Development and application of the method of analysis of selected sterols and steroid hormones in natural and wastewaters by liquid chromatography with tandem mass spectrometry

[Z. Jauković]

109 listova

Hemijske nauke - Analitička hemija i Hemija životne sredine / Chemical sciences - Analytical Chemistry and Environmental Chemistry

Prisustvo steroidnih jedinjenja u prirodnim i otpadnim vodama i njihov štetan uticaj na vodeni ekosistem su prepoznati u naučnoj zajednici nekoliko decenija. Istraživanje ekotoksičnosti ovih emergentnih zagađujućih materija pokazalo je da njihovi tragovi u vodenoj sredini mogu izazvati različite štetne efekte po vodene organizme. Steroli i steroidni hormoni su opsežno istraženi u različitim matricama životne sredine, te je dokazano da neki od njih čine prirodne konstituente ispitivanih matrica, ali u životnu sredinu u velikim količinama dospevaju iz netretiranih otpadnih voda iz domaćinstava, industrije i poljoprivrede. Metode procene zagađenja životne sredine komunalnim otpadnim vodama pri rutinskom monitoringu podrazumevaju upotrebu bakterijskih indikatora, međutim, ovi tradicionalni fekalni markeri mogu biti nepouzdani i mogu potceniti zdravstveni rizik, čak i kada ispunjavaju regulatorne standarde. Hemijski markeri, poput sterola, široko se koriste za praćenje fekalne kontaminacije životne sredine, podržavajući i dopunjujući konvencionalne indikatore zagađenja. Pored koncentracije koprostanola, upotrebom odnosa sterola moguće je odrediti nivo fekalne kontaminacije i razlikovati zagađenje poreklom od čoveka od biogenog unosa poreklom od viših biljaka ili životinja. U ovom radu opisan je razvoj i optimizacija analitičke metode za istovremenu ekstrakciju, određivanje i pouzdanu potvrdu dvadeset steroida različitih klasa (humani/životinjski i biljni steroli, prirodni i sintetski steroidni hormoni) u rečnim i otpadnim vodama, kao i u suspendovanom materijalu vodenih uzoraka. Uzorci vode su pripremljeni ekstrakcijom na čvrstoj fazi, a suspendovani materijal ultrazvučnoj ekstrakcijom. Dobijeni ekstrakti su analizirani metodom tečne hromatografije sa tandem masenom spektrometrijom, uz korišćenje linearnog jonskog trapa kao masenog analizatora. Razvijena je instrumentalna metoda za potpuno hromatografsko razdvajanje svih analita, a posebno onih sa identičnim reakcijama fragmentacije, zasnovana na upotrebi C8 kolone i acetonitrila kao organskog rastvarača u mobilnoj fazi. Optimizovana metoda je obezbedila visoke prinose analita sa relativnim standardnim devijacijama generalno manjim od 20%. Postignute su niske granice detekcije i kvantifikacije. Metoda je pokazala dobru linearnost u opsegu ispitivanih koncentracija. Rasprostranjenost steroidnih jedinjenja u životnoj sredini i nivo fekalnog zagađenja procenjeni su u oblasti ušća reka Dunava i Save u Beogradu, u kome se sirova komunalna otpadna voda ispušta u vodotokove bez tretmana. Posebna pažnja je posvećena raspodeli steroida između rastvorne i suspendovane faze uzoraka rečnih i otpadnih voda, s obzirom na afinitet sterola da se vezuju za suspendovane čestice. Takođe je procenjena efikasnost uklanjanja steroidnih jedinjenja u dva postrojenja za prečišćavanje otpadnih voda u Srbiji. Humani/životinjski steroli koprostanol i holesterol, kao i fitosterol β-sitosterol bili su dominantna jedinjenja u svim uzorcima vode. Zastupljenost sterola i raspodela između faza u rečnoj vodi bili su drugačiji od onih u sirovoj otpadnoj vodi, što ukazuje na izraženiji biogeni unos poreklom od viših biljaka, kao i veći uticaj otpadnih voda na sastav suspendovane faze. Utvrđena je izražena kontaminacija istraživanog područja, pri čemu je Dunav više zagađen ispuštanjem netretiranih otpadnih voda od Save zbog različitih hidrodinamičkih uslova koji dovode do značajno viših nivoa sterola u suspendovanim česticama. Takođe je pokazano da se svi detektovani humani/životinjski steroli i fitosteroli dominantno vezuju za čestice suspendovanog materijala uzoraka rečne i otpadne vode, u poređenju sa rastvornom fazom. Naglašena je potreba da se u praćenju zagađenja životne sredine steroidnim jedinjenjima uzmu u obzir i analiziraju i rastvorna i suspendovana faza kako bi se dobila realna slika o kontaminaciji steroidima i njihovoj sudbini u vodenoj sredini. Konačno, utvrđena je visoka stopa uklanjanja sterola u postojenjima za tretman otpadnih voda (> 98% za koprostanol i holesterol), kao i da je od izračunatih odnosa sterola samo koprostanol/(koprostanol + holestanol) dovoljno osetljiv da na njega utiče poboljšanje kvaliteta prečišćene otpadne vode.

