Determination of cocaine and its metabolites in human urine by gas chromatography/mass spectrometry after simultaneous use of cocaine and ethanol

Cocaethylene is an active metabolite produced when cocaine is consumed jointly with ethanol. The development of analytical techniques for determining cocaethylene and other cocaine metabolites is highly relevant for pharmacokinetic and toxicology studies of the cocaine and alcohol interaction in humans. The gas chromagraphy/mass spectrometry (GC/MS) method here reported is based on a single solid-phase extraction together with deuterated internal standards previously added to urine, followed by derivatization with pentafluoropropionic anhydride (hydroxyl and amine functions) and 1,1,1,3,3,3 hexafluor-2-propanol (carboxylic acid function) and injection into a capillary GC system coupled to a mass spectrometric detector in the selected ion monitoring acquisition mode. A sensitivity of 1–2 ng ml⁻¹ for the quantitative analysis of cocaine and its main metabolites (ecgonine methyl ester, benzoylecgonine, cocaethylene and norcocaine) was achieved. In addition, some other minor metabolites were easily extracted and detected.     

Gas chromatography analysis of urinary alkoxyacetic acids as biomarkers of exposure to aliphatic alkyl ethers

Analysis of alkoxyacetic acids has received considerable research interest in toxicology because these compounds have been reported as metabolites and biomarkers of exposure to widely used industrial chemicals such as alkyl-substituted ethylene glycols and other aliphatic ethers. This paper describes an improved method for the determination of methoxyacetic acid (MAA), ethoxyacetic acid (EAA), and butoxyacetic acid (BAA) in rat urine. Solid-phase extraction with Bakerbond(T) C18 bonded silica cartridges was successfully employed to isolate the acids from rat urine. The acids were then converted to methyl esters with diazomethane derivatization and analyzed using a gas chromatograph (GC) equipped with a mass spectrometry (MS) and a GC with flame ionization detector (FID). Employing GC-MS under selected ion monitoring detection, the lowest detection concentrations for MAA, EAA, and BAA were determined to be from 2 to 4 ng/mL urine in 1 mL of sample size. This method is 5 to 10 times more sensitive than that using GC-FID. The method described here is superior to the existing ones reported in the literature in that it employs an easy sample treatment procedure and gives much higher recoveries, making it suitable for routine assays. The utility of this new method was demonstrated in a toxicology study of aliphatic alkyl ethers.     

Identification and quantitative determination of a carboxylic and a mercapturic acid metabolite of etridiazole in urine of rat and man. Potential tools for biological monitoring

Etridiazole, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole, was orally administered to rats and human volunteers. Two metabolites of etridiazole were synthesized: 5-ethoxy-1,2,4-thiadiazole-3-carboxylic acid (ET-CA) and N-acetyl-S-(5-ethoxy-1,2,4-thiadiazol-3-yl-methyl)-l-cysteine (ET-MA). Selective and sensitive analytical procedures to determine etridiazole, the carboxylic acid ET-CA and the mercapturic acid ET-MA in urine were developed. The detection limit of etridiazole, applying GC with nitrogen selective detection (GC-NPD), was 36 (g/l urine (CV=15.4%,n=3). The detection limit of ET-CA, applying GC with sulphur selective detection (GC-FPD), was 100 g/l urine (CV=9.8%,n=3). In urine of rats orally treated with etridiazole, ET-CA and ET-MA were identified as metabolites of etridiazole, whereas in urine of humans given oral etridiazole, only ET-CA was identified. Unmetabolized etridiazole was excreted for less than 0.1% of the administered dose in rats. ET-CA, however, accounted for 22±9% of the administered dose of etridiazole in rats and for 13±6% in humans. ET-MA appeared to be a minor urinary metabolite of etridiazole. ET-CA is proposed as a possible biomarker for the biological monitoring of etridiazole.     

Detection of carboxylic acids and inhibition of hippuric acid formation in rats treated with 3-butene-1,2-diol, a major metabolite of 1,3-butadiene.

