Multivariate optimization of a derivatisation procedure for the simultaneous determination of nine anabolic steroids by gas chromatography coupled with mass spectrometry

The medical commission of the International Olympic Committee forbids the use of anabolic androgenic steroids to improve sporting performances. Nine anabolic steroids (androsterone (A), nandrolone, estradiol, testosterone propionate, nandrolone-17 propionate, dydrogesterone, testosterone, epitestosterone, boldenone) and alpha-cholestane as internal standard were studied by gas chromatography coupled with mass spectrometry (GC/MS). The derivatisation reagent employed for the derivatisation of anabolic steroids was a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and 2-mercaptoethanol (1000:2:6, v/w/v). Trimethylsilyl (TMS) derivatives were obtained. Anabolic steroids can be derivatised into one or two forms, mainly for androsterone into A-monoTMS and A-diTMS. The aim of this study was to research the optimization conditions of the derivatisation process (maximum yield of silylation reaction) of each anabolic steroid into only one form. A two-level factorial Doelhert design was used to determine the influence of different parameters and their interactions on each compound, thanks to response surface methodology. The parameters to be optimized were the reaction time and the temperature. The interaction "temperature-reaction time" is significant and has a positive effect on the improvement of the effectiveness of the derivatisation. Considering the large amount of information, often not convergent, a global desirability function was applied for multi-responses optimization. Thus, the optimized temperature and the reaction time of silylation were 85 degrees C and 24 min, respectively. Several GC/MS analytical parameters were also studied: linearity (regression coefficient upper than 0.99 for each compound, sensibility (range of concentration 0.05-0.30 microg/ml). Confirmatory experiments were applied to check the predicted values and to validate the model. The confirmatory assay responses are relatively close to the responses predicted. We observed satisfactory resolutions by GC/MS and a run lower than 12 min.     

Case Study: Doping Substances in Equestrian Food Supplements

In the course of investigations on equestrian supplemental products for the presence of doping substances, two products were found to contain forbidden substances. As reported earlier a plant extract (Mexican cactus extract) named “Energy 5” contained the anabolic androgenic steroids (AAS) stanozolol, 17β-hydroxy-17α-methyl-5α-androstane-3β-ol (3β,5α-THMT) as well as mestanolone not declared on the label. In the present study, a product called “Super Kalm Paste” was tested. Analysis by gas chromatography - mass spectrometry (GC-MS) revealed that the preparation contained the class I anti-arrhythmics quinine (trade names Kinidin^TM, Durules) and cinchonine. The samples were prepared according to a sample preparation procedure established for anabolic steroids in nutritional supplements for humans. The sample treatment comprised the extraction and purification of the analytes as well as the chemical conversion with N-methyl-N-trimethylsilyl-trifluoracetamide (MSTFA) to yield the trimethylsilyl (TMS)-derivatives. To verify whether the administration of such products could lead to positive doping tests, a pilot excretion study on “Energy 5” was conducted with two geldings, and urine samples were collected. Gas chromatography - high resolution mass spectrometry (GC-HRMS) after solid phase extraction and mixed derivatisation has demonstrated the presence of the stanozolol metabolite 16β-hydroxy-stanozolol in urine samples after “Energy 5” application.     

Thermal stability evaluation of doping compounds before GC-MS analysis by DSC

The Medical Commission of the International Olympic Committee forbids the use of anabolic androgenic steroids, β-agonists, stimulant and narcotic compounds to improve athletic performance. In this work, we evaluated the thermal stability of 17 compounds by the use of the DSC for their potential GC-MS analysis either under free form or under TMS derivative form. In DSC, esterified and unesterified anabolic steroids were characterized by a true melting peak, followed by a large exothermic peak at about 251–316°C due to oxidative degradation. They could be analysed by GC-MS mainly under TMS derivatives. Hydroxylated and unhydroxylated stimulant compounds (xanthines) seemed to be more stable at high temperature. As unhydroxylated xanthines were not silylated with BSTFA - TMCS, their GC analysis would be done under their free forms. TMS derivatisation of albuterol hemisulfate and codeine phosphate is preferable. In our conditions, to analyse by GC-MS all 17 doping compounds in the same GC-MS run, the optimal silylation temperature and best column initial temperature were determined at both 60°C.     