The presence of steroid compounds in natural waters and wastewater and their harmful effects on the aquatic ecosystem have been recognized by the scientific community for several decades. Ecotoxicity research of these emerging contaminants has shown that trace amounts in the aquatic environment can cause various adverse effects on aquatic organisms. Sterols and steroid hormones have been extensively investigated in various environmental matrices, and it has been shown that some of them are natural constituents of the investigated matrices, but they enter the environment in large quantities via untreated wastewater from households, industry and agriculture. Methods for assessing environmental pollution with municipal wastewater in routine monitoring involve the use of bacterial indicators, however, these traditional fecal markers can be unreliable and may underestimate the health risk, even when they meet regulatory standards. Chemical markers, such as sterols, are widely used to monitor fecal contamination of the environment, supporting and complementing conventional pollution indicators. In addition to the concentration of coprostanol, by using the sterol ratios it is possible to determine the level of fecal contamination and to distinguish between anthropogenic pollution and biogenic input from higher plants or animals. This paper describes the development and optimization of an analytical method for the simultaneous extraction, determination and reliable confirmation of twenty steroids of different classes (human/animal and plant sterols, natural and synthetic steroid hormones) in river water and wastewater, as well as in suspended material of water samples. Water samples were prepared by solid- phase extraction, and suspended material by ultrasonic extraction. The obtained extracts were analyzed by liquid chromatography with tandem mass spectrometry, using a linear ion trap as a mass analyzer. An instrumental method was developed for the complete chromatographic separation of all analytes, especially those with identical fragmentation reactions, based on the use of a C8 column and acetonitrile as an organic solvent in the mobile phase. The optimized method provided high analyte recoveries with relative standard deviations generally lower than 20%. Low limits of detection and quantification were achieved. The method showed good linearity in the range of tested concentrations. The distribution of steroid compounds in the environment and the level of fecal pollution were assessed in the confluence area of the Danube and the Sava rivers in Belgrade, where raw municipal wastewater is discharged into waterways without treatment. Special attention was paid to the distribution of steroids between the dissolved and suspended phases of river and wastewater samples, considering the affinity of sterols to bind to suspended particles. The efficiency of steroid removal in two wastewater treatment plants in Serbia was also evaluated. The human/animal sterols coprostanol and cholesterol as well as the phytosterol β-sitosterol were the dominant compounds in all water samples. The pattern of sterols and the distribution between phases in river water were different from those in raw wastewater, which indicates a more pronounced biogenic input originating from higher plants, as well as a greater influence of wastewater on the composition of the suspended phase. Extensive contamination of the investigated area was determined, whereby the Danube is more polluted by the discharge of untreated wastewater than the Sava due to different hydrodynamic conditions that lead to significantly higher levels of sterols in suspended particles. It was also shown that all detected human/animal sterols and phytosterols bind dominantly to suspended particles of river and wastewater samples, compared to the dissolved phase. The need to consider and analyze both the dissolved and suspended phases in the monitoring of environmental pollution with steroid compounds was emphasized in order to obtain a realistic picture of steroid contamination and their fate in the aquatic environment. Finally, a high removal rate of sterols in wastewater treatment was determined (> 98% for coprostanol and cholesterol), as well as that of the calculated sterol ratios, only coprostanol/(coprostanol + cholestanol) is sensitive enough to be affected by the improvement of the quality of treated wastewater.

steroidna jedinjenja, steroli, steroidni hormoni, rečna voda, otpadna voda, postrojenja za prečišćavanje otpadnih voda, suspendovana materija, tečna hromatografija-tandem masena spektrometrija, odnosi sterola, fekalna kontaminacija.

steroid compounds, sterols, steroid hormones, river water, wastewater, wastewater treatment plants, suspended material, liquid chromatography-tandem mass spectrometry, sterol ratios, fecal contamination.

543.645.2:628.312.2(043.3)

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