A column switching technique for the determination of lidocaine and its metabolites in horse urine

Summary A rapid method is described for the determination of lidocaine and its metabolites in horse urine using a column switching technique and HPLC analysis. This procedure offers a sensitive assay without the need for time consuming extractions.     

沉淀双点滴定法测定盐酸左旋咪唑

以Ag^＋作滴定剂,分别用双点滴定法和传统电位滴定法对盐酸左旋咪唑片进行了测定。试液的配制采用了过滤和不过滤两种方法。测定结果表明：所有方法测得的结果均在《药典》允许的范围之内;本法测定结果与《药典》规定方法的对照偏差约为2%-3%;采用过滤和不过滤两种方法配制试液的测定结果之间的偏差约为1%。     

Electromembrane extraction of levamisole from human biological fluids

Electromembrane extraction coupled with high-performance liquid chromatography (HPLC) and ultraviolet (UV) detection was developed for the determination of levamisole in some human biological fluids. Levamisole migrated from 4 mL of different acidized biological matrices, through a thin layer of 2-nitrophenyl octyl ether containing 5% tris-(2-ethylhexyl) phosphate immobilized in the pores of a porous hollow fiber, into a 20-μL acidic aqueous acceptor solution present inside the lumen of the fiber. The parameters influencing electromigration were investigated and optimized. Within 15 min of operation at 200 V, levamisole was extracted from different biological fluid samples with recoveries in the range of 59-65%, which corresponded to preconcentration factors in the range of 118-130. The calibration curves showed linearity in the range of 0.5-10, 0.2-10 and 0.1-10 μg/mL for plasma, urine and saliva, respectively. Limits of detection of 0.1, 0.07 and 0.05 μg/mL and limits of quantification of 0.5, 0.2 and 0.1 μg/mL were obtained for plasma, urine and saliva, respectively. The relative standard deviations of the analysis were found to be in the range of 5.6-9.7% (n = 3). Electromembrane extraction was successfully processed for determination of levamisole in plasma, urine and saliva samples.     

Metabolic studies of methenolone acetate in horses

Methenolone acetate (17β-acetoxy-1-methyl-5α-androst-1-en-3-one), a synthetic anabolic steroid, is frequently abused in human sports. It is preferred for its therapeutic efficiency and lower hepatic toxicity compared with its 17α-alkylated analogs. As with other anabolic steroids, methenolone acetate may be used to enhance performance in racehorses. Metabolic studies on methenolone acetate have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the investigation of in vitro and in vivo metabolism of methenolone acetate in racehorses.Studies on the in vitro biotransformation of methenolone acetate with horse liver microsomes were carried out. Methenolone (M1, 1-methyl-5α-androst-1-en-17β-ol-3-one) and seven other metabolites (M2-M8) were detected in vitro. They were 1-methyl-5α-androst-1-ene-3,17-dione (M2), 1-methyl-5α-androst-1-en-6-ol-3,17-dione (M3) and two stereoisomers of 1-methylen-5α-androstan-2-ol-3,17-dione (M4 and M5), 1-methyl-5α-androst-1-en-16-ol-3,17-dione (M6) and monohydroxylated 1-methyl-5α-androst-1-en-17-ol-3-one (M7 and M8). After oral administration of Primobolan^® (80 tablets × 5 mg of methenolone acetate each) to two thoroughbred geldings, the parent steroid ester was not detected in the post-administration urine samples. However, seven metabolites, namely M1, M6-M8, two stereoisomers of M7 (M9 and M10) and 1-methyl-5α-androst-1-en-17α-ol-3-one (M11), could be detected. The metabolic pathway for methenolone acetate is postulated. This study has shown that metabolite M1 could be targeted for controlling the abuse of methenolone acetate in horses.     