Metabolism of methyltestosterone in the greyhound

Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of methyltestosterone following oral administration to the greyhound. Several metabolites were identified including reduced, mono‐, di‐ and trihydroxylated steroids. The major metabolites observed were 17α‐methyl‐5β‐androstane‐3α‐17β‐diol, 17α‐methyl‐5β‐androstane‐3α,16α,17β‐triol, and a further compound tentatively identified as 17α‐methyl‐5z‐androstane‐6z,17β‐triol. The most abundant of these was the 17α‐methyl‐5β‐androstane‐3α,16α,17β‐triol. This metabolite was identified by comparison with a reference standard synthesised using a Grignard procedure and characterised using trimethylsilyl (TMS) and acetonide‐TMS derivatisation techniques. There did not appear to be any evidence for 16β‐hydroxylation as a phase I metabolic transformation in the greyhound. However, significant quantities of 16α‐hydroxy metabolites were detected. Selective enzymatic hydrolysis procedures indicated that the major metabolites identified were excreted as glucuronic acid conjugates. Metabolic transformations observed in the greyhound have been compared with those of other mammalian species and are discussed here. Copyright © 2009 John Wiley & Sons, Ltd.     

Aggregation of amyloid Aβ(1–40) peptide in perdeuterated 2,2,2‐trifluoroethanol caused by ultrasound sonication

Ultrasound sonication of protein and peptide solutions is routinely used in biochemical, biophysical, pharmaceutical and medical sciences to facilitate and accelerate dissolution of macromolecules in both aqueous and organic solvents. However, the impact of ultrasound waves on folding/unfolding of treated proteins, in particular, on aggregation kinetics of amyloidogenic peptides and proteins is not understood. In this work, effects of ultrasound sonication on the misfolding and aggregation behavior of the Alzheimer's Aβ_(1–40)‐peptide is studied by pulsed‐field gradient (PFG) spin–echo diffusion NMR and UV circular dichroism (CD) spectroscopy. Upon simple dissolution of Aβ_(1–40) in perdeuterated trifluoroethanol, CF₃‐CD₂‐OD (TFE‐d₃), the peptide is present in the solution as a stable monomer adopting α‐helical secondary structural motifs. The self‐diffusion coefficient of Aβ_(1–40) monomers in TFE‐d₃ was measured as 1.35 × 10^?10 m² s^?1, reflecting its monomeric character. However, upon ultrasonic sonication for less than 5 min, considerable populations of Aβ molecules (ca 40%) form large aggregates as reflected in diffusion coefficients smaller than 4.0 × 10^?13 m² s^?1. Sonication for longer times (up to 40 min in total) effectively reduces the fraction of these aggregates in ¹H PFG NMR spectra to ca 25%. Additionally, absorption below 230 nm increased significantly upon sonication treatment, an observation, which also clearly confirms the ongoing aggregation process of Aβ_(1–40) in TFE‐d₃. Surprisingly, upon ultrasound sonication only small changes in the peptide secondary structure were detected by CD: the peptide molecules mainly adopt α‐helical motifs in both monomers and aggregates formed upon sonication. Copyright © 2010 John Wiley & Sons, Ltd.     

Accelerated sample treatment for screening of banned doping substances by GC–MS: ultrasonication versus microwave energy

A comparison between ultrasonication and microwave irradiation as tools to achieve a rapid sample treatment for the analysis of banned doping substances in human urine by means of gas chromatography–mass spectrometry (GC–MS) was performed. The following variables were studied and optimised: (i) time of treatment, (ii) temperature, (iii) microwave power and (iv) ultrasonic amplitude. The results were evaluated and compared with those achieved by the routine method used in the World Anti-Doping Agency (WADA) accredited Antidoping Laboratory of Rome. Only under the effect of the ultrasonic field was it possible to enhance the enzymatic hydrolysis reaction rate of conjugated compounds. Similar reaction yield to the routine method was achieved after 10 min for most compounds. Under microwave irradiation, denaturation of the enzyme occurs for high microwave power. The use of both ultrasonic or microwave energy to improve the reaction rate of the derivatisation of the target compounds with trimethyliodosilane/methyl-N-trimethylsilyltrifluoroacetamide (TMSI/MSTFA/NH₄I/2-mercaptoethanol) was also evaluated. To test the use of the two systems in the acceleration of the reaction with TMSI, a pool of 55 banned substances and/or their metabolites were used. After 3 min of ultrasonication, 34 of the 55 compounds had recoveries similar to those obtained with the classic procedure that lasts for 30 min (Student’s t test, n = 5), 18 increased to higher silylation yields, and for the compounds 13β,17α-diethyl-3α,17β-dihydroxy-5α-gonane (norboletone metabolite 1), metoprolol and metipranolol the same results were obtained increasing the ultrasonication time to 5 min. Similar results were obtained after 3 min of microwave irradiation at 1,200 W. In this case, 30 of the 55 compounds had recoveries similar to the classic procedure (Student’s t test, n = 5) whilst 18 had higher silylation yields. For the compounds 3α-hydroxy-1α-methyl-5α-androstan-17-one (mesterolone metabolite 1), 17α-ethyl-5β-estrane-3α,17β,21-triol (norethandrolone metabolite 1), epioxandrolone, 4-chloro-6β,17β-dihydroxy-17α-methyl-1,4-androstadien-3-one (chlormetandienone metabolite 1), carphedon, esmolol and bambuterol the same results were obtained after 5 min under microwave irradiation.     

