鄰苯二甲酸二異壬酯(DINP)是一種廣泛使用的工業增塑劑。人們因使用DINP作為增塑劑的塑料產品而接觸到DINP。 DINP的暴露會對人體健康造成不良影響。因為DINP具有很高的人體暴露和毒性潛力，找到DINP的暴露指標來了解個人暴露狀況至關重要。通過LTQ-Orbitrap高分辨率質譜數據集，將Signal Mining Algorithm with Isotope Tracing (SMAIT), mass defect filtering (MDF)和XCMS Online軟件用於尿液中DINP暴露指標的尋找。使用SMAIT，MDF和XCMS方法分別獲得了16個，83個和139個可疑的DINP代謝物信號。利用LC-MS/MS鑑定結構後，三種代謝組學方法同時尋找到的14種可疑代謝物信號被確認為DINP代謝物。其中，在大鼠動物試驗中，有13種可疑的代謝物信號有劑量效應關係，確認為與暴露相關的標誌物。DINP代謝信息可以為進一步研究DINP毒性，毒代動力學，暴露評估和人類健康影響提供有價值的信息。由於人體會迅速排泄DINP代謝產物，因此在流行病學研究中使用單點尿分析法評估長期暴露可能會產生不一致的結果。頭髮分析在生物監測中非常有用，尤其是在評估某些化學品的長期暴露。在動物實驗中，成功在大鼠毛髮中測量到DINP的代謝物，且大鼠毛髮中DINP代謝物的濃度與暴露劑量呈正相關具劑量效應關係。毛髮和尿液之間的DINP代謝物MINP，MOINP和MHINP呈顯著正相關(分別為r = 0.86，r = 0.79和r = 0.74，p <0.05)。頭髮分析在評估DINP的長期暴露方面具有潛在的應用。在使用LTQ-Orbitrap 高分辨率質譜和非靶向代謝組學方法研究DINP時發現兩種新型代謝物，mono(hydroxyl isooctyl) phthalate (MHIOP, m/z 293.139) and mono(hydroxyl isodecyl) phthalate (MHIDP, m/z 321.170)。推測代謝物MHIOP(m/z 293.139)是來自代謝物mono-hydroxyisononyl phthalate (MHINP, m/z 307.155)的烷基鏈斷裂(-14.015 Da)，而MHINP是MHIDP (m/z 321.170)的烷基鏈斷裂。基於該觀察結果，提出了這些代謝產物是DINP的烷基鏈分解產物。從m/z 321.170開始推測了DINP的八種烷基鏈分解代謝產物。其中，大鼠尿液樣品中七個烷基鏈分解代謝產物(m/z 321.170、307.155、293.139、279.123、265.108、237.076和223.061)的信號呈現劑量效應關係。由於鄰苯二甲酸酯代謝過程的烷基鏈斷裂，將很難通過它們的代謝物來區分相似的鄰苯二甲酸酯，尤其是那些僅具有不同支鏈長度的鄰苯二甲酸酯，例如DINP和鄰苯二甲酸二異癸酯(DIDP)。

Di-iso-nonyl phthalate (DINP) is a widely used industrial plasticizer. People come into contact with this chemical by using plastic products made with it. Human health can be adversely affected by DINP exposure. Exposure marker discovery of DINPs is crucial, because of their high potential for human exposure and toxicity. SMAIT(Signal Mining Algorithm with Isotope Tracing), mass defect filtering (MDF), and XCMS Online software were used for DINP exposure marker discovery in urine with an LTQ-Orbitrap high-resolution mass spectrometry (HRMS) dataset. Sixteen, 83, and 139 suspected DINP metabolite signals were obtained using the SMAIT, MDF, and XCMS procedures, respectively. Fourteen suspected metabolite signals mined simultaneously by the three metabolomics approaches were confirmed as DINP metabolites by structural information provided by LC-MS/MS. Among them, 13 suspected metabolite signals were validated as exposure-related markers in a rat model. Because the body rapidly excretes DINP metabolites, the use of spot urine analysis to assess long-term exposure may produce inconsistent results in epidemiologic studies. Hair analysis has a useful place in biomonitoring, particularly in estimating long-term or historical exposure for some chemicals. DINP metabolites were measured in rat hair in animal experiments to evaluated long-term exposure to DINP. The levels of DINP metabolites in rat hair had a positive correlation with increasing administered dose. Significant positive correlations for MINP, MOINP and MHINP were found between hair and urine (r = 0.86, r = 0.79 and r = 0.74, respectively, p<0.05). The DINP metabolism information can provide valuable information for further investigations of DINP toxicity, toxicokinetics, exposure assessment, and human health effects. Hair analysis has potential applications in the assessment of long-term exposure to DINP. Three novel hydroxyl metabolites were identified as DINP exposure markers. Using an LTQ-Orbitrap HRMS and untargeted metabolomics approaches two novel metabolites of di-isononyl phthalate (DINP), mono(hydroxyl isooctyl) phthalate (MHIOP, m/z 293.139) and mono(hydroxyl isodecyl) phthalate (MHIDP, m/z 321.170) were identified. The m/z values derived from the two hydroxyl­metabolites