Development and Application of HPLC-MS/MS Method for Determination of Human Pharmaceuticals and Synthetic Hormones in River Water and Sewage Effluents

Pollution of the aquatic environment by residues of human pharmaceuticals and synthetic hormones has become a cause for increasing concern in recent years and acknowledged as one of the most emerging environmental issues. The occurrence of human pharmaceuticals and synthetic hormones in the Malaysian aquatic environment has never been reported. This lack of data could be attributed to the absence of an internationally recognized method for the analysis of pharmaceutical residues in water matrices. The main objective of this study is to develop a sensitive and selective method for the simultaneous determination of 23 pharmaceuticals of interest in environmental matrices such as river water and sewage effluents. The 23 pharmaceuticals consist of different therapeutic classes which include both top prescribed and over the counter (OTC)pharmaceuticals. The developed method was based on sample pre-treatment using Solid Phase Extraction (SPE) followed by analysis using High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS). The method was validated and tested against environmental samples. The validation results indicated that the method is able to simultaneously detect and quantify the targeted pollutants with good selectivity and sensitivity. The method performed well for the majority of the pharmaceuticals evaluated, with recoveries greater than 70% for most pharmaceuticals. To the researcher’s knowledge, 5 pharmaceuticals (i.e. amlodipine, chlorpheniramine, chlorothiazide, perindopril and gliclazide) out of the 23 pharmaceuticals had never been analyzed in environmental matrices. The other objectives of this study were to identify and quantify pharmaceuticals residues in environmental samples collected from the Langat River and in effluents of sewage treatment plants (STPs) using the developed method. Seventeen out of twenty-three targeted pharmaceuticals were detected in river water samples namely metformin, atenolol, acetaminophen, metoprolol, mefenamic acid, salicylic acid, salbutamol, perindopril, gliclazide, glibenclamide, loratadine, furosemide, levonorgestrel, cyproterone, diclofenac, chlorothiazide and nifedipine. The median concentration of detected pharmaceutical ranged between less than the Method Detection Limit (<MDL) for furosemide, loratadine and nifedipine to higher concentration of 112.7 ng/L for diclofenac. The highest concentration found was acetaminophen (346.3 ng/L). On the other hand, nineteen targeted pollutants were detected in effluent samples from STPs namely metformin, atenolol, acetaminophen, metoprolol, mefenamic acid, salicylic acid, salbutamol, perindopril, gliclazide, diclofenac, furosemide, chlorothiazide, glibenclamide, lovastatin, loratadine, amlodipine, 17-ethinylestradiol, norethindrone and cyproterone. The median concentration of the detected pollutants were less than the Method Detection Limit (<MDL) for lovastatin, loratadine, norethindrone, cyproterone, amlodipine and 17-ethinylestradiol to a higher concentration of 3270.6 ng/L for metformin. The highest concentration found was metformin (34228.6 ng/L). This study has confirmed that the Malaysian aquatic environment is impacted by pharmaceutical residues at varying levels. Mefenamic acid and Salicylic acid were found to be present in all the river water and STPs effluent samples, suggesting their widespread use and high degree of persistency in the tropical aquatic environment. However, chlorphernamine and simvastatin were not found in any sample for all matrices indicating their high degradability and low persistency in the tropical aquatic environment. Keywords: Human pharmaceuticals, synthetic hormones, water pollution, HPLC- MS/MS, Langat Rive

Multi-residue analytical method for human pharmaceuticals and synthetic hormones in river water and sewage effluents by solid-phase extraction and liquid chromatography–tandem mass spectrometry.

Pollutants such as human pharmaceuticals and synthetic hormones that are not covered by environmental legislation have increasingly become important emerging aquatic contaminants. This paper reports the development of a sensitive and selective multi-residue method for simultaneous determination and quantification of 23 pharmaceuticals and synthetic hormones from different therapeutic classes in water samples. Target pharmaceuticals include anti-diabetic, antihypertensive, hypolipidemic agents, β2-adrenergic receptor agonist, antihistamine, analgesic and sex hormones. The developed method is based on solid phase extraction (SPE) followed by instrumental analysis using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) with 30 min total run time. River water samples (150 mL) and (sewage treatment plant) STP effluents (100 mL) adjusted to pH 2, were loaded into MCX (3 cm3, 60 mg) cartridge and eluted with four different reagents for maximum recovery. Quantification was achieved by using eight isotopically labeled internal standards (I.S.) that effectively correct for losses during sample preparation and matrix effects during LC–ESI-MS/MS analysis. Good recoveries higher than 70% were obtained for most of target analytes in all matrices. Method detection limit (MDL) ranged from 0.2 to 281 ng/L. The developed method was applied to determine the levels of target analytes in various samples, including river water and STP effluents. Among the tested emerging pollutants, chlorothiazide was found at the highest level, with concentrations reaching up to 865 ng/L in STP effluent, and 182 ng/L in river water

The occurrence of human pharmaceuticals in wastewater effluents and surface water of Langat River and its tributaries, Malaysia.

