Intact Esters Research Articles

Detection of Over 110 Anabolic-Androgenic Steroids, Corticosteroids and/or Their Esters in Horse Hair Using Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry and Gas Chromatography-Tandem Mass Spectrometry After Solid-Supported Liquid Extraction.

Anabolic-androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their duration of action. As such, the detection of intact esters of endogenous steroids in hair is particularly advantageous, as it can provide unequivocal proof of their external origin. Compared with urine and blood matrices, hair in general allows a longer detection window as long as the administered drug can be incorporated into hair and has not degraded. This can be useful in doping control of equine sports, where the timing of sample collection may be critical due to the transient nature of AAS in other biological fluids. This paper describes the detection of AAS and/or their esters, as well as corticosteroids, in horse hair using ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) after solid-supported liquid extraction (SLE) at basic pH. The developed UPLC-HRMS and GC-MS/MS methods in combination allowed the detection of 117 AAS, corticosteroids and/or their esters with estimated limits of detection down to sub-ppb levels and with good inter-day precision. Method applicability has been demonstrated through the detections of (i) boldenone undecylenate, nandrolone phenylpropionate and trenbolone acetate in three proficiency testing hair samples and (ii) nandrolone as a metabolite in a post-administration hair sample collected from a castrated horse having been administered nandrolone decanoate.

New approach to derivatisation for oestradiol esters detection in animal blood plasma using negative chemical ionisation GC-MS

ABSTRACT In 1996, the EU prohibited the use of substances with anabolic action for food-producing animals (EU Directive 96/22/EC). In cases of illegal use of steroid hormones, these substances are usually applied to the animals in the form of esters. The reliable determination of intact steroid esters in animal tissues or body fluids is an unequivocal proof of illegal treatment of animals with EU prohibited anabolic substances. Previously our laboratory developed a sensitive method for determination of oestradiol benzoate and other steroid esters in blood plasma using LC-MS/MS, validated according to Commission Decision 2002/657/EC. This study describes a GC-MS method which has been developed for five oestradiol esters in blood plasma. The sample preparation procedure consisted of protein precipitation, phospholipids removal and cleaning on an alumina column. Oestradiol esters were derivatised with 2, 3, 4, 5, 6-pentafluorobenzoyl chloride (PFBCl) and pyridine in dichloromethane. The measurement of oestradiol esters was carried out by GC-MS/NCI with Cool On-Column injection. Methane was used as a negative chemical ionisation reagent gas. The method for determination of oestradiol esters in blood plasma has been validated according to Commission Decision 2002/657/EC. Decision limits for all analytes were observed below 0.05 ng mL−1. The method is robust for bovine and porcine plasma analyses and can be applied both for screening and confirmatory determination in routine residue monitoring.

Determination of Steroids in Bovine Serum: Validation of a Reliable LC-MS/MS Method and In Vivo Studies with Boldenone Undecylenate and Testosterone Propionate.

Serum analysis has received much attention in regulatory analysis of food-producing animals, especially for anabolic steroids. The possibility of confirming the parent drugs with minimum metabolization enables the detection of intact steroid esters, whose identification represents unequivocal proof of drug administration. This work involved the development and validation of a quantitative LC-MS/MS method to determine 30 steroids and steroid esters in bovine serum. Sensitivity was improved using microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was successfully conducted in accordance with the Decision 657/2002/EC guidelines. An in vivo experiment was performed on 12 crossbred steers in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections. The samples were collected over a period of 120 days, in which both intact esters were identified within 11 days postadministration. 17β-Boldenone was observed after 92 days for 2 steers and 56 days for the other animals. The applicability of a cut-off level to the ratio between 17β-testosterone and epitestosterone was evaluated in an attempt to differentiate testosterone abuse from endogenous production. It could be observed that a calculated ratio above this level is strong evidence of drug administration, although a high false-negative rate was obtained.