are only slightly different (0.036 u) from those carbonyl­metabolites, mono(carbonyl isohexyl) phthalate (MCIHP, m/z 293.103) and mono(carbonyl isooctyl) phthalate, (MCIOP, m/z 307.155), reported in the literature. In addition, the m/z 293.139 was the alkyl chain breakdown (-14.015 Da) from m/z 307.155, which, in turn, was the alkyl chain breakdown from m/z 321.170. Base on this observation, these metabolites are proposed the alkyl chain breakdown products of DINP. Eight alkyl chain breakdown metabolites of DINP were speculated starting at m/z 321.170. Among them, the signal intensity of seven alkyl chain breakdown metabolites (m/z 321.170, 307.155, 293.139, 279.123, 265.108, 237.076 and 223.061) in rat urine samples had a positive correlation with increasing administered dose of DINP in a rat model. Due to the alkyl chain breakdown of phthalate metabolic process, it will be difficult to distinguish between similar phthalates by their metabolites, especially those with only different branching length, such as DINP and di-isodecyl phthalate (DIDP).

Abb M, Heinrich T, Sorkau E, Lorenz W. 2009. Phthalates in house dust. Environ Int 35:965-970.
Agency GFE. 2011. Phthalates – new and updated reference values for monoesters and oxidised metabolites in urine of adults and children. Gesundheitsschutz 54:770-785.
AGPU. 2006. Plastizicers market data.
Allegrand J, Touboul D, Schmitz-Afonso I, Guerineau V, Giuliani A, Le Ven J, et al. 2010. Structural study of acetogenins by tandem mass spectrometry under high and low collision energy. Rapid Commun Mass Spectrom 24:3602-3608.
Anari MR, Sanchez RI, Bakhtiar R, Franklin RB, Baillie TA. 2004. Integration of knowledge-based metabolic predictions with liquid chromatography data-dependent tandem mass spectrometry for drug metabolism studies: Application to studies on the biotransformation of indinavir. Anal Chem 76:823-832.
Anderson WA, Castle L, Hird S, Jeffery J, Scotter MJ. 2011. A twenty-volunteer study using deuterium labelling to determine the kinetics and fractional excretion of primary and secondary urinary metabolites of di-2-ethylhexylphthalate and di-iso-nonylphthalate. Food Chem Toxicol 49:2022-2029.
Bellew M, Coram M, Fitzgibbon M, Igra M, Randolph T, Wang P, et al. 2006. A suite of algorithms for the comprehensive analysis of complex protein mixtures using high-resolution lc-ms. Bioinformatics 22:1902-1909.
Calafat AM, Ye X, Silva MJ, Kuklenyik Z, Needham LL. 2006. Human exposure assessment to environmental chemicals using biomonitoring. Int J Androl 29:166-171; discussion 181-165.
Calafat AM, Wong LY, Silva MJ, Samandar E, Preau JL, Jr., Jia LT, et al. 2011. Selecting adequate exposure biomarkers of diisononyl and diisodecyl phthalates: Data from the 2005-2006 national health and nutrition examination survey. Environ Health Perspect 119:50-55.
Chang YJ, Lin KL, Chang YZ. 2013. Determination of di-(2-ethylhexyl)phthalate (dehp) metabolites in human hair using liquid chromatography-tandem mass spectrometry. Clin Chim Acta 420:155-159.
Chen C, Gonzalez FJ, Idle JR. 2007. Lc-ms-based metabolomics in drug metabolism. Drug Metab Rev 39:581-597.
Chen J, Wang W, Lv S, Yin P, Zhao X, Lu X, et al. 2009. Metabonomics study of liver cancer based on ultra performance liquid chromatography coupled to mass spectrometry with hilic and rplc separations. Anal Chim Acta 650:3-9.
Chiang KP, Niessen S, Saghatelian A, Cravatt BF. 2006. An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling. Chem Biol 13:1041-1050.