Epidemiological studies have indicated that 1,3-butadiene exposure is associated with an increased risk of leukemia. In human liver microsomes, 1,3-butadiene is rapidly oxidized to butadiene monoxide, which can then be hydrolyzed to 3-butene-1,2-diol (BDD). In this study, BDD and several potential metabolites were characterized in the urine of male B6C3F1 mice and Sprague-Dawley rats after BDD administration (i.p.). Rats given 1420 micromol kg(-1) BDD excreted significantly greater amounts of BDD relative to rats administered 710 micromol kg(-1) BDD. Rats administered 1420 or 2840 micromol kg(-1) BDD excreted significantly greater amounts of BDD per kilogram of body weight than mice given an equivalent dose. Trace amounts of 1-hydroxy-2-butanone and the carboxylic acid metabolites, crotonic acid, propionic acid, and 2-ketobutyric acid, were detected in mouse and rat urine after BDD administration. Because of the identification of the carboxylic acid metabolites and because of the known ability of carboxylic acids to conjugate coenzyme A, which is critical for hippuric acid formation, the effect of BDD treatment on hippuric acid concentrations was investigated. Rats given 1420 or 2272 micromol kg(-1) BDD had significantly elevated ratios of benzoic acid to hippuric acid in the urine after treatment compared with control urine. However, this effect was not observed in mice administered 1420 or 2840 micromol kg(-1) BDD. Collectively, the results demonstrate species differences in the urinary excretion of BDD and show that BDD administration in rats inhibits hippuric acid formation. The detection of 1-hydroxy-2-butanone and the carboxylic acids also provides insight regarding pathways of BDD metabolism in vivo.     

Metabolism of nilvadipine,a new dihydropyridine calcium antagonist,in rats and dogs

1. The metabolic profiles of nilvadipine in the excreta of male rats and dogs were studied after i.v. and oral dosing. Six types of metabolites were isolated and identified from the urine of male rats and dogs or bile of rats.

2. Several metabolites were detected in the urine (12) and bile (17) of rats by two-dimensional t.l.c., after dosing with ¹⁴C-nilvadipine. The metabolic profiles in the excreta of rats and dogs were qualitatively similar but quantative differences were observed.

3. The main metabolites were products of (i) oxidation of the 1,4-dihydropyridine ring to the corresponding pyridine, (ii) hydrolysis of the 5-isopropyl ester or 3-methyl ester group to carboxylic acid, and/or (iii) hydroxylation of the 6-methyl group or methyl group of the isopropyl ester chain.

4. Minor metabolites were products of hydrolysis from the 5-isopropyl ester to the carboxylic acid having a dihydropyridine ring, or reduction of the 3-nitro group of the phenyl moiety having a pyridine ring.     

板蓝根脂溶性成分的GC-MS分析

目的:分析、鉴定板蓝根的脂溶性成分。方法:采用石油醚萃取板蓝根的脂溶性成分,进行甲酯化处理后用气相色谱-质谱联用技术分离和鉴定其组成和含量。结果:从石油醚萃取部位共鉴定出42种脂溶性成分,其中脂肪酸甲酯化产物占36.72%(饱和脂肪酸甲酯为8.67%、不饱和脂肪酸甲酯为28.05%);有35种成分在板蓝根脂溶性成分的研究中未见报道。结论:板蓝根脂溶性成分可为其综合利用以及开发抗氧化方面应用提供参考。     

Metabolism of nilvadipine, a new dihydropyridine calcium antagonist, in rats and dogs

1. The metabolic profiles of nilvadipine in the excreta of male rats and dogs were studied after i.v. and oral dosing. Six types of metabolites were isolated and identified from the urine of male rats and dogs or bile of rats. 2. Several metabolites were detected in the urine (12) and bile (17) of rats by two-dimensional t.l.c., after dosing with 14C-nilvadipine. The metabolic profiles in the excreta of rats and dogs were qualitatively similar but quantitative differences were observed. 3. The main metabolites were products of (i) oxidation of the 1,4-dihydropyridine ring to the corresponding pyridine, (ii) hydrolysis of the 5-isopropyl ester or 3-methyl ester group to carboxylic acid, and/or (iii) hydroxylation of the 6-methyl group or methyl group of the isopropyl ester chain. 4. Minor metabolites were products of hydrolysis from the 5-isopropyl ester to the carboxylic acid having a dihydropyridine ring, or reduction of the 3-nitro group of the phenyl moiety having a pyridine ring.     

