Determination of non polar metabolites in human plasma using innovative sample preparation strategies coupled with gas chromatography and mass spectrometry Maria Margarida Gonçalves 1, Sandra Sagredo 1, Maria Serrano Bernardo 1, Benilde Mendes 1, Maria Cristina Marques 2, Daniel Etlin 3, Ana Bragança 4 and Valentina Vassilenko 4 1 Environmental Biotechnology Unit, Faculty of Sciences and Technology, New University of Lisbon, Portugal; 2 Faculty of Pharmacy, University of Lisbon, Portugal; 3 Unicam Sistemas Analíticos, SA, Portugal; 4 Centre of Physics and Technological Research, New University of Lisbon, Portugal
Overview: Introduction: The main objective of this work is to develop simple, fast and selective extraction methods to isolate non polar metabolites or contaminants from human plasma. Solid phase extraction using a novel adsorbant (MonoTrap TM ) was tested for direct extration from diluted plasma samples or from the corresponding headspace. Both direct extraction and headspace extraction with MonoTrapTM revealed the presence of terphenyls and partially hydrogenated terphenyls in 39 human plasma samples. Dispersive liquid-liquid microextraction (DLLME) of plasma samples was tested using CCl 4 or using heptane. Conventional DLLME procedure was adapted to allow the use of non halogenated solvents. A new method of simultaneous derivatisation, extraction and concentration of plasma fatty acids is proposed. This method couples direct transesterification of plasma samples with methanolic KOH and homogeneous liquid-liquid microextraction (HLLME) with halogenated or non halogenated solvents. All steps take place in a single vial with an extraction time of min or less. References Metabolomics addresses the screening of compounds with medium to low molecular weight, with biological relevance as disease or exposure biomarkers [1]. Sample preparation is a key step of the process, because biological fluids are complex samples often available at limited amounts (1-2 ml in the case of blood plasma). The metabolic profile of plasma samples includes free aminoacids, organic acids, amines, sugar, steroids, nucleic acid bases and other substances [2]. The analysis of these metabolites by GC-MS requires the use of appropriated derivatization reactions. Non polar metabolites like aliphatic hydrocarbons have been determined in biological samples (breath samples) and related to lipid peroxydation processes occurring in cells of cancer patients or of smokers [3,4]. [1] Harin Kanani, Panagiotis K. Chrysanthopoulos, Maria I. Klapa Standardizing GC MS metabolomics Journal of Chromatography B, 871 (8) 191 1 [2] Kishore K. Pasikanti, P.C. Ho, E.C.Y. Chan Gas chromatography/mass spectrometry in metabolic profiling of biological fluids Journal of Chromatography B, 871 (8) 2 211 [3] Leiliane Coelho A. Amorim, Zenilda de L. Cardeal Breath air analysis and its use as a biomarker in biological monitoring of occupational and environmental exposure to chemical agents Journal of Chromatography B, 83 (7) 1 9 [4] Michael Phillips, Renee N. Cataneo, Andrew R.C. Cummin, Anthony J., Gagliardi, Kevin Gleeson, Joel Greenberg, Roger A. Maxfield, William N. Rom Detection of Lung Cancer With Volatile Markers in the Breath Chest 123 (3) 21-2123
Methods : Blood samples from volunteers ( smokers and non smokers) were collected and the corresponding plasma was isolated and kept at -ºC until analysis. Plasma deproteinization was performed adding methanol and separating the precipitates by centrifugation. Solid phase extraction with MonoTrap TM DSC18 disks (GL Sciences, Japan) Plasma samples (L) were diluted with distilled water and extracted with MonoTrap TM DSC18 disks using: a) Direct extraction (DE) MonoTrapTM disks are placed in direct contact with the plasma sample, at 6 ºC, for 1h, under stirring. b) Headspace extraction (HE) MonoTrap TM disks suspended in the headspace over the plasma sample, at 6 ºC, for 1h, under stirring. The SPE disks were extracted with dichloromethane, in an ultrasonic bath. The extract was dried, concentrated to µl and analysed by GC-MS. Homogeneous liquid-liquid microextraction coupled with direct transesterification. (HLLME-DT): Plasma samples (L) were diluted with methanolic KOH; a solution of organic solvent CCl 4 or heptane ( - µl) in acetone was added to the reaction mixture and an homogeneous solution was obtained. Phase separation was induced by adding 2 ml of water. After centrifugation the organic phase was recovered and injected in a GC-MS-MS (Focus GC, Polaris Q, Thermounicam).