Colacino J, Soliman A, Calafat A, Nahar M, Zijl. V, Hablas A, et al. 2011. Exposure to phthalates among premenstrual girls from rural and urban gharbiah, egypt: A pilot exposure assessment study. Environ Health 10:40.
Crews B, Wikoff WR, Patti GJ, Woo HK, Kalisiak E, Heideker J, et al. 2009. Variability analysis of human plasma and cerebral spinal fluid reveals statistical significance of changes in mass spectrometry-based metabolomics data. Anal Chem 81:8538-8544.
Crinnion WJ. 2010. Toxic effects of the easily avoidable phthalates and parabens. Altern Med Rev 15:190-196.
Dunn WB, Brown M, Worton SA, Davies K, Jones RL, Kell DB, et al. 2011. The metabolome of human placental tissue: Investigation of first trimester tissue and changes related to preeclampsia in late pregnancy. Metabolomics 8:579-597.
Ejsing CS, Moehring T, Bahr U, Duchoslav E, Karas M, Simons K, et al. 2006. Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: A study by quadrupole tof and linear ion trap-orbitrap mass spectrometry. J Mass Spectrom 41:372-389.
European Commission JRC. 2003. European union risk assessment report. 1,2-benzenedi- carboxylic acid, di-c8-10-branched alkyl esters, c9-rich and di-isononyl phthalate (dinp), eur 20784 en. Office for Official Publications of the European Communities.
Favretto D, Vogliardi S, Stocchero G, Nalesso A, Tucci M, Ferrara SD. 2011. High performance liquid chromatography-high resolution mass spectrometry and micropulverized extraction for the quantification of amphetamines, cocaine, opioids, benzodiazepines, antidepressants and hallucinogens in 2.5 mg hair samples. J Chromatogr A 1218:6583-6595.
Foster PM. 2006. Disruption of reproductive development in male rat offspring following in utero exposure to phthalate esters. Int J Androl 29:140-147; discussion 181-145.
Frederiksen H, Jørgensen N, Andersson AM. 2010. Correlations between phthalate metabolites in urine, serum, and seminal plasma from young danish men determined by isotope dilution liquid chromatography tandem mass spectrometry. J Anal Toxicol 34:400-410.
Frederiksen H, Kranich SK, Jorgensen N, Taboureau O, Petersen JH, Andersson AM. 2013. Temporal variability in urinary phthalate metabolite excretion based on spot, morning, and 24-h urine samples: Considerations for epidemiological studies. Environ Sci Technol 47:958-967.
Gray LE, Jr., Ostby J, Furr J, Price M, Veeramachaneni DN, Parks L. 2000. Perinatal exposure to the phthalates dehp, bbp, and dinp, but not dep, dmp, or dotp, alters sexual differentiation of the male rat. Toxicol Sci 58:350-365.
Hill JR. 2003. In vitro drug metabolism using liver microsomes:John Wiley & Sons, Inc.
Hsu JF, Peng LW, Li YJ, Lin LC, Liao PC. 2011. Identification of di-isononyl phthalate metabolites for exposure marker discovery using in vitro/in vivo metabolism and signal mining strategy with lc-ms data. Anal Chem 83:8725-8731.
Huang X, Chen YJ, Cho K, Nikolskiy I, Crawford PA, Patti GJ. 2014. X13cms: Global tracking of isotopic labels in untargeted metabolomics. Anal Chem 86:1632-1639.
Juan YY, Wei YC, Hung SC. 2011. Food scare windens: Tainted additives used for two decades: Manufacturer. Taipei Times (Taiwan) May 29.
Kato K, Silva MJ, Wolf C, Gray LE, Needham LL, Calafat AM. 2007. Urinary metabolites of diisodecyl phthalate in rats. Toxicology 236:114-122.
Kavlock R, Boekelheide K, Chapin R, Cunningham M, Faustman E, Foster P, et al. 2002. Ntp center for the evaluation of risks to human reproduction: Phthalates expert panel report on the reproductive and developmental toxicity of di-isononyl phthalate. Reprod Toxicol 16:679-708.
Kind T, Tolstikov V, Fiehn O, Weiss RH. 2007. A comprehensive urinary metabolomic approach for identifying kidney cancerr. Anal Biochem 363:185-195.
Koch HM, Angerer J. 2007. Di-iso-nonylphthalate (dinp) metabolites in human urine after a single oral dose of deuterium-labelled dinp. Int J Hyg Environ Health 210:9-19.