The metabolism of EGYT-475, a new antidepressant agent, in rats

The metabolites of EGYT-475 excreted by the kidney were studied in rats using radioactive analogues of the compound tested. 40% of the total radioactivity was found in rat urine which was collected over 48 h, pooled, centrifuged, membrane-filtered, and from which the metabolites were separated by combined chromatographic procedures or extracted after acidic hydrolysis. Gas chromatographic and GC/MS studies showed that 31.1% of the radioactivity found in urine proved to be picolinyl-piperazine, 3.7% was N-benzyl-piperazine and 28.5% of it was picolinic acid. Hippuric acid accounted for 53.2% of the radioactivity of the acid hydrolyzed urine.     

Extraction of omega- and omega-1-hydroxylauric acids with ethyl acetate results in formation of acetoxy products

In studies of the regiospecific metabolism of lauric acid by monooxygenase enzymes, incubation medium from the reaction of [14C] lauric acid and microsomes is routinely acidified and extracted with ethyl ether. Separation of products by HPLC shows the formation of omega(12)- and omega-1(11)-hydroxylauric acids by rat liver microsomes. In a comparison of several extraction solvents, we observed that the extraction of incubation media with ethyl acetate alone was associated with the appearance of a major new metabolite peak concomitant with loss of the 12- and 11-hydroxylaurate peaks by 62 and 23%, respectively. No difference was observed between extraction procedures in the per cent recovery of radiolabel. Similar results were observed with extraction of lauric acid metabolites formed by fish liver microsomes. Acidification of the incubation media prior to ethyl acetate extraction prevented formation of the new metabolite peak. In experiments to identify the new metabolite(s), 11- and 12-hydroxylaurate products formed by rat liver microsomes were acetylated and found to show an HPLC retention time similar to that of the unknown metabolite peak formed with ethyl acetate extraction. Mass spectrometric analysis further showed that the methyl esters of 11- and 12-acetoxylauric acids were very similar to that of the methyl ester derivative of the unknown metabolite fraction. Thus, data indicate that the acetoxy derivatives of 11- and 12-hydroxylauric acids were formed by direct reaction of ethyl acetate with the laurate products formed by microsomal monooxygenase enzymes. This extraction procedure can produce a major shift in the amount and ratio of omega- and omega-1-hydroxylaurates measured in the incubation media.     

In vivo metabolism of alpha-methyltryptamine in rats: identification of urinary metabolites

1. The in vivo metabolism of alpha-methyltryptamine (AMT), a psychoactive tryptamine analogue, was studied in rats. 2. Male Wistar rats were administered 10 mg kg(-1) AMT orally and 24-h urine fractions were collected. After enzymatic hydrolysis of the urine sample, the metabolites were extracted by liquid-liquid extraction and analysed by gas chromatography/mass spectrometry (GC/MS). 3. 2-Oxo-AMT, 6-hydroxy-AMT, 7-hydroxy-AMT and 1'-hydroxy-AMT were detected as metabolites of AMT.     

Determination of chloral hydrate metabolites in human plasma by gas chromatography-mass spectrometry

Chloral hydrate (CH) is a widely used sedative. Its pharmacological and toxicological effects are directly related to its metabolism. Prior investigations of CH metabolism have been limited by the lack of analytical techniques sufficiently sensitive to identify and quantify metabolites of CH in biological fluids. In this study a gas chromatography mass spectrometry (GC/MS) method was developed and validated for determining CH and its metabolites, monochloroacetate (MCA), dichloroacetate (DCA), trichloroacetate (TCA) and total trichloroethanol (free and glucuronidated form, TCE and TCE-Glu) in human plasma. Of these, DCA and MCA are newly identified metabolites in humans. The drug, its plasma metabolites and an internal standard, 4-chlorobutyric acid (CBA), were derivatized to their methyl esters by reacting with 12% boron trifluoride-methanol complex (12% BF3-MeOH). The reaction mixture was extracted with methylene chloride and analyzed by GC/MS, using a selected ion monitoring (SIM) mode. The quantitation limits of MCA, DCA, TCA, and TCE were between 0.12 and 7.83 microM. The coefficients of variation were between 0.58 and 14.58% and the bias values ranged between -10.03 and 14.37%. The coefficients of linear regression were between 0.9970 and 0.9996.     