This study provides the first investigation on the occurrence of human pharmaceuticals in the tropical aquatic environment of Malaysia. Water samples were collected at seven different sites along the Langat River and effluents from five sewage treatment plants (STPs) in Langat River Basin. Samples were extracted by solid phase extraction (SPE) and analyzed using liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) for 18 pharmaceuticals from six therapeutic classes and one metabolite. Fifteen out of these 19 pharmaceuticals were detected in the river water samples. Mefenamic acid, salicylic acid and glibenclamide were detected in all river water samples indicating their ubiquitous nature and resistance to degradation under the warm and humid tropical conditions. The median concentrations of detected pharmaceuticals ranged from less than the method detection limit (<MDL) for furosemide, loratadine, salbutamol, perindopril, metoprolol and nifedipine to 112.7 ng L−1 for diclofenac. A similar number of pharmaceuticals were detected in the STPs’ effluents samples. Several of these pharmaceuticals, namely salbutamol, atenolol, metoprolol, mefenamic acid, salicylic acid and furosemide were detected in all the STPs’ effluents samples. The median concentrations for the detected pollutants ranged between <MDL for lovastatin and amlodipine to 1994 ng L−1 for metformin. The highest concentration detected in the river water samples was for acetaminophen (346.3 ng L−1) and in STPs effluents was for metformin (34228 ng L−1). The concentrations of most analytes found in this study were comparable to those reported in the other parts of the world. However, this is the first time amlodipine is detected in the environmental samples

Anthropogenic waste indicators (AWIs), particularly PAHs and LABs, in Malaysian sediments: application of aquatic environment for identifying anthropogenic pollution

Polycyclic aromatic hydrocarbons (PAHs) and linear alkylbenzenes (LABs) were used as anthropogenic markers of organic chemical pollution of sediments in the Selangor River, Peninsular Malaysia. This study was conducted on sediment samples from the beginning of the estuary to the upstream river during dry and rainy seasons. The concentrations of ƩPAHs and ƩLABs ranged from 203 to 964 and from 23 to 113 ng g− 1 dry weight (dw), respectively. In particular, the Selangor River was found to have higher sedimentary levels of PAHs and LABs during the wet season than in the dry season, which was primarily associated with the intensity of domestic wastewater discharge and high amounts of urban runoff washing the pollutants from the surrounding area. The concentrations of the toxic contaminants were determined according to the Sediment Quality Guidelines (SQGs). The PAH levels in the Selangor River did not exceed the SQGs, for example, the effects range low (ERL) value, indicating that they cannot exert adverse biological effects

Quantification of selected steroid hormones (17b-Estradiol and 17a-Ethynylestradiol) in wastewater treatment plants in Klang Valley (Malaysia)

Steroid estrogens, such as 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) are potent and were categorized as "Watch List" in Directive 2013/39/EU because of their potential risks to aquatic environment. Commercialized enzyme-linked immunosorbent assay (ELISA) kits have been used to quantify steroid estrogens in wastewater samples due to their simplicity, rapid, cost-effectiveness, and validated assays. Hence, this study aims to determine the occurrence and removal of steroid hormones in Malaysian wastewater treatment plants (WWTPs) by ELISA, to identify the association of removal efficiency (E2 and EE2) with respect to WWTPs operating conditions, and to assess the potential risks of steroid estrogens to aquatic environment and human. Results showed E2 concentration ranged from 88.2 ± 7.0 ng/L to 93.9 ± 6.9 ng/L in influent and 35.1 ± 17.3 ng/L to 85.2 ± 7.6 ng/L in effluent, with removal of 6.4%-63.0%. The EE2 concentration ranged from 0.2 ± 0.2 ng/L to 4.9 ± 6.3 ng/L in influent and 0.02 ± 0.03 ng/L to 1.0 ± 0.8 ng/L in effluent, with removal of 28.3-99.3%. There is a correlation between EE2 removal with total suspended solid (TSS) and oxidation reduction potential (ORP), and was statistically significant. Despite the calculated estrogenic activity for E2 and EE2 was relatively high, dilution effects could lower estrogenic response to aquatic environment. Besides, these six selected WWTPs have cumulative RQ values below the allowable limit, except WWTP 1. Relatively high precipitation (129-218 mm) could further dilute estrogens concentration in the receiving river. These outputs can be used as quantitative information for evaluating the occurrence and removal of steroid estrogens in Malaysian WWTPs