Koch HM, Lorber M, Christensen KL, Palmke C, Koslitz S, Bruning T. 2013. Identifying sources of phthalate exposure with human biomonitoring: Results of a 48h fasting study with urine collection and personal activity patterns. Int J Hyg Environ Health 216:672-681.
Li J, Guo F, Wang Y, Liu J, Cai Z, Zhang J, et al. 2012. Development of extraction methods for the analysis of perfluorinated compounds in human hair and nail by high performance liquid chromatography tandem mass spectrometry. J Chromatogr A 1219:54-60.
Liang Y, Wang G, Xie L, Sheng L. 2011. Recent development in liquid chromatography/mass spectrometry and emerging technologies for metabolite identification. Curr Drug Metab 12:329-344.
Lin LC, Wu HY, Tseng VS, Chen LC, Chang YC, Liao PC. 2010. A statistical procedure to selectively detect metabolite signals in lc-ms data based on using variable isotope ratios. J Am Soc Mass Spectrom 21:232-241.
Lin LC, Wang SL, Chang YC, Huang PC, Cheng JT, Su PH, et al. 2011. Associations between maternal phthalate exposure and cord sex hormones in human infants. Chemosphere 83:1192-1199.
Ma S, Chowdhury SK, Alton KB. 2006. Application of mass spectrometry for metabolite identification. Curr Drug Metab 7:503-523.
Ma S, Chowdhury SK. 2011. Analytical strategies for assessment of human metabolites in preclinical safety testing. Anal Chem 83:5028-5036.
Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, et al. 2006. Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age. Environ Health Perspect 114:270-276.
Makarov A. 2000. Electrostatic axially harmonic orbital trapping: A high-performance technique of mass analysis. Anal Chem 72:1156-1162.
Miyaguchi H, Kakuta M, Iwata YT, Matsuda H, Tazawa H, Kimura H, et al. 2007. Development of a micropulverized extraction method for rapid toxicological analysis of methamphetamine in hair. J Chromatogr A 1163:43-48.
Miyaguchi H, Iwata YT, Kanamori T, Tsujikawa K, Kuwayama K, Inoue H. 2009. Rapid identification and quantification of methamphetamine and amphetamine in hair by gas chromatography/mass spectrometry coupled with micropulverized extraction, aqueous acetylation and microextraction by packed sorbent. J Chromatogr A 1216:4063-4070.
Mylchreest E, Cattley RC, Foster PM. 1998. Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl) phthalate: An antiandrogenic mechanism? Toxicol Sci 43:47-60.
Nomura DK, Long JZ, Niessen S, Hoover HS, Ng SW, Cravatt BF. 2010. Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis. Cell 140:49-61.
Pan G, Hanaoka T, Yoshimura M, Zhang S, Wang P, Tsukino H, et al. 2006. Decreased serum free testosterone in workers exposed to high levels of di-n-butyl phthalate (dbp) and di-2-ethylhexyl phthalate (dehp): A cross-sectional study in china. Environ Health Perspect 114:1643-1648.
Parks LG, Ostby JS, Lambright CR, Abbott BD, Klinefelter GR, Barlow NJ, et al. 2000. The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. Toxicol Sci 58:339-349.
Petrovic M, Farre M, de Alda ML, Perez S, Postigo C, Kock M, et al. 2010. Recent trends in the liquid chromatography-mass spectrometry analysis of organic contaminants in environmental samples. J Chromatogr A 1217:4004-4017.
Preau JL, Jr., Wong LY, Silva MJ, Needham LL, Calafat AM. 2010. Variability over 1 week in the urinary concentrations of metabolites of diethyl phthalate and di(2-ethylhexyl) phthalate among eight adults: An observational study. Environ Health Perspect 118:1748-1754.
Qiu Y, Cai G, Su M, Chen T, Liu Y, Xu Y, et al. 2010. Urinary metabonomic study on colorectal cancer. J Proteome Res 9:1627-1634.
Ramanathan R. 2011. Mass spectrometry in drug metabolism and pharmacokinetics:Wiley.
Roux A, Lison D, Junot C, Heilier JF. 2011. Applications of liquid chromatography coupled to mass spectrometry-based metabolomics in clinical chemistry and toxicology: A review. Clin Biochem 44:119-135.
Saravanabhavan G, Murray J. 2012. Human biological monitoring of diisononyl phthalate and diisodecyl phthalate: A review. J Environ Public Health 2012:810501.
Saykali M. Plasticisers update and reputation management within the vinyls value chain. In: Proceedings of the European Council for Plasticizers and Intermediates (ECPI), 17th April 2012 2012. Johannesburg.