Relative Abundance Relative Abundance Relative Abundance Relative Abundance Results : Detection of terphenyls and hydrogenated terphenyls in human plasma SPE with MonoTrap DSC18 disks (DE and HE) + GC-MS RT: 2.9-62.2 9 8 7 6 6 DE (Direct Extraction) 6.3 9.31 6.48 6.1E MonotrapC 18-1 Extracted Chromatograms with base peak = 2 m/z DE (Direct Extraction) HE (Headspace Extraction) Terphenyls 3.4 6.39 6.8 3.84 6.89 7.72 7.88 7. 4.8. 8.33 6.3 6.18 6.74 62.8 Diphenylcyclohexanes 3 4 6 7 8 9 6 61 62 RT: 2.9-61.6 9 8 7 6 6 HE (Headspace Extraction) RT: 3.4 AA: 99376 RT: 6.39 AA: 73826 RT: 6.3 AA: 19616 RT: 6.89 MA: 8434 RT: 7.72 AA: 438797 RT: 3.84 AA: 46 RT: 7.88 AA: 17 RT:. RT: 8.33 7. AA: 374 AA: 41344 3. 4.8 6.3 RT: 9.31 AA: 692 RT: 6.48 AA: 426 Partially hydrogenated terphenyls are produced in large amounts for industrial use they are included in the HPV (high production volume) chemicals list by US-EPA. They are mainly used as heat transfer fluids and polymer plasticizers. These compounds have been found in recycled paper and cardboard, as well as in food products packed with these recycled materials. Our results show the presence of these contaminants in human plasma samples of 39 volunteers, both smokers and non smokers. 6.18 6.8 3 4 6 7 8 9 6 61 6.74 6.1E MonotrapC 18-1 RT: 3.34-7.6 9 8 7 6 6 RT: 1.79 -.33 9 8 7 6 6 Extracted Chromatograms with base peak = 2 m/z RT: 4.2 AA: 938 RT: 4.26 AA: 794 RT: 4.4 AA: 161266 3. 4. 4... 6. 6. 7. 7. Extracted Chromatograms with base peak = 242 m/z RT: 2.89 AA: 8491 RT: 3.31 AA: 92 RT:.3 AA: 3933 RT: 4.28 AA: 384 RT: 6.7 AA: 3712 2. 2. 3. 3. 4. 4.. 1.39E6 2.- 236. F: MS ICIS MQ8366NF1 8.E4 241.- 242. F: MS ICIS MQ8366NF1 RT: 1.73-61.14 9 8 7 6 6 RT:.17-8.72 9 8 7 6 6 2 3 4 6 7 8 9 6 61.29 6.39 6.89 7.72 7.87 9.31 3.8 1.9 2.11 3. 3.9.11 6.33 6.41 7.4 8.33 8.43 9. 6.8 7.1 6.47.69.88 6.26 7.9 7.44 7.73 8. 8.33 8.3. 6. 6. 7. 7. 8. 8. 1.6E 2.- 236. F: MS MonotrapC18-1 2.84E4 241.- 242. F: MS MonotrapC18-1 Cyclohexylbiphenyls and diphenylcyclohexanes Dicyclohexylbenzenes
Peak area Results : Extraction of terphenyls and hydrogenated terphenyls using HLLME TIC profile from HLLME of a non smoker plasma RT: 1.1-62.99 9 8 7 6 3.76 9.7 6.2E6 dllme CCL4_ MQ836NF _18_6_2 Base peak 2 Peak nº Name Base peak area ( 2) MT-DE MT-HE HLLME 1 o-terphenyl 242932 24166 77 2 m-terphenyl 333761-1186 6 2.86 9.23 6.1 6.38 7.2 3.4 6. 7.16 2.1 7.69 3.99.7.7 61.1 61.89 2 3 4 6 7 8 9 6 61 62 Peak nº Name Base peak area ( 236) DE HE HLLME 3 1,2-diphenylcyclohexane 164368 17267 79147 4 1,3-diphenylcyclohexane 938 41 33 1,4-diphenylcyclohexane 794 981 61418 Base peak 236 Base peak 242 6 3-cyclohexylbiphenyl 3933 77 2872927 7 4-cyclohexylbiphenyl 3712 237143 181988 6 Monotrap - Direct Extraction Monotrap - Headspace HLLME Peak nº Name Base peak area ( 242) DE HE HLLME 8 1,3-dicyclohexylbenzene 8491 117248 23 9 phenyldicyclohexane 92 84 674 1,4-dicyclohexylbenzene 384 26736 62484 1 2 3 4 6 7 8 9 Peak nº