Sustainable production of biodiesel from microalgae by direct transesterification

This study addresses the question of the sustainable production of biodiesel (fatty acid ethyl esters) by direct transesterification from Chlorella sp. The lipid fractions from Chlorella sp. using chloroform: methanol, methanol, and ethanol were obtained and the acid value measurements were 39.4, 58.2, and 82.5 mg KOH/g, respectively. Following this, transesterification of algal lipid extraction and direct transesterification was undertaken in the presence of ethanol and the results were compared with those for methanol. Apart from the lower yield in extracted lipids obtained with ethanol, when ethanol was used for transesterification of the algal lipid extraction, surprisingly, similar yields of fatty acid ethyl esters and fatty acid methyl esters were obtained. Furthermore, direct transesterification from Chlorella sp. provided higher ethyl and methyl esters yields than was the case with the extraction–transesterification process. Ethanol, a renewable feedstock, is the most sustainable alternative, which, compared to methanol in direct transesterification, resulted in similar fatty acid methyl and ethyl ester yields (11.6% and 11.0%, respectively, in the same experimental conditions).     

Metabolism of cannabidiol by the rat

Metabolites of CBD excreted into the bile and perfusion fluid were examined in a rat liver perfusion preparation. Metabolites were extracted with ethyl acetate and identified by GC/MS as TMS derivatives. Four mono- and five di-hydroxy metabolites were identified with major sites of metabolic attack being at C-7 and C-4". A hydroxy-ketone was detected but not fully identified. All biliary metabolites were conjugated with glucuronic acid. Urinary metabolites were studied in rats with samples taken at times to 25 h after drug administration. Unmetabolized CBD and 13 metabolites were identified by GC/MS. Major metabolites were acids with beta-oxidation being a prominent pathway. The 6- and 7-hydroxy derivatives of 4",5"-bis,nor-CBD-3"-oic acid were the most abundant compounds but substantial concentrations of the di-acids, CBD-5",7-dioic acid and 4",5"-bis,nor-CBD-3",7-dioic acid were present. Concentrations of the more highly oxidized metabolites increased with time.     

Biotransformation of [(12)C]- and [(13)C]-tert-amyl methyl ether and tert-amyl alcohol.

tert-Amyl methyl ether (TAME) is intended for use as a gasoline additive to increase oxygen content. Increased oxygen content in gasoline reduces tailpipe emissions of hydrocarbons and carbon monoxide from cars. Due to possible widespread use of TAME, the toxicity of TAME is under investigation. We studied the biotransformation of TAME in rats and one human volunteer after inhalation of (12)C- or (13)C-labeled TAME. In addition, the biotransformation of [(13)C]-tert-amyl alcohol was studied in rats after gavage. Urinary metabolites were identified by GC/MS and (13)C NMR. Rats (two males and two females) were individually exposed to 2000 ppm [(12)C]- or [(13)C]TAME for 6 h, and urine was collected for 48 h. Free and glucuronidated 2-methyl-2,3-butanediol and a glucuronide of tert-amyl alcohol were identified by (13)C NMR, GC/MS, and LC/MS/MS as major urinary metabolites on the basis of the relative intensities of the (13)C NMR signals. The presence of several minor metabolites was also indicated by (13)C NMR; they were identified as tert-amyl alcohol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid. One human volunteer was exposed to an initial concentration of 27 000 ppm [(13)C]TAME by inhalation for 4 min from a 2 L gas sampling bag, and metabolites of TAME excreted in urine were analyzed by (13)C NMR. All TAME metabolites identified in rats were also present in the human urine samples. To study tert-amyl alcohol biotransformation, male rats (n = 3) were treated with 250 mg/kg [(13)C]-tert-amyl alcohol dissolved in corn oil by gavage, and urine was collected for 48 h. (13)C NMR of the urine samples showed the presence of metabolites identical to those in the urine of [(13)C]TAME-treated rats. Our results suggest that TAME is extensively metabolized by rats and humans to tert-amyl alcohol which may be further oxidized to diols and carboxylic acids. These reactions are likely mediated by cytochrome P450-dependent oxidations.     

Biotransformation of nifedipine in rat and dog.

Following oral and/or intraduodenal administration, the biotransformation of 14C-labelled nifedipine (dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-pyridine-3, 5-dicarboxylate, Bay a 1040, Adalat, CAS 21829-25-4) has been reinvestigated in rats and dogs (dose: 5 mg/kg body weight in both species) to complete the metabolic data. Thirteen metabolites were isolated from the perfusate and bile of the isolated perfused rat liver model. Their structures were elucidated by spectroscopic methods (FAB-MS, combined GC/MS, NMR). The analyzed samples were used for the chromatographic (HPLC) comparison with urine and bile from the in vivo studies. The metabolites identified in rat urine (oral dose) account for 47.4% of the dose administered. 82.8% (rat) and 62.8% (dog) of the dose, resp., could be attributed to known structures in urine and bile following intraduodenal administration. Based on the structures identified the following biotransformation steps occurred: dehydrogenation of the 1,4-dihydropyridine system, hydroxylation of the methyl groups at 2- or 6-position followed by glucuronidation or by subsequent oxidation to the carboxylic acid, and oxidative ester cleavage.     