Detection of testosterone,nandrolone and precursors in horse hair

Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the hair strand or segment with methanol/water (1:1), milling, extraction of the hair material in an ultrasonic bath using methanol, purification by liquid–liquid extraction (n-pentane/methanol, 25:1) and HPLC cleanup, derivatisation of the relevant LC fractions with MSTFA, and measurement using GC-MS/MS technique. The first objective of our study was the detection of exogenous nandrolone (nortestosterone, NT) in the horse hair; therefore nandrolone-associated compounds [nandrolone dodecanoate administered intramuscularly (i.m.) and a mixture of 4-estrenediol and 4-estrenedione, transdermal] were administered to four geldings. The highest concentrations of NT following i.m. treatment were measured after 10 days in a 2-cm hair segment (up to 18 pg/mg); NT was detectable for up to 120 days and in some cases up to 330 days in tail hair (limit of detection 0.3 pg/mg). Following transdermal application, nandrolone as well as the administered prohormones were identified in tail and mane until the latest sampling at 3 months. Furthermore, untreated stallions (128) were investigated to estimate the range of endogenous levels of NT and testosterone (T) in hair. Maximum values of 3 pg/mg (NT) and 1 pg/mg (T) were quantified originating from endogenous formation in the male horse. Additionally, a possible relationship between steroid concentrations in hair specimens and the age of stallions was appraised. NT and T were not detected in hair samples of control geldings. Following nandrolone treatment of geldings, highest values in hair exceeded the endogenous amount detected in untreated stallions. Therefore comparison of concentrations measured in control samples with the estimated endogenous levels could give a clue to exogenous application in cases of abnormally high amounts of NT or T. The possibility of the evaluation of threshold values is discussed as a means to verify an exogenous administration of NT and T in hair samples. Furthermore, the detection of a synthetic substance in hair, e. g. the parent steroid ester by itself, would be unequivocal proof of an exogenous origin of NT or T and the previous medication of the stallion.     

Determination of benzimidazoles and levamisole residues in milk by liquid chromatography–mass spectrometry: Screening method development and validation

The screening method for the determination of residues of 19 benzimidazoles (parent drugs and their metabolites) and levamisole in bovine milk has been developed and validated. Milk samples were extracted with ethyl acetate, sample extracts were cleaned up by liquid–liquid partitioning with hexane and acidic ethanol. Liquid chromatography–single-quadrupole mass spectrometry was used for the separation and determination of analytes. The method was validated in bovine milk, according to the CD 2002/657/EC criteria. An alternative approach to the validation of the method was applied (“sum MRL” substances). The method was successfully verified in CRL proficiency test.     

First mass spectrometric detection of boldenone in horse mane samples

Summary In order to investigate the detection of boldenone in horse mane samples, a boldenone study was conducted on two horses. The analytical procedure consisted in a hydrolysis using the Sorensen buffer, a liquid-liquid extraction using diethyl ether and a PFPA derivatization. The instrumental method was a gas chromatography sequential mass spectrometry performed on an ion trap instrument in full scan mode. The limit of detection was estimated to 1 pg mg⁻¹. The detection of boldenone in the mane was made possible for up to 12 months after administration.     

High-performance liquid chromatographic-UV detection analysis of ceftiofur and its active metabolite desfuroylceftiofur in horse plasma and synovial fluid after regional intravenous perfusion and systemic intravenous injection of ceftiofur sodium