Development and validation of a multi-residue method for the detection of a wide range of hormonal anabolic compounds in hair using gas chromatography-tandem mass spectrometry

The monitoring of anabolic steroid residues in hair is undoubtedly one of the most efficient strategies to demonstrate the long-term administration of these molecules in meat production animals. A multi-residue sample preparation procedure was developed and validated for 28 steroids. A 100 mg hair sample was grinded into powder and extracted at 50 degrees C with methanol. After acidic hydrolysis and extraction with ethyl acetate, phenolsteroids, such as estrogens, resorcyclic acid lactones and stilbens in one hand, are separated from androgens and progestagens in the other hand. Solid phase extractions were performed before applying a specific derivatisation for each compound sub-group. Detection and identification were achieved using gas chromatography-tandem mass spectrometry with acquisition in the selected reaction monitoring mode after electron ionisation. The method was validated according to the 2002/657/EC guideline. Decision limits (CCalpha) for main steroids were in the 0.1-10 microg kg(-1) range.     

Ultrasonic energy as a tool in the sample treatment for polymer characterization through matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Different ultrasonic devices including ultrasonic bath with dual frequency, sonoreactor and ultrasonic probe, were tested for their viability in the sample treatment for polymer characterization by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The effect of sonication frequency (35 kHz, 40 kHz and 130 kHz), sonication amplitude, and sonication time on the polymer's number-average molecular weight (M_n) and weight-average molecular weight (M_w) were investigated. The effect of those variables in the molecular mass distribution of three polymer standards, poly(styrene) 2000 Da and 10,000 Da and poly(ethylene glycol) 1000 Da, was evaluated. In addition, the influence of ultrasonic energy on the sample treatment as a function of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) matrix was also studied through two common standard matrices, dithranol and 2,5-dihydroxybenzoic acid. The results obtained show that the ultrasonic bath at 35 kHz is the best option for the purpose of fast sample treatment for polymer characterization. The M_n and M_w values obtained for this ultrasonic device and for the three polymers tested using dithranol as MALDI matrix, were not statistically different from the ones acquired with vortex mixing and also were in concordance with the values recommended by the polymer manufacturers.     

Gas chromatography coupled to mass spectrometry‐based metabolomic to screen for anabolic practices in cattle: identification of 5α‐androst‐2‐en‐17‐one as new biomarker of 4‐androstenedione misuse

The use of anabolic steroids as growth promoters for meat‐producing animals is banned within the European Union. However, screening for the illegal use of natural steroid hormones still represents a difficult challenge because of the high interindividual and physiological variability of the endogenous concentration levels in animals. In this context, the development of untargeted profiling approaches for identifying new relevant biomarkers of exposure and/or effect has been emerging for a couple of years. The present study deals with an untargeted metabolomics approach on the basis of GC‐MS aiming to reveal potential biomarkers signing a fraudulent administration of 4‐androstenedione (AED), an anabolic androgenic steroid chosen as template. After a sample preparation based on microextraction by packed sorbent, urinary profiles of the free and deglucurono‐conjugates urinary metabolites were acquired by GC‐MS in the full‐scan acquisition mode. Data processing and chemometric procedures highlighted 125 ions, allowing discrimination between samples collected before and after an administration of 4‐AED. After a first evaluation of the signal robustness using additional and independent non‐compliant samples, 17 steroid‐like metabolites were pointed out as relevant candidate biomarkers. All these metabolites were then monitored using a targeted GC‐MS/MS method for an additional assessment of their capacity to be used as biomarkers. Finally, two steroids, namely 5α‐androstane‐3β,17α‐diol and 5α‐androst‐2‐en‐17‐one, were concluded to be compatible with such a definition and which could be finally usable for screening purpose of AED abuse in cattle. Copyright © 2012 John Wiley & Sons, Ltd.     