Isolation and characterization of the urinary metabolites of arbaprostil in the male dog after intravenous administration

The profile of urinary metabolites of 3H-arbaprostil was characterized in the male dog after intravenous administration. The major metabolites were purified and their structures deduced by gas chromatography/mass spectrometry (GC/MS) studies after conversion to the methyl ester-methoxime-trimethylsilyl ether derivatives, aided by GC with simultaneous radioactivity monitoring. The identified metabolites accounted for 96% of the urinary excretion products. beta-Oxidation of the carboxy side-chain of arbaprostil to 15-methyl-2,3,4,5-tetranor PGE1, via the 15-methyl-2,3-dinor PGE2 intermediate, appeared to be the most significant metabolic pathway. In contrast to the rat, the following were observed in the dog: glucuronic acid conjugation of the 15-methyl-2,3,4,5-tetranor PGE, and PGA metabolites; detection of the 15-methyl-2,3-dinor PGE2 intermediate; absence of 19-hydroxyl-15-methyl-2,3,4,5-tetranor PGA, and PGB metabolites; oxidation at C-20; and excretion of some parent drug.     

In vivo metabolism of 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in the rat: identification of urinary metabolites

The in vivo metabolism of 4-bromo-2,5-dimethoxyphenethylamine (2C-B), a ring-substituted psychoactive phenethylamine, in the rat was studied. Male Wistar rats were administered 10 mg/kg of 2C-B hydrochloride orally, and 24 h urine fractions were collected. After enzymatic hydrolysis of the urine samples, the metabolites were extracted by liquid-liquid extraction and analyzed by gas chromatography-mass spectrometry. 2-(4-Bromo-2,5-dimethoxyphenyl)-ethanol, 4-bromo-2,5-dimethoxyphenylacetic acid, 2-(2-hydroxy-4-bromo-5-methoxyphenyl)-ethylamine, 2-(2-methoxy-4-bromo-5-hydroxyphenyl)-ethylamine, 1-acetoamino-2-(2-hydroxy-4-bromo-5-methoxyphenyl)-ethane, and 1-acetoamino-2-(2-methoxy-4-bromo-5-hydroxyphenyl)-ethane were identified as 2C-B metabolites. These findings suggest that at least two metabolic pathways for 2C-B are operative in rats. The first pathway leads to the corresponding aldehyde metabolite by deamination, which is subsequently reduced or oxidized, to give the corresponding alcohol and carboxylic acid metabolites. The second pathway leads to the corresponding 2-O-desmethyl or 5-O-desmethyl metabolites in which the amino group is subsequently acetylated.     

Increased Endobiotic Fatty Acid Methyl Esters Following Exposure to Methanol

Human exposure to methanol is likely to increase in the futuredue to its proposed use as an alternate automobile fuel. Sincealcohols are known to esteri1 the fatty acids in the body andsome of those esterifled esters are toxic, we studied the formationof fatty acid esters of methanol in Long-Evans male rats givena single oral dose of 3.5 g/kg body weight of methanol in saline.Animals given an equal volume of saline served as control. Threerats were euthanized at 1, 3, 6, 12, and 24 hr following thetreatment. Fatty acid methyl esters, extracted from whole blood,liver, pancreas, and brown fat were separated by thin-layerchromatography and quantitated by gas chromatography (GC). Theiridentity was then confirmed by GC-mass spectrometry. Averagelevels as high as 596, 5293, 2239, 1106, 9665, 7728, 562, and2792 µg/g (wet weight basis) of 14:0, 16:0, 16:1, 18:0,18:1, 18:2, 18:3, and 20:4 fatty acid methyl esters, respectively,were found in the pancreas of methanol-treated rats. The averageconcentration of total fatty acid methyl esters was computedto be 4513, 29594, 22871, 18956, 17014, and 9702 µg/gin the pancreas compared to 1.9, 25.4, 36.8, 18.5, 18.9, and14.2 µg/g in the liver at 0, 1, 3, 6, 12, and 24 hr, respectively,following methanol exposure. On dry lipid weight basis, thelevels were significantly higher again in pancreas followedby brown fat and liver. In whole blood, only low levels of 16:0,18:0, and 20:4 fatty acid methyl esters could be detected atall time points. The highest concentration of total fatty acidmethyl esters in the pancreas, liver, and brown fat was detectedat 1, 3, and 24 hr, respectively. Most of the fatty acid methylesters found in the liver and pancreas decreased after 6 hrof methanol exposure. The fatty acid methyl esters of higherconcentrations were 16:0 in the whole blood, 18:0, 18:1, 18:2,and 20:4 in liver, 18:1, and 18:2 in pancreas and 16:0, 18:1,and 18:2 in brown fat. These fatty acid methyl esters were alsodetected in the tissues of control rats indicating their endogenousformation. Significant increase in methylation of the fattyacids during methanol exposure, as found in this study, mayserve as a defense mechanism for preventing available methanolfrom oxidative metabolism to render toxicity. However, the biologicalsignificance of these fatty acid methyl esters is yet to beunderstood.     