Impact of thermooxidation of phytosteryl and phytostanyl fatty acid esters on cholesterol micellarization in vitro

The effects of thermooxidation of a phytosteryl/-stanyl and a phytostanyl fatty acid ester mixture on cholesterol micellarization were investigated using an in vitro digestion model simulating enzymatic hydrolysis by cholesterol esterase and subsequent competition of the liberated phytosterols/-stanols with cholesterol for incorporation into mixed micelles. As a first step, relationships between different doses of the ester mixtures and the resulting micellarized cholesterol were established. Subsequent subjection of the thermooxidized ester mixtures to the in vitro digestion model resulted in three principal observations: (i) thermal treatment of the ester mixtures led to substantial decreases of the intact esters, (ii) in vitro digestion of cholesterol in the presence of the thermooxidized ester mixtures resulted in significant increases of cholesterol micellarization, and (iii) the extents of the observed effects on cholesterol micellarization were strongly associated to the remaining contents of intact esters. The loss of efficacy to inhibit cholesterol micellarization due to thermally induced losses of intact esters corresponded to a loss of efficacy that would have been induced by an actual removal of these amounts of esters prior to the in vitro digestion. The obtained results suggest that in particular oxidative modifications of the fatty acid moieties might be responsible for the observed increases of cholesterol micellarization.

Occurrence of 3-monochloropropanediol esters and glycidyl esters in commercial infant formulas in the United States

ABSTRACTThis work presents occurrence data for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD) and glycidol in 98 infant formula samples purchased in the United States. These contaminants are considered potentially carcinogenic and/or genotoxic, making their presence in refined oils and foods a potential health risk. Recently, attention has focused on methodology to quantify MCPD and glycidyl esters in infant formula for risk-assessment purposes. Occurrence data for 3-MCPD and glycidyl esters were produced using a procedure for extracting fat from infant formula and an LC-MS/MS method for analysing fat extracts for intact esters. Infant formulas were produced by seven manufacturers, five of which use palm oil and/or palm olein in their formulations. In formulas containing palm/palm olein, concentrations for bound 3-MCPD and glycidol ranged from 0.021 to 0.92 mg kg–1 (ppm) and from < LOQ to 0.40 mg kg–1 (ppm), respectively. Formulas not containing palm/palm olein, bound 3-MCPD and glycidol concentrations ranged from 0.072 to 0.16 mg kg–1 (ppm) and from 0.005 to 0.15 mg kg–1 (ppm), respectively. Although formulas from manufacturers A and G did not contain palm/palm olein, formulas from manufacturer E (containing palm olein) had the lowest concentrations of bound 3-MCPD and glycidol, demonstrating the effectiveness of industrial mitigation strategies.

Use of dried blood spots in doping control analysis of anabolic steroid esters

Dried blood spot (DBS) sampling, a technique for whole blood sampling on a piece of filter paper, has more than 50-years tradition, particularly in the diagnostic analysis of metabolic disorders in neonatal screening. Due to the minimal invasiveness, straightforwardness, robustness against manipulation and fastness DBS sampling recommends itself as an advantageous technique in doping control analysis. The present approach highlights the development of a screening assay for the analysis of eight anabolic steroid esters (nandrolone phenylpropionate, trenbolone enanthate, testosterone acetate, testosterone cypionate, testosterone isocaproate, testosterone phenylpropionate, testosterone decanoate and testosterone undecanoate) and nandrolone in DBS. The detection of the intact esters allows an unequivocal proof of the administration of conjugates of exogenous testosterone and its derivatives. Precise, specific and linear conditions were obtained by means of liquid chromatography high resolution/high accuracy mass spectrometry. Sensitivity in the low ppb range was accomplished by the preparation of the methyloxime derivatives of the target compounds. Labeled internal standards (d3-nandrolone, d3-nandrolone caproate and d3-nandrolone undecanoate) were applied to compensate for the broad range in chain length of the esters.The assay presented here outlines the application of DBS for the analysis of anabolic steroid esters in doping controls for the first time providing great potential to simplify the proof of exogenous administration of testosterone.