Ultrasonic‐assisted derivatization of estrogenic compounds in a cup horn booster and determination by GC‐MS

Cup horn boosters are miniaturized ultrasound baths that maximize efficiency and precision. The optimization of an ultrasonic‐assisted derivatization step by means of a cup horn booster and the determination of estrone, 17β‐estradiol, estriol, 17α‐ethynyl estradiol and mestranol was developed by GC‐MS. Different derivatization reagents and solvents were studied for maximizing the di‐derivatization of 17α‐ethynyl estradiol under ultrasound energy. Only N,O‐bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane in pyridine gave satisfactory results and this mixture was further used in the optimization of the ultrasound assisted derivatization. The experiment designs included sonication time (1–10 min), sonication power (20–80%), sonication cycles (1–9), derivatization reagent volume (25–125 μL) and solvent volume (25–125 μL). Once the optimum conditions were fixed, the effect of organic matter and the frequency of the water bath change were studied. Finally, the validation of the analytical method was carried out using spiked natural and synthetic waters. Recoveries (natural (138–70%) and synthetic (112–89%)), the LODs (0.35–1.66 ng/L), and LOQs (1.16–5.52 ng/L) and the precision (0.2–5.3%) of the method were studied. This is the first work in the literature where a cup horn booster is used with the aim of minimizing derivatization time during the determination of estrogenic compounds.     

Simultaneous determination of seven fluoroquinolones in royal jelly by ultrasonic‐assisted extraction and liquid chromatography with fluorescence detection

A method for the quantitative determination of seven fluoroquinolone antibacterial agents (FQs) used in beekeeping, viz. ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, danofloxacin, enrofloxacin, and difloxacin, in royal jelly samples was developed on the basis of high performance liquid chromatography with fluorescence detection. Sample preparation included deproteination, ultrasonic‐assisted extraction with a mixed inorganic solution of monopotassium phosphate (KH₂PO₄) and ethylenediaminetetraacetic acid disodium salt (Na₂EDTA), and clean‐up on a solid‐phase extraction cartridge. The extraction procedure was optimized with regard to the amount of inorganic solvent and the duration of sonication for royal jelly as a complicated matrix. Overall recoveries for FQs ranged from 85.9 to 99.1% for royal jelly with standard deviations between 2.79 and 6.27%. Limits of quantification were 2–40 ng/g for seven FQs in royal jelly. A total of 57 real royal jelly samples collected from beekeepers and supermarkets were analyzed. The three most abundant honeybee‐use FQs, i. e. ofloxacin, ciprofloxacin, and norfloxacin, were determined in some royal jelly samples in concentrations ranging from 11.9 to 55.6 ng/g. Unexpectedly, however, difloxacin was found at concentrations of about 46.8 ng/g in one sample although it is rarely used in beekeeping. The presented method was successfully applied to quantify FQs in real royal jelly samples.     

Hair analysis of anabolic steroids in connection with doping control-results from horse samples

Doping control of anabolic substances is normally carried out with urine samples taken from athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair material is preferred in cases of limited availability or complicated collection of urine samples, e.g. from horses. In this work, possible ways of interpretation of analytical results in hair samples are discussed and illustrated by practical experiences. The results demonstrate the applicability of hair analysis to detect anabolic steroids and also to obtain further information about previous abuse. Moreover, the process of incorporation of steroids into hairs is described and the consequences on interpretation are discussed, e.g. on the retrospective estimation of the application date. The chosen examples deal with the detection of the anabolic agent testosterone propionate. Hair samples of an application study, as well as a control sample taken from a racing horse, were referred to. Hair material was investigated by a screening procedure including testosterone, nandrolone and several esters (testosterone propionate, phenylpropionate, decanoate, undecanoate, cypionate; nandrolone decanoate, dodecanoate and phenylpropionate; limits of detection (LODs) between 0.1 and 5.0 pg/mg). Confirmation of testosterone propionate (LOD 0.1 pg/mg) was carried out by an optimised sample preparation. Trimethylsilyl (TMS) and tert-butyl dimethylsilyl derivatives were detected by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).     