Determination of phenolalkylamines, narcotic analgesics, and beta-blockers by gas chromatography/mass spectrometry

An analytical procedure for determination of phenolalkylamines, narcotic analgesics, and beta-blockers in urine by gas chromatography/mass spectrometry (GC/MS) is described. The detection of phenolalkylamines, narcotic analgesics, and beta-blockers is based on acid hydrolysis, liquid-liquid extraction, and selective derivatization. For screening of phenolalkylamines the m/e 179 and 267 ions were monitored by GC/MS. With narcotic analgesics, the extracted ion corresponded to the molecular ion (M+) of the drug and two additional characteristic ions. Beta-blockers were analyzed as the selectively derivatized forms of the parent molecule and its metabolites by GC/MS with selected ion monitoring. The ions monitored for screening of beta-blockers containing an isopropylamine group were m/e 284 and 129 ions. The ion at m/e 86 was monitored to characterize the tert-butylamine group of beta-blockers.     

Synthesis and evaluation of B-, C-, and D-ring-substituted estradiol carboxylic acid esters as locally active estrogens

We have synthesized derivatives of estradiol that are structurally modified to serve as "soft" estrogens and act within a geographically limited area of the body; estrogens without systemic action. We have previously shown with 16alpha-substituted analogues of estradiol that carboxylates proximal to the steroid ring neither bind to the estrogen receptor nor activate estrogen-responsive genes. However, when the carboxylic acid is masked as an ester, they bind to the receptor and stimulate estrogenic responses. Enzymatic hydrolysis through nonspecific esterases can inactivate these estrogens and thereby limit their area of action. Here, we describe our continued studies to design "soft" estrogens by synthesizing carboxylic acid esters of estradiol at the 7alpha-, 11beta-, and 15alpha-positions in the steroid nucleus at which bulky substituents are accommodated by the estrogen receptor. These compounds were tested for estrogen receptor binding (estrogen receptors alpha and beta), stimulation of an estrogen sensitive gene in Ishikawa cells in culture, and as substrates for enzymatic hydrolysis. Likely candidates were tested in in vivo assays for systemic and local estrogenic action. The biological studies showed that regardless of the point of attachment, all of the short-chain carboxylic acids, C-1 to C-3, were devoid of hormonal action, while many of the esters were estrogenic. The site on the steroid nucleus had great influence on hormonal activity and esterase hydrolysis. Formate esters at 7alpha and 15alpha were good estrogens, but lengthening the chain to acetate dramatically decreased hormonal activity. However, the 7alpha-formate esters were not enzymatically hydrolyzed. At 11beta, the acetate (methyl ester) was an effective estrogen, but increasing the chain length to propionate dramatically reduced hormonal activity. In general, the length of the alcohol from methyl to butyl had only a small effect on receptor binding, and as the size of the alcohol increased, so did esterase hydrolysis. One exception was the 11beta-acetate esters where increasing the alcohol moiety from methyl to ethyl eliminated estrogenic activity (Ishikawa cells) without affecting estrogen receptor binding. Several of the esters were tested in vivo, and two, the methyl and ethyl esters of estradiol-15alpha-formate, appeared to have the requisite properties (high local and low systemic activity) of superior "soft" estrogens.