandin F 1a (C); 2,3-dinor-8-iso-prostaglandin F 2a (D); 6-keto-prostaglandin F 1a (E); 2,3-dinor-11b-prostaglandin F 2a (F); 8-iso-15- keto-prostaglandin F 2a (G); 8-iso-15(R)-prostaglandin F 2a (H); 8-iso-prostaglandin F 2a (I); 11-dehydro-thromboxane B 2 (J); 11b-prostaglandin F 2a (K); U (L), and U (M). was calculated as previously described. [12] The ratio [(final concentration initial concentration)/added concentration] was determined for three different concentrations between the lower and upper limits of the previously estimated linear range of the standards (0.2, 150, and 330 ng ml 1 ). It was calculated in quintuplicate (n = 5) for each concentration level. Quantification of IsoPs was assayed previously without preliminary extraction (only centrifugation and filtration) and the results were not satisfactory, showing much lower and irregular recoveries for some analytes (10 98% as a general range). Therefore, matrix-related ion-suppression effects were found to be dramatic for some compounds (overall for Tetranor-PGEM and Tetranor-PGFM). In order to solve this problem, recovery tests with different extraction cartridges were performed: the Strata X-AW cartridges showed the highest recovery (93 107%), surpassing the C18 Sep-Pak (59 67%) and Oasis HLB (60 71%) cartridges. Based on these data, the Strata X-AW cartridge was chosen, as it showed the highest average recoveries for the 13 compounds tested. These results corresponded to the total recovery, since they provided information about the extraction method and the analytical signal of the UPLC/MS/MS. fitted by the linear regression equation y=ax, the correlation coefficients (R 2 ) being higher than 0.99 within the concentration range of 0.16 to 330 ng ml 1, for each separate analyte (Table 3). These R 2 values indicate an adequate linearity of the analytical procedure. The standard curves were tested separately or in a pool of all standards, with identical, satisfactory results. Recovery of the SPE cartridges and the matrix effect The recovery may be affected by several factors, including chromatography, ionization at the source, and selectivity of the SPE cartridges. It involves the efficiency of the extraction procedure and is determined by spiking the baseline urine samples with known amounts of standards. The recovery Sensitivity, precision, and accuracy The sensitivity of an analytical method is the capability of the technical procedure to discriminate differences in concentration or mass of the compounds as well as to provide a linear range indication. The LOD and LOQ values of the compounds analyzed ranged between and ng ml 1 and and 1.281, respectively: 8-Iso-PGF 2a and 11b-PGF 2a were the IsoPs with the lowest LOD (0.021 ng ml 1 ) and tetranor-pgem the one which showed the highest value (0.640 ng ml 1 ). Regarding the LOQ, 8-Iso-PGF 2a and tetranor- PGEM exhibited the lowest and highest LOQ values: and 1.281, respectively (Table 3). The intra-day coefficients of variation (CV%) for each compound ranged from 0.1 to 7%. The inter-day CV%, corresponding to the eicosanoids, ranged from 2.3 to 12% Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

6 S. Medina et al. Table 2. Precursor ions, product ions (for qualitative confirmation of the compound), and preferential MRM transitions for quantification of the eicosanoids by UPLC/QqQ-MS/MS Analyte Precursor ion (m/z) Product ions (m/z) Quantification transition 8-iso-prostaglandin F 2a ; ! iso-15(R)-prostaglandin F 2a ; ! b-prostaglandin F 2a ; ! ,3-dinor-8-iso-prostaglandin F 2a ; 227.0; ! U ; 223.3; ! U ; ! dehydro-thromboxane B ; 305.1; 243.1; ! ,3-dinor-6-keto-prostaglandin F 1a ; ! ,3-dinor-11b-prostaglandin F 2a ; 227.0; ! keto-prostaglandin F 1a ; 207.1; 163.1; ! iso-15-keto-prostaglandin F 2a ; 315.1; ! tetranor-pgfm ; ! tetranor-pgem ; 291.2; 264.9; ! iso-prostaglandin F 2a -d ; ! dehydro-thromboxane B 2 -d ; 309.2; ! Table 3. Analytical features of the method. The linearity, limit of detection (LOD), and limit of quantification (LOQ) for each eicosanoid Analyte Calibration equation Coefficient of regression (R 2 ) LOD (ng ml 1 ) LOQ (ng ml 1 ) 8-iso-prostaglandin F 2a y = x iso-15(R)-prostaglandin F 2a y = 97968x b-prostaglandin F 2a y = x ,3-dinor-8-iso-prostaglandin F 2a y = x U y = x U y = x dehydro-thromboxane B 2 y = 79209x ,3-dinor-6-keto-prostaglandin F 1a y = 20674x ,3-dinor-11b-prostaglandin F 2a y = x keto-prostaglandin F 1a y = 77036x iso-15-keto-prostaglandin F 2a y = x tetranor-pgfm y = x tetranor-pgem y = x Table 4. Urinary isoprostanes (ng 24 h 1 ) Z determined in 24 healthy volunteers (n = 12 women and n = 12 men) Analytes Eicosanoids (mean SD) Women Men 8-iso-PGF 2a iso-15(R)-PGF 2a b-PGF 2a ,3-dinor-8-iso-PGF 2a dehydro-TXB ,3-dinor-11b-PGF 2a tetranor-pgfm tetranor-pgem Z The volume of urine excreted by the women and men volunteers was and ml per 24 h, on average, respectively. Concentrations of eicosanoids in the urine of healthy human volunteers The analysis of the urine samples of 24 healthy volunteers (12 women and 12 men) using the new method allowed the detection of eight prostanoids, one of which has not been quantified previously in human biological samples. This IsoP, 8-iso-15(R)-PGF 2a, was quantified and identified according to its specific transitions from the precursor ion m/z [M H] to the product ions m/z [M H] and The transition m/z 353.0! was used to quantify the 8-iso-15(R)-PGF 2a in the urine samples: the average concentrations were 1593 and 2775 ng 24 h 1 for women and men, respectively (Table 4). In addition to this previously non-quantified IsoP, the new method developed in the present work allowed the identification and quantification of seven other prostanoids in urine, as listed in Table 4, which ranged from 685 to 3480 ng 24 h 1 and from 864 to 7511 ng 24 h 1 in urine from women and men, respectively. wileyonlinelibrary.com/journal/rcm Copyright 2012 John Wiley & Sons, Ltd.

7 Simultaneous analysis of 13 eicosanoids in human urine by UPLC/MS/MS DISCUSSION The aim of this work was to develop an analytical method for the simultaneous determination of 13 eicosanoids, in order to distinguish between isomeric forms of F 2a IsoPs without previous derivatization and implement the clinical utility of existing methods. The importance of the IsoPs derives from their biologicalactivity,aswellastheiruseasbiomarkersofdegenerative conditions (aging and disabling degenerative diseases); hence, they are useful for the early diagnosis of pathologies linked to oxidative stress (OS). [17,18] Furthermore, their measurement constitutes a suitable method for the evaluation of the influence of clinical treatments and environmental or nutritional factors able to modulate the OS and, consequently, the production of IsoPs, in the context of clinical or nutritional trials. The selection of the analyzed IsoPs and 11-dehydro-TXB 2 was based on their link to the initiation and progression of cardiovascular diseases. The 11-dehydro-TXB 2 concentration in urine has been correlated with different pathological conditions including diabetes mellitus, homocystinuria, hypertension, and obesity, [12] indicating its relevance as a biomarker for such conditions. Furthermore,theisoprostane IsoP 6-keto-PGF 1a was analyzed because of its close relationship with vasodilatation events and kidney function, [30] despite being an inactive form of the IsoP PGI. A method aimed at the measurement of IsoPs which are of use as biomarkers of OS in clinical applications should be specific, sensitive, reproducible, and robust. The GC/MS technique was the first one used for the