Occurrence of 3-MCPD and glycidyl esters in edible oils in the United States

Fatty acid esters of 3-monochloropropanediol (3-MCPD) and glycidol are processing contaminants found in a wide range of edible oils. While both 3 MCPD and glycidol have toxicological properties that at present has concerns for food safety, the published occurrence data are limited. Occurrence information is presented for the concentrations of 3-MCPD and glycidyl esters in 116 retail and/or industrial edible oils and fats using LC-MS/MS analysis of intact esters. The concentrations for bound 3-MCPD ranged from below the limit of quantitation (<LOQ) to 0.09 mg kg−1 (ppm) in 22 unrefined oils and from 0.005 to 7.2 mg kg−1 (ppm) in 94 refined oils. The concentrations for bound glycidol ranged from <LOQ to 0.03 mg kg−1 (ppm) in unrefined oil samples and from <LOQ to 10.5 mg kg−1 (ppm) in processed oil samples. The highest concentrations for both 3-MCPD and glycidol were seen in refined palm oil and palm olein samples. Palm olein samples also contained a higher percentage of 3-MCPD in mono-ester form than any other type of oil.

Inverse antagonist activities of parabens on human oestrogen-related receptor γ (ERRγ): In vitro and in silico studies

Parabens are p-hydroxybenzoic acid esters that have been used extensively as preservatives in foods, cosmetics, drugs and toiletries. These intact esters are commonly detected in human breast cancer tissues and other human samples, thus arousing concern about the involvement of parabens in human breast cancer. In this study, an in vitro nuclear receptor coactivator recruiting assay was developed and used to evaluate the binding activities of parabens, salicylates and benzoates via antagonist competitive binding on the human oestrogen-related receptor γ (ERRγ), which is known as both a diagnostic biomarker and a treatment target of breast cancer. The results showed that all of the test parabens (methyl-, ethyl-, propyl-, butyl- and benzylparaben) possessed clear inverse antagonist activities on ERRγ, with a lowest observed effect level (LOEL) of 10−7M and the 50% relative effective concentrations (REC50) varying from 3.09×10−7 to 5.88×10−7M, whereas the salicylates possessed much lower activities and the benzoates showed no obvious activity. In silico molecular docking analyses showed that parabens fitted well into the active site of ERRγ, with hydrogen bonds forming between the p-hydroxyl group of parabens and the Glu275/Arg316 of ERRγ. As the paraben levels reported in breast cancer tissues are commonly higher than the LOELs observed in this study, parabens may play some role via ERRγ in the carcinogenesis of human breast cancer. In addition, parabens may have significant effects on breast cancer patients who are taking tamoxifen, as ERRγ is regarded as a treatment target for tamoxifen.

Fate of dietary phytosteryl/-stanyl esters: analysis of individual intact esters in human feces

The objective was to investigate the metabolic fate of phytosteryl/-stanyl fatty acid and ferulic acid esters upon consumption by healthy humans. A capillary gas chromatographic methodology was employed to follow a randomized, single-blind three group crossover clinical trial and to quantify simultaneously individual intact esters, liberated phytosterols/-stanols and their metabolites in feces. Skimmed milk drinking yogurts enriched with complex mixtures of phytosteryl/-stanyl fatty acid esters and ferulates, respectively, were employed as food carriers. On average, 73 % of total plant stanyl fatty acid esters and 80 % of total plant steryl fatty acid esters were hydrolyzed. Among the individuals, the hydrolysis rates ranged from 40 to 96 %. In addition, there were subject-dependent discrepancies between the amounts of phytosterols/-stanols actually determined in the feces and the calculated hydrolysis rates. On average, 69 % of the amounts of sterols/stanols expected from the amounts of remaining intact esters were found. The study revealed large interindividual variability regarding the recoveries of dietary phytosteryl/-stanyl esters upon gastrointestinal passage in healthy humans. Nevertheless, there was a significant impact of the acid moiety (oleate=linoleate=linolenate>eicosanoate>palmitate>ferulate) on the hydrolysis rates; the influence of the phytosterol/-stanol moiety was less pronounced.

Parabens enable suspension growth of MCF‐10A immortalized, non‐transformed human breast epithelial cells

Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben resulted in a greater number of colonies per dish (P < 0.05 in each case) and an increased average colony size (P < 0.001 in each case). Dose-responses showed that concentrations as low as 10(-6) M methylparaben, 10(-7) M n-propylparaben and 10(-7) M n-butylparaben could increase colony numbers (P = 0.016, P = 0.010, P = 0.008, respectively): comparison with a recent measurement of paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis.