Relative Abundance Relative Concentration (% peak area) Results : direct transesterification + HLLME CCl 4 or heptane Tic profile, FFA methyl esters from smoker plasma RT:.8 -.1 9 8 7 6 6 4.4 9.36 9. 64.6 8..42E7 dllme CCL4_ MQCF_ 23_6_1 Smoker, CCl4 Smoker, Heptane Non smoker, CCl4 Non smoker, Heptane 6.26 64. 69.29 38. 41.96 3.3 6.9 47.2 7.72 69.66 79. 88.6 71.43 79.96 49.66 6.8 73.86 76.37 6 6 7 8 C12: C14: C16: C16:1 C18: C18:1 C18:2 C18:3 C:3 C:4 C: C22: C22:6 Fatty Acid Fatty acid profile can be obtained with direct transesterification and HLLME either with heptane or with CCl 4. Comparison of 13 reference fatty acid showed no significant differences between smokers and non smokers. Although heptane extracts are less concentrated than CCl4 extracts we could identify 26 fatty acid methyl esters. 26 Fatty Acid Methyl Esters Identified (C12:) Lauric acid C14:) Myristic acid (C14:) 12-Methyltetradecanoic acid (C16:1) Palmitoleic acid (C16:) Palmitic acid (C16:) -methylhexadecanoic acid (C16:) 14-methylhexadecanoic acid (C17:), Heptadecanoic acid (C18:3), Linolenic acid (C18:2), Linoleic acid (C18:1), Oleic acid (C18:1) Elaidic acid (C18:) Stearic acid (C:) Eicosapentaenoic acid C:4) Eicosatetraenoic acid (3 isomers) (C:3) Eicosatrienoic acid (2 isomers) ( (C:2)Eicosadienoic acid (C:1) Eicosenoic acid (C:) Eicosanoic acid (C22:6) Docosahexaenoic (2 isomers) (C22:) Docosapentaenoic (C22:4) Docosatetraenoic
Conclusions : Terphenyls and partially hydrogenated terphenyls were extracted from human plasma. Extracts of the materials in contact with the plasma samples and blanks of the analytical process revealed no contamination with these compounds. SPE of deproteinized and diluted plasma with a new monolithic adsorbant (MonoTrapTM), both in direct contact or from the headspace, allows the selective extraction of these plasma contaminants without significant co-extraction of plasma metabolites. The method with direct contact provides better extraction yield than the headspace extraction. To the best of our knowledge this is the first report on the use of non halogenated solvents for any kind of liquidliquid microextraction. Although showing smaller enrichment factors then halogenated solvents, the non halogenated solvents are generally less toxic and therefore can be a environmentally safer alternative. When performing DLLME with plasma samples diluted in water the addition of the organic phase (heptane or CCl 4 ), causes the formation of a gel like structure that prevents a good phase separation. A new method for the direct derivatization (transesterification) of plasma lipids and simultaneous extraction and concentration of the corresponding methyl esters was developed. The method uses organic solvents in the microliter range, is performed in a single vial and has a total derivatization + extraction time of minutes or less. The homogeneous liquid-liquid microextraction coupled with direct transesterification allows the fast and sensitive determination of up to fatty acid methyl esters derivatives of plasma lipids. Terphenyls and partially hydrogenated terphenyls were found, at variable concentrations in 39 out of human plasma samples analysed. No significant differences were found between the smokers and the non smokers group in what concerns fatty acid profile or aromatic contaminants level. Aknowledgements: The authors thank Dr. Etsudo Ikeda (GL Sciences, Japan) for the kind offer of MonoTrap TM samples. Thanks are also due to Dr. Carlos Marques (Nova Era Clinical Analysis Laboratory, Lisbon) for supplying the plasma samples.