Benzotriazole and 5-methylbenzotriazole in recycled water, surface water and dishwashing detergents from Perth, Western Australia: analytical method development and application.

A simplified and sensitive liquid chromatography mass spectrometry (LC-MS) method without requiring sample pre-concentration was successfully developed for detecting the occurrence of ultra-low (ng L(-1)) concentrations of benzotriazole (BTri), and its derivative 5-methyl benzotriazole (5-MeBT) in various Western Australian environmental water samples. The method detection limit was 2 ng L(-1), providing similar detection limits to other more process intensive methods where pre-concentration using solid phase extraction (SPE) was employed. The method was used to assess the occurrence of BTri and 5-MeBT in wastewater and surface water samples. Over a period of 12 months, BTri and 5-MeBT concentrations in secondary treated wastewater were measured, with the highest BTri and 5-MeBT concentrations observed during winter months at 78 ng L(-1) and 21 ng L(-1), respectively. The method was also used to assess the removal efficiency of BTri and 5-MeBT through an advanced water recycling plant (AWRP). While BTri was more persistent than 5-MeBT, both compounds were removed from the AWRP to <10 ng L(-1) (BTri) and <2 ng L(-1) (5-MeBT), with reverse osmosis (RO) providing the most effective treatment process for their removal.

Fate of benzotriazole and 5-methylbenzotriazole in recycled water recharged into an anaerobic aquifer: column studies.

The fate of benzotriazole (BTri) and 5-methylbenzotriazole (5-MeBT) was investigated under anaerobic conditions at nano gram per litre concentrations in large-scale laboratory columns to mimic a managed aquifer recharge replenishment strategy in Western Australia. Investigations of BTri and 5-MeBT sorption behaviour demonstrated mobility of the compounds with retardation coefficients of 2.0 and 2.2, respectively. Degradation processes over a period of 220 days indicated first order biodegradation of the BTri and 5-MeBT under anaerobic aquifer conditions after a biological lag-time of approximately 30-60 days. Biodegradation half-lives of 29 ± 2 and 26 ± 1 days for BTri and 5-MeBT were respectively observed, with no threshold effect to biodegradation observed at the 200 ng L(-1). The detection of degradation products provided further evidence of BTri and 5-MeBT biodegradation. These results suggested that if BTri and 5-MeBT were present in recycled water recharged to the Leederville aquifer, biodegradation during aquifer passage is likely given sufficient aquifer residence times or travel distances between recycled water injection and groundwater extraction.

A urinary profile study of dietary phytoestrogens. The identification and mode of metabolism of new isoflavonoids.

The metabolic fate of the dietary isoflavones daidzein and genistein was investigated in human volunteers challenged with soya. Urinary diphenols, isolated by partition chromatography on Sephadex LH-20, were characterized and identified by profile capillary gas chromatography (GC) and electron ionization mass spectrometry (GC-EIMS) analysis of the trimethylsilyl ether (TMS) derivatives. Novel isoflavonic phytoestrogens found in the urine of volunteers were those of tetrahydrodaidzein, dihydrogenistein, 6'-hydroxy-O-demethylangolesin and 2-dehydro-O-demethylangolensin. Other known diphenols identified were those of equal, dehydrodaidzein, O-demethylangolensin, daidzein, genistein, glycitein, and the lignan enterolactone. Two other urinary isomers with a fragmentation pattern closely resembling that of the persilylated TMS ethers of cis/trans-isomers of tetrahydrodaidzein, were characterized based on the elucidation of fragments associated with the loss of a non-phenolic-OTMS functional group in ring-C. These are fragments presented in the persilylated mass spectra of isoflavan-4-ols and isoflav-3-ene-4-ols, demonstrated here by a combination of simple and tandem mass spectrometry study of the deuterated persilylated TMS ethers of dihydrodaidzein. In a similar study we also present the data on the structural identification and fragment elucidation of the keto/enol tautomers of the TMS ether derivatives of the dihydro derivatives of daidzein and genistein, observed in the urine of volunteers and considered probable products of the derivatization process. Finally, the GC and GC-MS data of two unknown isoflavonoids and that of a lignan-like compound are presented together with those of dihydrodaidzein, dihydrogenistein, tetrahydrodaidzein and 2-dehydro-O-demethylangolensin. The latter four were obtained here as products of small scale chemical synthesis in a preliminary study on the tentative identification of urinary isoflavonoids in human volunteers challenged with soya.

Metabolites of dietary (soya) isoflavones in human urine.