Parabens detection in different zones of the human breast: consideration of source and implications of findings

This article is a discussion of the recent study by Barr, Metaxas, Harbach, Savoy and Darbre (2012; J. Appl. Toxicol. 32; doi: 10.1002/jat.1786) reporting residues of five paraben esters in the human breast, at concentrations up to the microgram per gram tissue range and with highest concentrations in the axilla area (closest to the underarm). The conclusion is that the detection of intact esters that have escaped the action of esterases is consistent with a local (dermal) exposure source since the metabolic capacity of the gut and liver would produce p-hydroxybenzoic acid as the common metabolite. Whereas the zone concentration differences (propylparaben was found at highest concentrations in the axilla) support an underarm exposure model, seven subjects reportedly never used underarm cosmetics, and other exposure sources, including other cosmetic product types, are discussed. The findings are placed into context with the limited regulatory toxicology database on parabens, oestrogenic action of the parabens, and status of the parabens, cosmetics and human health debate.

Analysis of methyloxime derivatives of intact esters of testosterone and boldenone in equine plasma using ultra high performance liquid chromatography tandem mass spectrometry

Analysis of equine plasma samples to detect the abuse of anabolic steroids can be complicated when the parent steroid is endogenous to the animal. Anabolic steroids are usually administered intramuscularly as synthetic esters and therefore detection of the exogenous esters provides unequivocal proof of illegal administration. An ultra high performance liquid chromatography tandem mass spectrometric (UPLC-MSMS) method for the analysis of esters of testosterone (propionate, phenylpropionate, isocaproate, and decanoate) and boldenone (undecylenate) in equine plasma has been developed. Esters were extracted from equine plasma using a mixture of hexane and ethyl acetate and treated with methoxyamine hydrochloride to form methyloxime derivatives. Metenolone enanthate was used as an internal standard. After chromatographic separation, the derivatized steroid esters were quantified using selected reaction monitoring (SRM). The limit of detection for all of the steroid esters, based on a signal to noise ratio (S/N) of 3:1, was 1-3 pg/mL. The lower limit of quantification (LLOQ) for the all of the steroid esters was 5 pg/mL when 2 mL of plasma was extracted. Recovery of the steroid esters was 85-97% for all esters except for testosterone decanoate which was recovered at 62%. The intra-day coefficient of variation (CV) for the analysis of plasma quality control (QC) samples was less than 9.2% at 40 pg/mL and less than 6.0% at 400 pg/mL. The developed assay was used to successfully confirm the presence of intact testosterone esters in equine plasma samples following intramuscular injection of Durateston® (mixed testosterone esters).

Feasibility of desorption electrospray ionization mass spectrometry for rapid screening of anabolic steroid esters in hair

Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionization (DESI) mass spectrometry (MS). In this work the feasibility of DESI application for the rapid screening of intact esters of anabolic steroids in bovine hair has been studied. Using a linear ion trap both full scan and data-dependent collision induced dissociation MS n spectra were acquired in minutes for testosterone cypionate, testosterone decanoate and estradiol benzoate standard solutions deposited on a glass or PTFE surface. However direct analysis of incurred hair failed due to inefficient desorption ionization and the minute quantities of steroid esters present. Therefore a simplified ultrasonic liquid extraction procedure was developed, allowing rapid DESI analysis of a few microliters of the concentrate and a total analysis time of 2–4 h per batch instead of 3 days. The potential of this DESI approach is clearly demonstrated by MS 3 data from hair samples incurred with high levels (300–800 μg kg −1) of steroid esters, levels which do occur in samples from controlled- and illegally treated animals. For much lower levels state-of-the-art ultra high performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) screening methods remain the method of choice and might benefit from the proposed simplified extraction as well.