A method was optimalised for the quantitative determination of ceftiofur and its active metabolite desfuroylceftiofur in horse plasma and synovial fluid.The principle of the method was that bound desfuroylceftiofur is first released by dithioerythritol, a reducing agent, followed by the derivatization of the free sulfhydryl group with iodoacetamide. The stable derivative—desfuroylceftiofuracetamide—was then further purified using an Oasis HLB solid-phase extraction column. Chromatography was performed using a PLRP-S polymeric column (100 Å, d_p: 5 μm,  mm i.d.), with a mixture of 0.1% trifluoro acetic acid in water and acetonitrile as the mobile phase. Gradient elution was performed. The flow-rate was 0.4 ml/min and the UV detector was set at a wavelength of 266 nm. The method was validated in plasma and synovial fluid (linearity, precision, trueness, LOQ, LOD, specificity, susceptibility to interferences). Calibration graphs were prepared over a concentration range of 0-20 μg/ml and good linearity was achieved (r≥0.99, g≤10%). A limit of quantification of 0.5 μg/ml was obtained for ceftiofur in both matrices. Limits of detection were 0.36 and 0.27 μg/ml for ceftiofur in plasma and synovial fluid, respectively. The results of the within-run and between-run precision and the trueness fell within the ranges specified.The main advantage of our method, compared to previously reported methods, was that the sample preparation procedure was less time consuming, resulting in a higher sample throughput (up to 40 samples a day). In addition, the analysis cost was reduced due to the consumption of a lower amount of solvents and reagents and of only one solid-phase extraction column per sample. The method was successfully applied during a pharmacokinetic study in horses after the administration of ceftiofur sodium via regional intravenous perfusion and systemic intravenous injection.     

Glass transition and state diagram for freeze-dried horse mackerel muscle

Glass transition temperatures of freeze-dried horse mackerel muscle conditioned at various water activities at 25 °C were determined by differential scanning calorimetry (DSC). High moisture content (>0.33 g/g, d.b.) samples obtained by adding liquid water into freeze-dried samples, were also analyzed. The state diagram was composed of the freezing curve and the glass transition line, which were fitted according to Clausius–Clapeyron model and Gordon–Taylor model, respectively. The state diagram yielded maximally freeze-concentrated solutes at 0.786 solids with the characteristic temperature of glass formation being −83.1 °C. The state diagram of horse mackerel muscle developed in this work could be used to predict the stability during frozen storage and in dried conditions as well as in designing drying and freezing processes.     

Sample preparation for metalloprotein analysis: A case study using horse chestnuts

In the present work, 11 different procedures for protein and metalloprotein extraction from horse chestnuts (Aescullus hippocastanum L.) in natura were tested. After each extraction, total protein was determined and, after protein separation through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), those metals belonging to the protein structure were mapped by synchrotron radiation X-ray fluorescence (SRXRF). After mapping the elements (Cr, Fe and Mn) in the protein bands (ca. 33 and 23.7 kDa), their concentrations were determined using atomic absorption spectrometry (ET AAS).

Good results were obtained for protein extraction using a combination of grinding and sonication. However, this strategy was not suitable to preserve metal ions in the protein structure. In fact, there was 42% decrease on Mn concentration using this procedure, compared to that performed with sample agitation in water (taken as reference). On the other hand, when grinding and agitation with an extracting buffer was used, there was a 530% increase of Mn concentration, when compared to the reference procedure.

These results indicate agreement between metal identification and determination in proteins as well as the great influence of the extraction procedure (i.e., the sample preparation step) for preserving metals in the protein structures.     

Antibodies conjugated with new highly luminescent Eu and Tb chelates as markers for time resolved immunoassays. Application to simultaneous determination of clenbuterol and free cortisol in horse urine

Highly luminescent Eu³⁺ and Tb³⁺ complexes of 10-[4-(3-isothiocyanatopropoxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane-1,4,7 triacetic acid Eu³⁺ ⊂ 1 and Tb³⁺ ⊂ 1 were conjugated with a goat anti-rabbit IgG and a rabbit anti-mouse IgG, respectively, and applied as markers in a time resolved immunoassay for simultaneous quantitative determination of anabolic compounds clenbuterol (CL) and hydrocortisone (HC). The assay was performed in horse urine, using a monoclonal antibody specific to CL and a rabbit polyclonal antibody specific to the free HC. These lanthanide chelates are very stable and highly luminescent in aqueous solution and allowed to reach 10 μg L⁻¹ and 40 μg L⁻¹ sensitivities for CL and for HC, respectively. Application to the horse urine, that is a very complex matrix, has a considerable interest in the control of illegal use of these compounds.     