Organocatalytic Asymmetric Formation of Steroids

A novel and simple one‐step approach for the construction of optically active steroids in a highly stereoselective manner by using organocatalysis is presented. The reaction of (di)enals with cyclic dienophiles in the presence of a TMS‐protected prolinol catalyst leads to the construction of important 14 β‐steroids. This new reaction allows an easy access to optically active steroids with a variety of substituents in the A ring in high yields and up to greater than 99 % ee. The reaction has been extended to include the construction of B‐ and D ‐homosteroids as well as steroids containing heteroatoms in the B ring. The angular substituent at C13 can be varied and alkyl, ester, and sulfone functionalities are introduced with excellent stereoselectivities. Simple synthetic procedures provide access to a range of naturally occurring steroids such as estrone and related analogues.     

An improved approach to determining the yield of derivatization reaction and its application to the investigation of the silylation of some anabolic steroids

An approach to determining the yield of derivatization reaction is based on a comparison of chromatographic peak areas of the derivative and native (underivatizied) compound. In contrast to the previous publication [J. Anal. Chem., 2011, vol. 66, no. 12, pp. 1186–1189], ratio of the sensitivity coefficients of the derivative and native forms of the analyte was calculated using only experimental data obtained upon varying the derivatization conditions (solution containing equal amounts of underivatized compound and a respective derivative was analyzed previously). The approach was used to investigate the influence of the reaction time and the type of an external action on the yield of the derivatization (silylation) reaction for some anabolic steroids (methyltestosterone, methandienone, oxandrolone and oral-turinabol) containing a hindered tertiary hydroxyl group at C17. The amounts of the derivatized steroids were equal to about 20–60 ng (depending on the component). Steroids were derivatized with a mixture of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane. The derivatization reaction was carried out for 15, 30 or 45 min under conventional heating, sonication at room temperature, and sonication at elevated temperature.     

Development of a HPTLC method to profile the phytochemicals in Allanblackia parviflora (tallow tree) kernel and seed cakes

HPTLC is a useful and practical analytical tool to characterize plant compositions. This study was focused on exploring the results of high-performance thin-layer chromatography (HPTLC) analysis, particularly as a useful tool for the authentication of Allanblackia parviflora seed and kernel cakes. Bulked samples from sixteen different Ghanaian communities were analysed by HPTLC and their fingerprints were compared. The optimum experimental conditions were established: sample weight of 2.0 g, methanol:water (80:20 v/v) as extraction solvent, 30 min extraction time and twice extraction, ethyl acetate:methanol:water (100:16.5:13.5 v/v) as mobile phase, vanillic acid as derivatisation agent and 7 min of plate heating time after derivatisation. The HPTLC profile generated from extracts across 16 communities and 157 trees was very reproducible and demonstrates the robustness of the technique in characterising the profile.

     

Screening of glycoside isomers in P. scrophulariiflora using ionic liquid‐based ultrasonic‐assisted extraction and ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

A powerful ionic liquid‐based ultrasonic‐assisted extraction (ILUAE) method combined with ultra‐performance liquid chromatography coupled to electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC/ESI‐QTOFMSⁿ) was employed in the rapid simultaneous screening of iridoid glycosides, phenylethanoid glycosides, and cucurbitacin glycosides from P. scrophulariiflora. The ILUAE procedure was optimized over several ultrasonic parameters, including the ultrasonic power, concentration of the ionic liquid, and solid–liquid ratio. A comparison with conventional heat‐reflux extraction and regular UAE demonstrated that the optimized approach yielded a high extraction efficiency (Picroside I, 2.84%; Picroside II, 3.57%; 6‐O‐E‐feruloyl catalpol, 2.20%) within a short extraction time of 30 min. Negative ion mode ESI‐QTOFMS² analysis of the fragmentation reactions of the [M–H]^– ions was conducted to characterize the diagnostic ions related to the glycosyl moieties, aglycone units, and the type and substituted position of the ester groups. Interestingly, the positional isomers of the iridoid glycosides could be easily discriminated based on the characteristic ions. A total of 15 glycosides, including three groups of iridoid glycoside isomers and two groups of phenylethanoid glycoside isomers, were conveniently identified within 13.5 min. Moreover, 6'‐O‐vanilloyl catalpol was identified in P. scrophulariiflora for the first time. The method developed here was further validated by measuring the recovery, correlation coefficient (R²), and reproducibility (RSD, n = 5) of three iridoid glycosides: 89.60%–109.02%, 0.9991–0.9998, and 0.93%–1.44%, respectively. This study demonstrated the capabilities of ILUAE combined with UPLC/ESI‐QTOFMSⁿ for the rapid screening of glycosides in P. scrophulariiflora. This method offers an approach to similar studies on other natural plants. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of residual anabolic steroid in meat by gas chromatography-ion trap-mass spectrometer