Phytosterol Ester Constituents Affect Micellar Cholesterol Solubility in Model Bile

Plant sterols and stanols (phytosterols) and their esters are nutraceuticals that lower LDL cholesterol, but the mechanisms of action are not fully understood. We hypothesized that intact esters and simulated hydrolysis products of esters (phytosterols and fatty acids in equal ratios) would differentially affect the solubility of cholesterol in model bile mixed micelles in vitro. Sodium salts of glycine- and taurine-conjugated bile acids were sonicated with phosphatidylcholine and either sterol esters or combinations of sterols and fatty acids to determine the amount of cholesterol solubilized into micelles. Intact sterol esters did not solubilize into micelles, nor did they alter cholesterol solubility. However, free sterols and fatty acids altered cholesterol solubility independently (no interaction effect). Equal contents of cholesterol and either campesterol, stigmasterol, sitosterol, or stigmastanol (sitostanol) decreased cholesterol solubility in micelles by approximately 50% compared to no phytosterol present, with stigmasterol performing slightly better than sitosterol. Phytosterols competed with cholesterol in a dose-dependent manner, demonstrating a 1:1 M substitution of phytosterol for cholesterol in micelle preparations. Unsaturated fatty acids increased the micelle solubility of sterols as compared with saturated or no fatty acids. No differences were detected in the size of the model micelles. Together, these data indicate that stigmasterol combined with saturated fatty acids may be more effective at lowering cholesterol micelle solubility in vivo.

Hydroperoxide Formation of Steryl Ester

Phytosterols and -stanols are added to food products because of their known ability to lower serum cholesterol levels. They are applied either in their free or esterified forms, i.e. as fatty acid esters. Sterols are known to form variety of oxidation products under exposure to heat, light and metal contaminants, for example in food processing conditions. Since these oxides may have adverse health effects, the oxidation process needs to be studied. Until recently, sterol oxidation studies have concentrated on following the formation of secondary oxidation products in free sterol and steryl esters, but little is known about the oxidation of steryl esters as intact molecules. The aim of this experiment was to study primary autoxidation of intact steryl ester by measuring hydroperoxide formation in bulk cholesteryl ester. Cholesteryl linoleate was maintained at 60°C for 0-72 h after which formed hydroperoxides were determined with normal phase high performance liquid chromatography connected to diode array detector (HPLC-DAD). Also peroxide value (PV) was measured to indicate the total amount of formed hydroperoxides. With HPLC method steryl ester -OOH's could be analysed as intact esters, without saponification. A gradient elution was performed with 0.3-10% methyl-tert-butyl ether (MTBE) in heptane followed by cleanup with 30% MTBE. Compounds were detected with DAD at wavelengths 206 nm and 234 nm. Peroxide value indicated that the formation of hydroperoxides reached the maximum after 12 h of prolonged heating. According to HPLC data, at this time point less than 10% of the hydroperoxide groups were located in the sterol moiety and more than 90% in the fatty acid chain. The proportion of sterol-OOH's increased as the heating continued; at 24 h 20% and at 48 h 30%. However, after 72 h no hydroperoxides were observed. In conclusion, oxidation of cholesteryl linoleate started in the fatty acid moiety and as the reaction progressed more of the sterol -OOH's were observed, though at all time points fatty acid -OOH's were dominating.

Paraben esters: review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks

This toxicology update reviews research over the past four years since publication in 2004 of the first measurement of intact esters of p-hydroxybenzoic acid (parabens) in human breast cancer tissues, and the suggestion that their presence in the human body might originate from topical application of bodycare cosmetics. The presence of intact paraben esters in human body tissues has now been confirmed by independent measurements in human urine, and the ability of parabens to penetrate human skin intact without breakdown by esterases and to be absorbed systemically has been demonstrated through studies not only in vitro but also in vivo using healthy human subjects. Using a wide variety of assay systems in vitro and in vivo, the oestrogen agonist properties of parabens together with their common metabolite (p-hydroxybenzoic acid) have been extensively documented, and, in addition, the parabens have now also been shown to possess androgen antagonist activity, to act as inhibitors of sulfotransferase enzymes and to possess genotoxic activity. With the continued use of parabens in the majority of bodycare cosmetics, there is a need to carry out detailed evaluation of the potential for parabens, together with other oestrogenic and genotoxic co-formulants of bodycare cosmetics, to increase female breast cancer incidence, to interfere with male reproductive functions and to influence development of malignant melanoma which has also recently been shown to be influenced by oestrogenic stimulation.