Metabolic studies of mesterolone in horses

Mesterolone (1α-methyl-5α-androstan-17β-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration.In vitro biotransformation studies of mesterolone were performed by incubating the steroid with horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after acylation or silylation. Five metabolites (M1-M5) were detected. They were 1α-methyl-5α-androstan-3α-ol-17-one (M1), 1α-methyl-5α-androstan-3β-ol-17-one (M2), 1α-methyl-5α-androstane-3α,17β-diol (M3), 1α-methyl-5α-androstane-3β,17β-diol (M4), and 1α-methyl-5α-androstane-3,17-dione (M5). Of these in vitro metabolites, M1, M3, M4 and M5 were confirmed using authentic reference standards. M2 was tentatively identified by mass spectral comparison to M1.For the in vivo metabolic studies, Proviron^® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that mesterolone was extensively metabolised and the parent drug was not detected in urine. Three metabolites detected in the in vitro studies, namely M1, M2 and M4, were also detected in post-administration urine samples. In addition, two stereoisomers each of 1α-methyl-5α-androstane-3,17α-diol (M6 and M7) and 1α-methyl-5α-androstane-3,16-diol-17-one (M8 and M9), and an 18-hydroxylated metabolite 1α-methyl-5α-androstane-3,18-diol-17-one (M10) were also detected. The metabolic pathway for mesterolone is postulated. These studies have shown that metabolites M8, M9 and M10 could be used as potential screening targets for controlling the misuse of mesterolone in horses.     

Metabolic Studies of Clostebol Acetate in Horses

Clostebol acetate (4-chlorotestosterone acetate) is a synthetic anabolic steroid which may be used to enhance performance in racehorses. Studies on the in vitro biotransformation of clostebol acetate with horse liver microsomes were carried out. Six metabolites (C1 – C6) were detected. They were 4-chlorotestosterone (C1), 4-chloroandrost-4-en-3-ol-17-one (C2), 4-chloroandrost-4-ene-3,17-diol (C3), 4-chloroandrost-4-ene-3,17-dione (C4), 4-chloroandrost-4-en-6-ol-3,17-dione (C5) and 6-hydroxy-4-chlorotestosterone (C6). Clostebol acetate (350 mg) was administered orally to 2 thoroughbred geldings. The parent drug was not detected in post-administration urine, and only three metabolites C1, C3, and 4-chloroandrostane-3,17-diol (C7) were observed. The metabolic pathway for clostebol acetate is postulated. These studies have shown that metabolites C3 and C7 could be used as potential screening targets for controlling the abuse or misuse of clostebol acetate in racehorses.     

Computer simulation of ecdysone metabolism and of the HPLC analysis of the metabolites

Summary Computer simulation of ecdysone metabolism in insects has been done by the a software called HPLC-Metabolexpert®, that served to generate the metabolic pathways of ecdysone in a retrospective manner. Some of the generated metabolites have already been detected, others are to be confirmed. Lists of the applied metabolic transformations, the predicted metabolites and their HPLC elution times are also given.     

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: Applications and constraints

LC coupled to single (LC–MS) and tandem (LC–MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC–MS/MS. Analyses were performed using a previously published LC–MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT–tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their “enhanced” product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.     

Enantioselective capillary electrophoresis provides insight into the phase II metabolism of ketamine and its metabolites in vivo and in vitro