quantification of these compounds. [19] Among the handicaps reported regarding this technique [20] (extensive sample preparation and weak specificity), an inability to isolate isomers of IsoPs can be highlighted. Later, other techniques, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), were developed for the determination of IsoPs in biological samples. [21] The immunological methods were chosen because they are relatively inexpensive, reproducible, and easy to perform. However, the cross-reactivity among the different IsoPs and the over-estimation shown by these methods constituted significant disadvantages, making them inappropriate for clinical application. [22,23] In order to overcome the difficulties associated with GC/MS and immunoassays, detection and quantification of IsoPs were performed by LC/MS. [24] LC/MS is more specific because it allows the separation and identification of regioisomers and stereoisomers, and reduces the interferences of some classes of IsoPs. In addition, it constitutes a more sensitive method, since LC coupled with MS allows the detection of low levels of IsoPs in complex biological matrices. [20,21] LC/MS involves fewer steps than GC/MS given the omission of derivatization and therefore is less prone to artifact generation. [25] However, the LC/MS technique also shows limitations, due to the restricted number of transitions that can be monitored. This handicap may be overcome by chromatography devices coupled to a tandem mass spectrometer (MS/MS), such as UPLC/MS/MS, which improves peak shapes and achieves more reproducible chromatographic retention times, for shorter times of analysis. [26] The greater clinical utility of UPLC/MS/MS prompted us to develop and validate a new method for the analysis of IsoPs in human urine. The separation of the compounds using the chromatographic conditions described was satisfactory (Figs. 2 and 3). For the indubitable identification and quantification of the target compounds, a minimum of two transitions were used to identify each separate compound. The most intense transition was selected for IsoPs quantification, whereas the additional complementary MRM transitions were employed to confirm the identity of the compounds. Some MRM transitions were not exclusive to a single IsoP. This was the case for the transition 325.2! 237.1, corresponding to 2,3-dinor-8-iso-PGF 2a and 2,3-dinor-11b-PGF 2a. Both compounds also showed equal complementary qualitative transitions (325.2! and 325.2! 227.0), but were differentiated by their distinct retention times (2.266 and min, respectively). The same happened regarding the transition 349.2! 219.1, which is characteristic of the IsoPs U and U-46619, and they were distinguished by their retention times: and min, respectively (Table 2). Currently, the use of an SPE method for extraction of IsoPs and thromboxanes is mandatory to clean up, concentrate, and decreasethematrixeffectattheesiofthesample.themost common SPE techniques, for the detection of these compounds by LC or UPLC coupled to MS, have involved C18 Sep-Pak, [12,27] Oasis HLB, [10] and Strata X-AW cartridges. [12,28] Thus, comparison of the efficiencies of these types of cartridge for extraction of IsoPs is a key point for improving the analysis of the target eicosanoids, by UPLC/MS/MS. According to our analysis, the Strata X-AW cartridge provided the best results due to its higher recovery rates in urine samples. The weak ionic-exchange interaction of the Strata X-AW resin also provided the best reproducibility, in terms of percentage and reliable extraction of the analytes described in this study, compared to the other types of cartridge, which showed irregular extraction, depending on the nature of the IsoPs. Previous studies have measured concentrations of IsoPs in human urine [10] and human colonic mucosae [29] by UPLC/MS/MS. These were limited to the study of one (8-iso-PGF 2a,usually)orvery-fewIsoPsand/orTX(amaximum of 4). In comparison, our method establishes the conditions to increase significantly the number of eicosanoids that can be detected with high reproducibility and selectivity in a single injection. In addition, the sensitivity is improved. The relevance of the increased number of IsoPs analyzed derives from the fact that the dominant IsoPs formed in the tissues might not be the most abundant IsoPs in the urine: [24] the new method will lead to a better understanding of the metabolic pathways involved in the processing of the IsoPs present in tissues, prior to their excretion in urine. The method described by Blatnik and colleagues for the determination of the concentrations of the IsoPs tetranor-pgem, 6-keto-PGF 1a, and 2,3-dinor-6-keto-PGF 1a in urine by UPLC/MS/MS [30] gave LOD values (0.03 ng ml 1 for the three IsoPs) which were lower than those found with our method ( ng ml 1 ), due to a previous derivatization of the carbonyl groups of these compounds with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBOA), at 60 C for 30 min. For methods not involving derivatization, other studies showed that the LOD for 8-iso-PGF 2a in human urine was higher than that given by our method. [12,31,32] However, the LOD values of raw, underivatized samples were higher than in the present work (1 ng ml 1 for tetranor-pgem and 6-keto-PGF 1a, and 20 ng ml 1 in the case of 2,3-dinor-6- keto-pgf 1a ) Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

8 1256 The application of our method to the qualitative and quantitative determination of IsoPs, in the urine of healthy volunteers (n = 24) allowed us to contrast the utility of the method: eight prostanoids were detected and quantified, all at concentrations much higher than the LOQ, while another five were not detected. It is known that large amounts of urinary IsoPs are excreted as conjugates with glucuronic acid. In this work, all the urine samples were pre-treated enzymatically, which resulted in an increase in the concentrations of the prostanoids detected. However, other IsoPs were not detected (U-46619, U-44069, 2,3-dinor-6-keto-PGF 1a,6-keto-PGF 1a, and 8-iso-15-keto-PGF 2a ). This demonstrates the specificity of these compounds, depending on the type of individual and related factors. Thus, levels of IsoPs can be influenced by different factors, as described by Basu et al. [7] In our study, the influence of these factors on the concentrations of IsoPs was minimal, due to the fact that all volunteers were healthy, sedentary non-smokers and followed the same diet. The major metabolite of 8-iso-PGF 2a in humans is 2,3-dinor-8-iso-PGF 2a, which appears as the most-abundant eicosanoid in urine. [33] The concentrations of both of these IsoPs given by our method are in agreement with the range present in the literature, obtained using LC/MS/MS: ng ml 1 and ng ml 1 for 2,3-dinor-8-iso-PGF 2a and 8-iso-PGF 2a, respectively. [32,34] Recent work describing the concentrations of IsoPs in human urine expressed the relative data as ng per mg of urinary creatinine. However, when working with the IsoPs contents of 24 h urine, given that it constitutes an absolute value, its expression in relative form to creatinine concentration may be unnecessary. [3] In order to compare the results obtained with those published previously, the data were normalized regarding the average creatinine concentration (mg 24 h 1 ) in urine [35] (data not shown). This showed that the amount of 11-dehydro-TXB 2 present in baseline urine was similar to that obtained by Weber and colleagues, by GC/NICI-MS. [36] The detection of 11b-PGF 2a by our method showed 5-fold greater sensitivity compared to