Substituted aminoalcohol ester analogs of indomethacin with reduced toxic effects

Synthesis and evaluation of five different N,N-disubstituted aminoethanol ester derivatives of indomethacin bearing structural resemblance to the aminoethanol ester class of anticholinergics are reported herein. The anticholinergic activity was incorporated into the intact esters to overcome the gastric toxicity of indomethacin, not only by blocking the acidic functionality but also by decreasing gastric secretions and motility. These derivatives exhibited in vitro stability in buffers of pH 2.0 and 7.4 for 6 h and were readily hydrolyzed by human plasma esterases to liberate the parent drug. All the derivatives were significantly less irritating to the gastric mucosa than the parent drug. Though only two esters showed antiinflammatory activity similar to that of the parent drug at equivalent dose levels, all the esters were equipotent to indomethacin in the mouse acetic acid–induced writhing assay for analgesic action. The present evaluation indicates that the combined pharmacological properties of these ester derivatives may prove useful for design and development of novel gastric sparing antiinflammatory molecules with potentially important therapeutic applications.

Estimation of sucrose esters (E473) in foods using gas chromatography-mass spectrometry

A method was developed for estimating the content in foods of the emulsifying additive E473, sucrose esters of fatty acids. The analytical approach taken to estimate the complex mixtures that comprise this additive involved, selective solvent extraction of the intact esters using a mixture of tetrahydrofuran and ethyl acetate, alkaline hydrolysis of the esters to liberate sucrose, and then GC-MS measurement of the liberated sucrose using GC-MS after acidic hydrolysis to glucose and fructose and then silylation. The method was developed to aid future estimates of intake of this food additive. The method determines the total sucrose esters content of a food sample and does not attempt to discrimination between individual sucrose esters when present as a mixture in a food sample. A single (average) factor is used to convert the liberated sucrose content into sucrose ester content. The method was applied to analysis of eight different food types (including bakery wares, sugar confectionery, dairy product, margarine, meat pies and a sauce) spiked with 0.5–1% of a mixture of three sucrose esters that spanned the hydrophilic/lipophilic balance (HLB) range 1–16. The limit of quantification was around 50 mg kg−1, which is more than adequate for these additives. The analytical recovery rate was 73–106% with an average of 91%. The precision of the method (RSD) was 6–18% (n = 3–20 for each food type) with an average RSD of 11%. The main analytical uncertainty is the conversion factor used to express sucrose ester content from the amount of sucrose liberated. The method is also applicable to sucroglycerides (E474).

Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.

Evaluation of the role of intestinal and liver metabolism in the conversion of two different ester prodrugs of sanfetrinem to the parent drug in vitro and in vivo using different rat tissues and a surgically prepared rat model

To improve oral absorption of sanfetrinem, a broad-spectrum, beta-lactamase-stable antibiotic, two different ester prodrugs have been selected. Both prodrugs proved to be readily hydrolyzed after absorption before reaching the systemic circulation. The objective of this study was to evaluate the role of intestinal and liver metabolism in the conversion of the two prodrugs into the active compound. In vitro experiments were performed in different rat tissues involved in the absorption process. Moreover data obtained with in vitro experiments have been integrated with data obtained in vivo using a surgically prepared rat model which allows for the measurement of the amount of intact prodrug that overcomes the intestinal mucosa and its presence in the portal vein. Both prodrugs proved to be readily cleaved by jejunum and liver microsomes. The rates of ester hydrolysis with these two tissues were 10- to 30-fold higher than those calculated in intestinal juice at pH 7.4 and about 100-fold higher than in buffer at pH 5.5. These data suggest that both the intestinal wall and liver could play an important role in the conversion of the two prodrugs in active parent compound. In the in vivo experiment, relative to sanfetrinem levels, very low concentrations of intact esters were measured in the portal vein blood, indicating that the two prodrugs are nearly completely hydrolyzed to the active drug by the intestinal wall. In conclusion this study demonstrated that the intestinal epithelium plays a major role in the conversion of the two prodrugs into sanfetrinem. The liver, despite its high esterase activity seems to be only marginally involved.