This study was undertaken to better understand the metabolic fate of dietary isoflavones in humans. Twelve volunteers were challenged with soya flour and urinary diphenol levels were then determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The presence of previously described urinary diphenols was confirmed, i.e. the isoflavones, daidzein and genistein; the isoflavonoid metabolites, equol, dihydrodaidzein (Int-O-D), O-desmethyl-angolensin (O-Dma); the lignan, enterolactone. Diphenols detected for the first time were the isoflavone, glycitein and five novel isoflavonoid metabolites which are tentatively identified as 6'-hydroxy-O-desmethylangolensin (6' OH-O-Dma), dihydrogenistein (Int-O-G), dehydro-O-desmethylangolensin (dehydro-O-Dma) and two isomers of tetrahydrodaidzein. Urinary excretion rates of the three isoflavones (daidzein, genistein, glycitein) over a 3-day period following soya challenge showed moderate variation (4x, 6x and 12x, respectively) between the 12 individuals suggesting some individual variabilities in ability to deconjugate and to absorb dietary isoflavones. However, urinary excretion rates of each of three major isoflavonoid metabolites (equol, O-Dma, 6' OH-O-Dma) showed more marked variation (922x, 17x, 15x, respectively); while some of this variability may reflect varying individual ability to ferment dietary isoflavones per se, an inverse relationship was found between urinary levels of equol and both O-Dma and 6' OH-O-Dma suggesting individual variability in the preferred metabolic pathways of dietary isoflavones.

15 beta-hydroxysteroids (Part IV). Steroids of the human perinatal period: the synthesis of 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one and its A/B-ring configurational isomers.

In recent years several 15 beta-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3 alpha,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3 xi,5 xi-isomers, namely 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (3), 3 beta,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (7) and 3 beta,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3 beta,15 beta-Diacetoxy-17 alpha-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15 beta,17 alpha-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5 alpha-pregnan-20-one (13) a common intermediate for the synthesis of the 3 beta(and alpha),5 alpha-isomers. Hydrolysis of the 15 beta-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15 beta-acetoxy-17 alpha-hydroxy-5 alpha-pregnan-3,20-dione (14) which on reduction with L-Selectride and hydrolysis of the 15 beta-acetate gave 3. Finally, hydrogenation of 4 gave 15 beta, 17 alpha-dihydroxy-5 beta-pregnan-3,20-dione (10) which on reduction with L-Selectride gave 8.

15 beta-hydroxysteroids (Part III). Steroids of the human perinatal period: the synthesis of 3 beta, 15 beta, 17 alpha-trihydroxy-5-pregnen-20-one. Application of n-butyl boronic acid protection of a 17,20-glycol.

We report the synthesis of 3 beta,15 beta,17 alpha-trihydroxy-5-pregnen-20- one (1) from 3 beta,15 beta-dihydroxy-5,16-pregnadien-20-one (11) in 7 steps using boronate derivatives as a means of protecting the 17,20-glycol side-chain of steroid intermediates. 16 alpha,17 alpha-Epoxy-3 beta,15 beta-dihydroxy-5- pregnen-20-one (12), an intermediate in the synthesis was prepared by epoxidation of 11 using a mixture of sodium hydroxide and hydrogen peroxide. Reduction of 12 with lithium aluminium hydride gave the two isomers of 5-pregnene-3 beta, 15 beta,17 alpha,20 (S+R)-tetrol (13a and 13b) which on subsequent reaction with n-butyl boronic acid gave 5-pregnene-3 beta,15 beta,17 alpha, 20(S+R)-tetrol 17 alpha,20-butyl boronate (15a and 15b). Acetylation with acetic anhydride and pyridine yielded 3 beta,15 beta-diacetoxy-5-pregnene-17 alpha,20(S+R)-diol 17 alpha,20(S+R)-butyl boronate (15c and 15d). Oxidative cleavage of the boronic ester using sodium hydroxide and hydrogen peroxide gave 3 beta,15 beta-diacetoxy-5-pregnene-17 alpha,20(S+R)-diol (13c and 13d). After isolation of these latter two products, dibromide protection of the C-5,6 olefin of 13d and oxidation with N-bromosuccinimide gave 3 beta,15 beta-diacetoxy-17 alpha-hydroxy-5-pregnen-20-one (16) which on deacetylation gave in good yield (35%) the desired product 3 beta,15 beta,17 alpha-trihydroxy-5-pregnen-20-one (1) in an overall yield of 24% from 11.

15 beta-hydroxysteroids (Part V). Steroids of the human perinatal period: the synthesis of 3 beta, 15 beta, 17 alpha-trihydroxy-5-pregnen-20-one from 15 beta, 17 alpha-dihydroxy-4-pregnen-3,20-dione.