that obtained by Sullivan and colleagues using immunoenzymatic assays. [37] Similarly, regarding the previously-reported 2,3-dinor-11b-PGF 2a concentration in human urine determined by GC/MS, [38] the sensitivity was 8-times lower than that found by our method. These results were due to the greater suitability and selectivity of MS techniques for the quantification of these compounds. Finally, tetranor-pgfm and -PGEM were also detected in the baseline urines analyzed, and the concentrations obtained in our study were lower than those published by Hambert and colleagues. [39,40] These previous reports quantified the IsoPs contents, by immunoassays, as ng 24 h 1 and ng 24 h 1 for tetranor-pgfm and -PGEM, respectively. The differences in the prostanoid contents between the two approaches could derive from the lower specificity of the immunological quantification, which may cause an over-estimation of the values. Indeed, the hydrolysis of the urines allowed higher concentrations of eicosanoids to be found, in comparison with the analyses of non-hydrolyzed urines in previous reports. CONCLUSIONS In recent years, IsoPs and TX have emerged as non-invasive markers of the oxidative stress associated with the development of several degenerative diseases. Their quantification has been proposed as a tool for the evaluation of antioxidant therapy. In biological fluids and tissues, co-eluting isomeric forms of IsoPs are usually found at low concentrations, which complicate their analysis. This prompted us to develop a rapid, accurate, and validated analytical UPLC/MS/MS method that is more specific and selective, and capable of the quantification and identification of twelve IsoPs and a TX (plus two deuterated compounds: 8-iso-PGF 2a -d4 and 11-dehydro-TXB 2 -d4) in a single injection of processed urine samples. Indeed, in comparison with previously described methods, the one proposed here is the first that allows the simultaneous measurement of 13 eicosanoids, with a 13.5 min run time, and their satisfactory separation, regardless of the different or common MRM transition used. The advantages of this analysis, in a unique assay, are linked to a greater control of the variability arising from the subjects, operator, laboratory, and analytical instruments, as well as to an important reduction in solvent consumption. It could be helpful for the early diagnosis of diseases related to oxidative stress, avoiding complications in the experimental and clinical settings. Furthermore, the Strata X-AW cartridge was shown to be the most adequate for the SPE of whole eicosanoids analyzed in this work. This new analytical method yielded concentrations of urinary IsoPs higher than those described previously by other authors. This may have been due, in part, to the greater sensitivity of the device used, the hydrolysis of the urine samples, the optimized solid-extraction procedure prior to analysis, and the performance of the analyses by UPLC/MS/MS. Acknowledgements The authors are grateful to the CICYT (Project AGL ), CONSOLIDER-INGENIO 2010 Fun-C-Food Project CSD , Grupo de excelencia de la Región de Murcia 04486/ GERM/06, and the Intramural Project (Ref E041). SME and RDP were supported by pre-doctoral grants from the JAE Program (Council for Spanish Research and European Social Fund) and a contract from the Spanish Ministry of Science and Innovation (MICINN), respectively. REFERENCES S. Medina et al. [1] S. Kaviarasan, S. Muniandy, R. Qvist, I. S. Ismail. J. Clin. Biochem. Nutr. 2009, 45, 1. [2] M. Comporti, C. Signorini, B. Arezzini, D. Vecchio, B. Monaco, C. Gardi. Mol. Aspects Med. 2008, 29, 43. [3] M. G. Nikolaidis, A. Kyparos, I. S. Vrabas. Prog. Lipid Res. 2011, 50, 89. [4] L. J. Roberts, II, J. D. Morrow. Cell. Mol. Life Sci. 2002, 59, 808. [5] S. S. Davies, L. J. Roberts, II. Free Radical Biol. Med. 2011, 50, 559. [6] J. D. Morrow, L. J. Roberts. Prog. Lipid Res. 1997, 36, 1. [7] S. Basu, J. Helmersson. Antioxid. Redox Signal. 2005, 7, 221. wileyonlinelibrary.com/journal/rcm Copyright 2012 John Wiley & Sons, Ltd.

