Novel analytical methods for the determination of antiretroviral drugs and their metabolites in environmental water samples.

Thesis (PhD)--Stellenbosch University, 2021.ENGLISH SUMMARY: The occurrence and persistence of pharmaceuticals in aquatic environments is a topical public health issue because of their perpetual discharge into the environment as intact- or biotransformed products. The environmental effects for most these compounds are known, but much concern relates to the recently introduced pharmaceutical classes such as antiretroviral drugs (ARVDs), as well as their metabolites and transformation products. The potential presence of transformation products and metabolites of most ARVDs in wastewater samples is currently largely unknown. Most of the analytical methods developed to monitor ARVDs in aqueous environmental samples are targeted at selected compounds of a few therapeutic classes, warranting the need to expand their scope. ARVDs vary significantly in terms of molecular size, pKa and polarity, which further complicates the development of universal analytical methods for ARVD determination. The first task of this study was to develop a liquid chromatography-tandem mass spectrometric (LC-MS/MS) multiresidue method for the analysis of multiclass ARVDs and selected metabolites in wastewater samples. This necessitated the development of two separate sample preparation methods: one based on reversed phase (RP) solid phase extraction (SPE) sample clean-up and pre-concentration, and a second based on direct injection to correct for losses of polar ARVDs due to low breakthrough volumes on SPE. Application of the developed method to samples obtained from two wastewater treatment plants (WWTPs) in the Western Cape differing by advanced tertiary treatment processes allowed the first confirmation and quantification of two phase I ARVD metabolites, 8,14-dihydroxy efavirenz and 12-hydroxynevirapine. Furthermore, quantitative results confirm that levels of ARVDs in South African wastewaters are generally comparable to the rest of the world, and that they were mostly effectively removed by established treatment processes, with the exception of efavirenz and nevirapine. High concentrations were measured in the dry season, and the polar ARVDs were measured at high concentrations. WWTP treatment by uv-irradiation was found to be more effective at removing ARVDs compared to chlorination. In the second part of the study, supercritical fluid chromatography (SFC)-MS/MS was evaluated as an alternative to LC-MS/MS for the analysis of ARVDs in wastewater. Although the scope of the developed SFC-MS/MS method was more limited compared to LC, there was a high level of agreement in the quantitative data (recoveries, repeatability, and reproducibility) obtained by the two methods. These findings demonstrate for the first time the suitability of SFC-MS/MS for environmental analysis of ARVDs and their metabolites. Finally, a non-targeted LC-high resolution MS (HR-MS) method incorporating ion mobility spectrometry (IMS) was developed to screen for additional ARVDs and metabolites in wastewater samples. Based on IMS-filtered HR-MS low and high collision energy spectra and IMS collision cross section values, and using a suitable mass defect filter, several novel hydroxylated-, sulphated, carboxylated and glucuronidated metabolites for efavirenz, nevirapine, ritonavir and abacavir were identified for the first time in wastewater.AFRIKAANS OPSOMMING: Die voorkoms en hardnekkigheid van farmaseutiese produkte in akwatiese omgewings is 'n aktuele openbare gesondheids kwessie as gevolg van hul storting in die omgewing, beide as onveranderde- en biogetransformeerde produkte. Die omgewingsimpak van die meeste van hierdie verbindings is reeds bekend, maar heelwat kommer bestaan in verband met onlangs bekendgestelde farmaseutiese produkte soos antiretrovirale middels (ARVDs) asook hul metaboliete en transformasieprodukte. Die potensiële teenwoordigheid van hierdie transformasieprodukte en metaboliete van die meeste ARVDs in afvalwatermonsters is tans grotendeels onbekend. Meeste van die analitiese metodes wat ontwikkel is om ARVDs in waterige omgewingsmonsters te monitor, is gemik op geselekteerde verbindings van beperkte terapeutiese klasse en dit regverdig die behoefte aan die uitbreiding daarvan. ARVDs wissel aansienlik in terme van molekulêre grootte, pKa en polariteit en dit bemoeilik die ontwikkeling van universele analitiese metodes vir ARVD-bepaling verder. Die eerste mikpunt van hierdie studie was om 'n vloeistofchromatografie-tandem massaspektrometriese (LC-MS/MS) multiresidu-metode te ontwikkel vir die analise van multi-klas ARVDs en geselekteerde metaboliete in afvalwatermonsters. Dit het die ontwikkeling van twee afsonderlike monstervoorbereidingsmetodes genoodsaak: een is gebaseer op omgekeerde fase (RP) soliede fase ekstraksie (SPE) skoonmaak en voorkonsentrasie van die monster, en 'n tweede gebaseer op direkte inspuiting om verliese van polêre ARVDs, as gevolg lae deurbreekvolumes met SPE, te vermy. Die toepassing van die ontwikkelde metodes op monsters verkry vanaf twee afvalwaterbehandelingsaanlegte in die Wes-Kaap, wat verskil in hul gevorderde tersiêre behandelingsprosesse, het die eerste bevestiging en kwantifisering van twee fase I ARVD-metaboliete, 8,14-dihydroxy efavirenz en 12-hydroxynevirapine, moontlik gemaak. Verder bevestig kwantitatiewe resultate dat vlakke van ARVDs in Suid-Afrikaanse afvalwater oor die algemeen vergelykbaar is met die res van die wêreld, en dat hulle meestal effektief verwyder word deur gevestigde behandelingsprosesse, met die uitsondering van efavirenz en nevirapine. Hoër konsentrasies is in die droë seisoen gemeet en die polêre ARVDs is ook in hoër konsentrasies gevind. Daar is waargeneem dat behandeling deur uv-bestraling meer effektief is vir verwydering van ARVDs as chlorinering. In die tweede deel van die studie is superkritiese vloeistofchromatografie (SFC)-MS/MS geëvalueer as 'n alternatief tot LC-MS/MS vir die analise van ARVDs in afvalwater. Alhoewel die omvang van die ontwikkelde SFC-MS/MS-metode meer beperk was in vergelyking met LC, was daar 'n hoë mate van ooreenstemming in die kwantitatiewe data (herwinbaarheid, herhaalbaarheid en reproduseerbaarheid) wat deur die twee metodes verkry is. Hierdie bevindings toon vir die eerste keer die geskiktheid van SFC-MS/MS vir die analise van ARVDs en hul metaboliete in die omgewing. Ten slotte is 'n ongeteikende LC-hoë resolusie MS (HR-MS) metode wat ioonmobiliteitspektrometrie (IMS) insluit ontwikkel om addisionele ARVDs en hul metaboliete in afvalwatermonsters te probeer opspoor. Gebaseer op IMS-gefiltreerde HR-MS lae en hoë botsingsenergiespektra en IMS-botsingsdeursnee-waardes, en met behulp van 'n geskikte massa-defekfilter, is verskeie nuwe gehidroksileerde, gesulfateerde, gekarboksileerde en geglukuroniseerde metaboliete van efavirenz, nevirapien, ritonavir en abacavir vir die eerste keer in afvalwater geïdentifiseer.Master