Schutze A, Otter R, Modick H, Langsch A, Bruning T, Koch HM. 2017. Additional oxidized and alkyl chain breakdown metabolites of the plasticizer dinch in urine after oral dosage to human volunteers. Arch Toxicol 91:179-188.
Silva MJ, Kato K, Wolf C, Samandar E, Silva SS, Gray EL, et al. 2006a. Urinary biomarkers of di-isononyl phthalate in rats. Toxicology 223:101-112.
Silva MJ, Reidy JA, Preau JL, Jr., Needham LL, Calafat AM. 2006b. Oxidative metabolites of diisononyl phthalate as biomarkers for human exposure assessment. Environ Health Perspect 114:1158-1161.
Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G. 2006. Xcms: Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78:779-787.
Takayama N, Iio R, Tanaka S, Chinaka S, Hayakawa K. 2003. Analysis of methamphetamine and its metabolites in hair. Biomed Chromatogr 17:74-82.
Tautenhahn R, Patti GJ, Rinehart D, Siuzdak G. 2012. Xcms online: A web-based platform to process untargeted metabolomic data. Anal Chem 84:5035-5039.
Tolonen A, Turpeinen M, Pelkonen O. 2009. Liquid chromatography–mass spectrometry in in-vitro drug metabolite screening. Drug discovery today 14:120-133.
TURI. 2006. Five chemicals alternatives assessment study.
Tyl RW, Myers CB, Marr MC, Fail PA, Seely JC, Brine DR, et al. 2004. Reproductive toxicity evaluation of dietary butyl benzyl phthalate (bbp) in rats. Reprod Toxicol 18:241-264.
Waterman SJ, Ambroso JL, Keller LH, Trimmer GW, Nikiforov AI, Harris SB. 1999. Developmental toxicity of di-isodecyl and di-isononyl phthalates in rats. Reprod Toxicol 13:131-136.
Wilson VS, Lambright C, Furr J, Ostby J, Wood C, Held G, et al. 2004. Phthalate ester-induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis. Toxicol Lett 146:207-215.
Wittassek M, Koch HM, Angerer J, Bruning T. 2011. Assessing exposure to phthalates - the human biomonitoring approach. Mol Nutr Food Res 55:7-31.
Wormuth M, Scheringer M, Vollenweider M, Hungerbuhler K. 2006. What are the sources of exposure to eight frequently used phthalic acid esters in europeans? Risk Anal 26:803-824.
Xu YJ, Wang C, Ho WE, Ong CN. 2014. Recent developments and applications of metabolomics in microbiological investigations. Trends Anal Chem.
Yan ZY, Caldwell GW. 2004. Stable-isotope trapping and high-throughput screenings of reactive metabolites using the isotope ms signature. Anal Chem 76:6835-6847.
Yang K, Zhao Z, Gross RW, Han X. 2007. Shotgun lipidomics identifies a paired rule for the presence of isomeric ether phospholipid molecular species. PLoS One 2:e1368.
Zhang H, Zhang D, Ray K. 2003. A software filter to remove interference ions from drug metabolites in accurate mass liquid chromatography/mass spectrometric analyses. J Mass Spectrom 38:1110-1112.
Zhang H, Yang Y. 2008. An algorithm for thorough background subtraction from high-resolution lc/ms data: Application for detection of glutathione-trapped reactive metabolites. J Mass Spectrom 43:1181-1190.
Zhang H, Zhu M, Ray KL, Ma L, Zhang D. 2008. Mass defect profiles of biological matrices and the general applicability of mass defect filtering for metabolite detection. Rapid Commun Mass Spectrom 22:2082-2088.
Zhang H, Zhang D, Ray K, Zhu M. 2009. Mass defect filter technique and its applications to drug metabolite identification by high-resolution mass spectrometry. J Mass Spectrom 44:999-1016.
Zhu M, Ma L, Zhang D, Ray K, Zhao W, Humphreys WG, et al. 2006. Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data. Drug Metab Dispos 34:1722-1733.
Zhu M, Zhang H, Humphreys WG. 2011. Drug metabolite profiling and identification by high-resolution mass spectrometry. J Biol Chem 286:25419-25425.
Zhu P, Ding W, Tong W, Ghosal A, Alton K, Chowdhury S. 2009. A retention-time-shift-tolerant background subtraction and noise reduction algorithm (bgs-nora) for extraction of drug metabolites in liquid chromatography/mass spectrometry data from biological matrices. Rapid Commun Mass Spectrom 23:1563-1572.