A simple three-step synthetic method is reported on the conversion of delta 4-3-ketosteroids to the corresponding 3 beta-hydroxy-delta 5-steroid analogues. 17 alpha-Hydroxy-4-pregnen-3,20-dione (10a) was used as a model to develop a method for the synthesis of 3 beta, 17 alpha-dihydroxy-5-pregnen-20-one (16). The major problem being the synthesis of 3,17 alpha-diacetoxy-3,5-pregnadien-20-one (14) was solved by acetylating using a mixture of acetic anhydride and perchloric acid. The conversion of 15 beta, 17 alpha-dihydroxy-4-pregnen-3, 20-dione (8), product of Penicillium citrinum fermentation, to the desired 3 beta,15 beta,17 alpha-trihydroxy-5-pregnen-20-one (1), is described using a modification of this method. Reaction of 8 with acetic anhydride and perchloric acid in ethyl acetate gave 3,15 beta,17 alpha-triacetoxy-3,5-pregnadien-20-one (17) which on reduction with sodium borohydride gave 5-pregnen-3 beta,15 beta,17 alpha, 20(S + R)-tetrols (18a and 18b); however, reduction of 17 with a mixture of sodium borohydride and potassium bicarbonate gave after basic hydrolysis with methanolic sodium hydroxide the desired product 3 beta,15 beta,17 alpha-trihydroxy-5-pregnen-20-one (1) in good yield (54%).

15 beta-hydroxysteroids (Part VI). steroids of the human perinatal period: the preparation and reactions of 3 beta-hydroxy-5,15-androstadien-17-one. The synthesis of 3 beta,15 beta-dihydroxy-5-androsten-17-one and derivatives.

A successful approach to the synthesis of 3 beta,15 beta-dihydroxy-5-androsten-17-one (14d) has been developed using trichloroethoxy ethers as intermediates in the synthesis of the corresponding alcohols. 3 beta-Methoxymethoxy-5,15-androstadien-17-one (10c) was prepared by a selenation/dehydroselenation strategy from 3 beta-methoxymethoxy-5-androsten-17-one (14c). Base-catalyzed reaction of trichloroethanol with 10c gave 3 beta-methoxymethoxy-15 beta-trichloroethoxy-5-androsten-17-one (14g). Under the same conditions, 3 beta-acetoxy-5,15-androstadien-17-one (10b) gave 3 beta-hydroxy-15 beta-trichloroethoxy-5-androsten-17-one (14f) which was characterized after conversion to 14g. Cleavage of the trichloroethoxy group in 14f with zinc or zinc/copper couple gave 14d. The acid-catalyzed hydrolysis of 17,17-ethylenedioxy-5,15-androstadien-3 beta-ol (15) gave 3 beta-hydroxy-5,15-androstadien-17-one (10a) as the major product along with 14d. However, addition of water to 10a in the presence of acid gave the desired product 14d in poor yield (15%).

15 beta-Hydroxysteroids (Part VII). Steroids of the human perinatal period: the synthesis of steroid markers and their radioactive tracers.

We report the synthesis of 10 novel steroids obtained from 3 beta, 15 beta-diacetoxy-17 alpha-hydroxy-5-pregnen-20-one (1c) as intermediates in the synthesis of 15 beta-acetoxy-20,20-ethylenedioxy-17 alpha-hydroxy-4-pregnen-3-one (6a) and its tritiated tracer 15 beta-acetoxy-20,20-ethylenedioxy-17 alpha-hydroxy-1,2,6,7-3H-pregn-4-en-3-one (6d). The one pot interconversion of intermediate (6a) to 3 beta, 15 beta, 17 alpha-trihydroxy-5-pregnen-20-one (1a) and 3 alpha, 15 beta, 17 alpha-trihydroxy-5 beta-pregnan-20-one (2a) provides a new and efficient approach to the synthesis of diagnostically important metabolites of the human neonate and a possible route in the synthesis of the tritated tracers 3 beta, 15 beta, 17 alpha-trihydroxy-1,2,7-3H-pregn-5-en-20-one (1d) and 3 alpha, 15 beta, 17 alpha-trihydroxy-1,2,6,7-3H-5 beta-pregnan-20-one (2b) for the development of new immunoassays. We also report in this investigation an alternative route in the synthesis of 15 beta, 17 alpha-dihydroxy-4-pregnen-3,20-dione (7a) an intermediate in the synthesis of human 15 beta-hydroxysteroid metabolites.

15 beta-hydroxysteroids (part I). Steroids of the human perinatal period: the synthesis of 3 beta,15 beta, 17 alpha-trihydroxy-5-pregnen-20-one.