Glucuronidation catalyzed by uridine‐5′‐diphospho‐glucuronosyl‐transferases (UGTs) is the most important reaction in phase II metabolism of drugs and other compounds. O‐glucuronidation is more common than N‐glucuronidation. The anesthetic, analgesic and antidepressive drug ketamine is metabolized in phase I by cytochrome P450 enzymes to norketamine, hydroxynorketamine (HNK) diastereomers and dehydronorketamine (DHNK). Equine urine samples collected two hours after ketamine injection were treated with β‐glucuronidase and analyzed with three enantioselective capillary electrophoresis assays. Concentrations of HNK diastereomers and norketamine were significantly higher in comparison to untreated urine and an increase of ketamine and DHNK levels was found in selected but not all samples. This suggests that O‐glucuronides of HNK and N‐glucuronides of the other compounds are formed in equines. N‐glucuronidation of norketamine was studied in vitro with liver microsomes of different species and the single human enzyme UGT1A4. With equine liver microsomes (ELM) a stereoselective N‐glucuronidation of norketamine was found that compares well to the results obtained with urines collected after ketamine administration. No reaction was observed with canine liver microsomes, human liver microsomes and UGT1A4. Incubation of ketamine and DHNK with ELM did not reveal any glucuronidation. Enantioselective CE is suitable to provide insight into the phase II metabolism of ketamine and its metabolites.     

Quantitation of 17beta-nandrolone metabolites in boar and horse urine by gas chromatography-mass spectrometry

A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-butylmethylether. The dried organic extracts were derivatized with MSTFA:NH4I:2-mercaptoethanol to obtain the TMS derivatives, and were subjected to analysis by gas chromatography mass spectrometry (GC/MS). The procedure was validated in boar and horse urine for the following metabolites: norandrosterone, noretiocholanolone, norepiandrosterone, 5beta-estran-3alpha, 17beta-diol, 5alpha-estran-3beta, 17beta-diol, 5alpha-estran-3beta, 17alpha-diol, 17alpha-nandrolone, 17betaN, 5(10)-estrene-3alpha, 17alpha-diol, 17alpha-estradiol and 17beta-estradiol in the different metabolic fractions. Extraction recoveries were higher than 90% for all analytes in the free fraction, and better than 80% in the glucuronide and sulphate fractions, except for 17alpha-estradiol in the glucuronide fraction (74%), and 5alpha-estran-3beta, 17alpha-diol and 17betaN in the sulphate fraction (close to 70%). Limits of quantitation ranged from 0.05 to 2.1 ng mL(-1) in the free fraction, from 0.3 to 1.7 ng mL(-1) in the glucuronide fraction, and from 0.2 to 2.6 ng mL(-1) in the sulphate fraction. Intra- and inter-assay values for precision, measured as relative standard deviation, and accuracy, measured as relative standard error, were below 15% for most of the analytes and below 25%, for the rest of analytes. The method was applied to the analysis of urine samples collected after administration of 17betaN laureate to boars and horses, and its suitability for the quantitation of the metabolites in the three fractions has been demonstrated.     

A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome

A parallel chiral/achiral LC-MS/MS assay has been developed and validated to measure the plasma and urine concentrations of the enantiomers of ketamine, (R)- and (S)-Ket, in complex regional pain syndrome (CRPS) patients receiving a 5-day continuous infusion of a sub-anesthetic dose of (R,S)-Ket. The method was also validated for the determination of the enantiomers of the Ket metabolites norketamine, (R)- and (S)-norKet and dehydronorketamine, (R)- and (S)-DHNK, as well as the diastereomeric metabolites hydroxynorketamine, (2S,6S)-/(2R,6R)-HNK and two hydroxyketamines, (2S,6S)-HKet and (2S,6R)-Hket. In this method, (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK and the diastereomeric hydroxyl-metabolites were separated and quantified using a C₁₈ stationary phase and the relative enantiomeric concentrations of (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK were determined using an AGP-CSP. The analysis of the results of microsomal incubations of (R)- and (S)-Ket and a plasma and urine sample from a CRPS patient indicated the presence of 10 additional compounds and glucuronides. The data from the analysis of the patient sample also demonstrated that a series of HNK metabolites were the primary metabolites in plasma and (R)- and (S)-DHNK were the major metabolites found in urine. The results suggest that norKet is the initial, but not the primary metabolite and that downstream norKet metabolites play a role in (R,S)-Ket-related pain relief in CRPS patients.