KiSS1 gene as a novel mediator of TGFß pro-invasive effects in triple negative breast cancer

The attainment of invasive and metastatic phenotype by tumors ushers in transition from indolent to aggressive disease with poor survival and high recurrence rates. TGF-β is a member of the TGF-β cytokine super family which regulates development and homeostasis. TGF-β exerts tumor suppressive effects that cancer must elude for malignant evolution. Yet, paradoxically, TGF-β modulates processes like cell migration and invasion and epithelium to mesenchymal transition. To dissect the molecular basis of TGF-β pro-carcinogenic effects in breast cancer, we screened human microarray chip to analyze the genomic profile of triple negative breast tumor in response to TGF-β. Interestingly, we found the gene KiSS1 is up-regulated by TGF-β. KiSS1 encodes the secreted proteins kisspeptines. While kisspeptines play anti metastatic role in various types of cancers, its role in breast cancer remain controversial.Our study aims to comprehend the relationship between TGFβ and KiSS1 In breast cancer. We validated our RNA microarray data by real Time Q-PCR, which confirmed the upregulation and demonstrated the pathway involved in this regulation Then, with help of short interference RNA (SiRNA), we examined KiSS1 biological function downstream TGFβ with different In Vitro assays in triple negative breast cancer cell line. To explain the cell response to KiSS1, we explored its effect on a panel of genes that are regulated by TGFβ and known to be associated with aggressive cancer phenotype. The last part of the study, focused on exploring KiSS1 clinical utility as, both, prognostic biomarker and therapeutic target.L'acquisition de propriétés invasives et métastatiques d'une tumeur annonce la transition d'une affection dormante vers une maladie agressive avec un faible taux de survie et un haut taux de récurrence. Le TGF-ß est un membre de la super famille de cytokines TGF-ß, qui régule le développement et l'homéostasie. Le TGF-ß exerce sur les tumeurs un effet anti-tumoral auquel le cancer doit échapper pour évoluer vers des phénotypes plus agressifs. Paradoxalement, le TGF-ß promeut aussi des procédés tels que la migration cellulaire, l'invasion, et la transition épithélio-mésenchymateuse.Afin de décortiquer les effets pro-carcinogènes du TGF-ß dans le cancer du sein, nous avons analysé le profil génétique de cancers triple-négatifs à l'aide de puces à ADN en réponse au TGF-ß. Nous avons trouvé que le gène KiSS1 est positivement régulé par le TGF-ß. KISS1 code pour les protéines sécrétées kisspeptines. Les kisspeptines sont connues pour jouer un rôle anti-métastatique dans différents types de cancer, cependant leur rôle dans le cancer du sein reste controversé.Notre étude vise à mieux comprendre la relation entre le TGF-ß et KiSS1 dans le cancer du sein. Nous avons validé les données obtenues de la puce par réaction de polymérase en chaîne quantitative en temps réel. Nous avons ensuite utilisé des siRNAs pour examiner les fonctions biologiques du KiSS1 en aval du TGF-ß par plusieurs analyses in vitro dans les lignées triple-négatives du cancer du sein. Pour expliquer la réponse cellulaire suivant une stimulation par KiSS1, nous avons exploré ses effets sur une gamme de gènes qui sont régulés par le TGF-ß et connus pour être impliqués dans la progression vers un phénotype agressif. La dernière partie de l'étude explore l'utilité clinique de KiSS1 comme marqueur pronostique et comme cible thérapeutique

Selective benzylic C–H monooxygenation mediated by iodine oxides

A method for the selective monooxdiation of secondary benzylic C–H bonds is described using an N-oxyl catalyst and a hypervalent iodine species as a terminal oxidant. Combinations of ammonium iodate and catalytic N-hydroxyphthalimide (NHPI) were shown to be effective in the selective oxidation of n-butylbenzene directly to 1-phenylbutyl acetate in high yield (86%). This method shows moderate substrate tolerance in the oxygenation of substrates containing secondary benzylic C–H bonds, yielding the corresponding benzylic acetates in good to moderate yield. Tertiary benzylic C–H bonds were shown to be unreactive under similar conditions, despite the weaker C–H bond. A preliminary mechanistic analysis suggests that this NHPI-iodate system is functioning by a radical-based mechanism where iodine generated in situ captures formed benzylic radicals. The benzylic iodide intermediate then solvolyzes to yield the product ester