15-Hydroxysteroids have long provided information about fetal well-being and fetal steroidogenesis. 3 beta,15 beta,-17 alpha-Trihydroxy-5-pregnen-20-one (1a) is a major 15 beta-hydroxylated metabolite unique to the human perinatal period. The synthesis of 3 beta, 15 beta, 17 alpha-trihydroxy-5-pregnen-20-one (1a) is reported here in the first of a series of publications on the chemical synthesis of 15 beta-hydroxylated steroids for use in the (a) development of new immunoassay techniques for application to newborn screening programs and fetal well-being; (b) development of new anti-androgenic drugs; and (c) study of androgen/estrogen interaction in late pregnancy. To this end, a method for the introduction of the 15 beta-hydroxy group onto the steroid nucleus was developed resulting in a nine-step stereoselective synthesis of 1a with an overall yield of 26%. A high yielding selenation-dehydroselenation procedure was developed for the synthesis of 3 beta-hydroxy-5,15-androstadien-17-one (8) which avoided the previously reported Baeyer-Villiger rearrangement. The introduction of the 15 beta-hydroxy group and the side chain was achieved by the addition of 2-lithio-2-methyl-1,3-dithiane to give 20,20-trimethylenedithio-5,15-pregnadien-3 beta, 17 beta-diol (9a) followed by its acid-catalyzed rearrangement to give 20,20-trimethylenedithio-5, 16-pregnadien-3 beta,15 beta-diol (10a). Acetylation and cleavage of the dithioacetal gave 3 beta,15 beta-diacetoxy-5,16-pregnadien-20-one (11b) which was hydrogenated to give 3 beta,15 beta-diacetoxy-5-pregnen-20-one (12b). Reaction of the ketone (12b) with oxygen and then basic hydrolysis gave the desired product 1a.

15 beta-hydroxysteroids (part II). Steroids of the human perinatal period: the synthesis of 3 alpha,15 beta, 17 alpha-trihydroxy-5 beta-pregnan-20-one.

Steroids hydroxylated at C-15 have long provided useful information about the well-being of the fetus and feto-placental unit in human pregnancy. In an attempt to develop a new and reliable immunoassay method for use in newborn screening programs for congenital adrenal hyperplasia, we report the chemical synthesis of 3 alpha,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (2) from 3 alpha-hydroxy-5 beta-androstan-17-one (4) in 9 steps. In brief, 3 alpha-hydroxy-5 beta-androst-15-en-17-one (6), was obtained from 4 by phenylselenation yielding 3 alpha-hydroxy-16 alpha-phenylseleno-5 beta- androstan-17-one (5a) which on dehydroselenation gave 6. Introduction of the 15 beta-hydroxy group and the side-chain was achieved by the addition of 2-lithio-2-methyl-1,3- dithiane followed by an acid-catalyzed rearrangement to give 20,20-trimethylenedithio-5 beta-pregn-16-en- 3 alpha,15 beta-diol (8a). Acetylation then cleavage of the dithioacetal gave 3 alpha,15 beta-diacetoxy-5 beta-pregn-16-en- 20-one (9) which on hydrogenation gave 3 alpha,15 beta-diacetoxy-5 beta-pregnan-20-one (10). Reaction of base and oxygenation of 10 gave a mixture of products which on basic hydrolysis gave 3 alpha,15 beta,17 alpha-trihydroxy-5 beta- pregnan-20-one (2) in an overall yield of 8.8%.

The variable metabolic response to dietary isoflavones in humans.

The aim of this study was to better understand the metabolic fate of dietary estrogenic isoflavones in humans. Twelve volunteers were challenged with soya flour and urinary levels of the isoflavones daidzein (Da), genistein (Gen), and glycitein (Gly), and the isoflavonoid metabolites equol (Eq) and O-desmethylangolensin (O-Dma) determined by GC and GC-MS. Prior to challenge, Da, Gen, and Gly were present in the urine of all participants at low levels and Eq and O-Dma were present in 9/12 and 10/12 participants, respectively. Urinary levels of all five diphenols were increased in each individual on the day following challenge, returning to approximately prechallenge levels on the second or third day post-challenge. Mean post-challenge peak levels of the five diphenols compared with pre-challenge levels were Da (4x), Gen (8x), Gly (5x), Eq (45x), and O-Dma (66x). However, there was considerable individual variation in this metabolic response with peak levels of Eq showing the highest variation (1527x). An inverse relationship between Eq and O-Dma excretion was also found post-challenge suggesting individual variability in the preferred metabolic pathways of dietary isoflavones.