9 Simultaneous analysis of 13 eicosanoids in human urine by UPLC/MS/MS [8] X. Wu, H. Cai, Y. B. Xiang, Q. Cai, G. Yang, D. Liu, S. Sanchez, W. Zheng, G. Milne, X.O. Shu. Cancer Epidemiol. Biomarkers Prevention 2010, 19, 947. [9] S. Basu. Prostaglandins Leukot. Essent. Fatty Acids 2010, 82, 219. [10] H. Zhang, D. Il yasova, J. Sztaray, S. P. Young, F. Wang, D. S. Millington. Anal. Biochem. 2010, 399, 302. [11] A. W. Taylor, R. S. Bruno, M. G. Traber. Lipids 2008, 43, 925. [12] V. Cavalca, F. Minardi, S. Scurati, F. Guidugli, I. Squellerio, F. Veglia, L. Dainese, A. Guarino, E. Tremoli, D. Caruso. Anal. Biochem. 2010, 397, 168. [13] Z. Yan, E. Mas, T. A. Mori, K. D. Croft, A. E. Barden. Anal. Biochem. 2010, 403, 126. [14] M. L. Langhorst, M. J. Hastings, W. H. Yokoyama, S. C. Hung, N. Cellar, K. Kuppannan, S. A. Young. J. Agric. Food Chem. 2010, 58, [15] International Conference on Harmonization (ICH). Validation of Analytical Method: Definitions and Terminology. ICH Q2A, Geneva, [16] FDA, US Department of Health and Human Services. Guidance for industry: bioanalytical method validation, May, Available: [17] Q. Cai, Y. T. Gao, W. H. Chow, X. O. Shu, G. Yang, B. T. Ji, W. Wen, N. Rothman, H. L. Li, J. D. Morrow, W. Zheng. J. Clin. Oncol. 2006, 24, [18] L. M. Dong, X. O. Shu, Y. T. Gao, G. Milne, B. T. Ji, G. Yang, H. L. Li, N. Rothman, W. Zheng, W. H. Chow, C. C. Abnet. Cancer Epidemiol. Biomarkers Prevention 2009, 18, [19] J. D. Morrow, K. E. Hill, R. F. Burk, T. M. Nammour, K. F. Badr, L.J. Roberts, II. Proc. Natl. Acad. Sci. USA 1990, 87, [20] B. Zhang, K. Saku. J. Lipid Res. 2007, 48, 733. [21] C. M. Spickett, I. Wiswedel, W. Siems, K. Zarkovic, N. Zarkovic. Free Radical Res. 2010, 44, [22] D. M. Callewaert, C. Sloan. Methods Mol. Biol. 2010, 610, 435. [23] Y. H. Teng, C. W. Wang, Y. T. Liao, M. W. Yang, T. Y. Liu. J. Clin. Lab. Anal. 2010, 24, 237. [24] H. Li, J. A. Lawson, M. Reilly, M. Adiyaman, S. W. Hwang, J. Rokach, G. A. FitzGerald. Proc. Natl. Acad. Sci. USA 1999, 96, [25] D. Sircar, P. V. Subbaiah. Clin. Chem. 2007, 53, 251. [26] C. Mesaros, S. H. Lee, I. A. Blair. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009, 877, [27] M. Korecka, C. M. Clark, V. M. Y. Lee, J. Q. Trojanowski, L. M. Shaw. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2010, 878, [28] A. W. Taylor, M. G. Traber. Anal. Biochem. 2010, 396, 319. [29] M. Mal, P. K. Koh, P. Y. Cheah, E. C. Y. Chan. Rapid Commun. Mass Spectrom. 2011, 25, 755. [30] M. Blatnik, R. C. Steenwyk. Prostaglandins Other Lipid Mediat. 2010, 93, 8. [31] J. H. Dahl, R. B. van Breemen. Anal. Biochem. 2010, 404, 211. [32] Y. Liang, P. Wei, R. W. Duke, P. D. Reaven, S. M. Harman, R. G. Cutler, C. B. Heward. Free Radical Biol. Med. 2003, 34,10. [33] J. Nourooz-Zadeh, M. B. Cooper, D. Ziegler, D. J. Betteridge. Biochem. Biophys. Res. Commun. 2005, 330, 731. [34] W. Yan, G. D. Byrd, M. W. Ogden. J. Lipid Res. 2007, 48, [35] G. L. Blackburn, B. R. Bistrian, B. S. Maini, H. T. Schlamm, M. F. Smith. J. Parenteral Enteral Nutr. 1977, 1, 11. [36] C. Weber, J. Beetens, F. De Clerck, F. Tegtmeier. J. Chromatogr. Biomed. Appl. 1992, 577, 1. [37] S. O Sullivan, B. Dahlén, S. E. Dahlén, M. Kumlin. J. Allergy Clin. Immunol. 1996, 98, 421. [38] S. O Sullivan, M. J. Mueller, S. E. Dahlén, M. Kumlina. Prostaglandins Other Lipid Mediat. 1999, 57, 149. [39] M. Hamberg. Biochem. Biophys. Res. Commun. 1972, 49, 7. [40] M. Hamberg. Anal. Biochem. 1973, 55, Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

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