The use of natural and synthetic anabolic steroids in animal fattening has been prohibited in Taiwan and many countries because of their potential toxic effect on public health. This paper describes a newly developed gas chromatography-ion trap-mass spectrometry (GC-IT-MS) method for the quantitative determination of various residual anabolic steroids in meat. Anabolic steroid was derivatized with N-methyl-N-trimethylsilytrifluoroacetamide prior to GC-IT-MS analysis. MS² was employed for quantitative measurement. In addition, 2d-estradiol was used as an internal standard. Quantitative determination was based on the ratio of peak area of steroid derivative to peak area of internal standard derivative. Good linearity of each compound, 0.03-1.0 μg/ml, was determined. Solvent extraction was used to extract residual anabolic compounds in meat samples and a solid phase extraction (SPE) procedure was utilized for sample cleanup and pre-concentration. The limits of detection of anabolic compounds approximately ranged from 0.1 to 0.4 μg/kg. The detection limit was comparable with or better than reported methods and was below the minimum required performance limits (MRPLs) established by the European Community (EC). The application of this newly developed method was demonstrated by analyzing various beef, pork, chicken and several animal internal organ samples from local markets.     

Comparative study of matrices for their use in the rapid screening of anabolic steroids by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry

New data on sample preparation and matrix selection for the fast screening of androgenic anabolic steroids (AAS) by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) is presented. The rapid screening of 15 steroids included in the World Anti‐Doping Agency (WADA) prohibited list using MALDI was evaluated. Nine organic and two inorganic matrices were assessed in order to determine the best matrix for steroid identification in terms of ionisation yield and interference by characteristic matrix ions. The best results were achieved for the organic matrices 2‐(4‐hydroxyphenylazo)benzoic acid (HABA) and trans‐3‐indoleacrylic acid (IAA). Good signals for all the steroids studied were obtained for concentrations as low as 0.010 and 0.050 µg/mL on the MALDI sample plate for the HABA and IAA matrices, respectively. For these two matrices, the sensitivity achieved by MALDI is comparable with the sensitivity achieved by gas chromatography/mass spectrometry (GC/MS), which is the conventional technique used for AAS detection. Furthermore, the accuracy and precision obtained with MALDI are very good, since an internal mass calibration is performed with the matrix ions. For the inorganic matrices, laser fluences higher than those used with organic matrices are required to obtain good MALDI signals. When inorganic matrices were used in combination with glycerol as a dispersing agent, an important reduction of the background noise was observed. Urine samples spiked with the study compounds were processed by solid‐phase extraction (SPE) and the screening was consistently positive. Copyright © 2009 John Wiley & Sons, Ltd.     

Fast Analysis of Free Amino Acids in Tobacco by HPLC with Fluorescence Detection and Automated Derivatization

A fast, simple, and sensitive HPLC method for the determination of free amino acids in tobacco was described. A fully automated sample processor performed precolumn derivatization of both primary and secondary amino acids with o‐phthalaldehyde/3‐mercaptopropionic acid and 9‐fluorenylmethyl chloroformate (FMOC‐Cl), respectively. All reactions were fully automated by means of an injector programme and accomplished in 10 min. Sample preparation consisted of a single step of extraction with 0.1 mol/L HCl at ambient temperature (assisted by sonication) in 30 min, followed by filtration of an aliquot and derivatization. By optimization of sample preparation and HPLC conditions, separation of 20 amino acids in 30 min was achieved. Detection limits ranged from 0.50 to 1.40 μg/g; coefficients of variation ranged from 1.8% to 3.9%; recoveries ranged from 84.6% to 108.5%. The method was applied to the analysis of amino acids contents of tobacco leaves in different varieties and flue‐curing period.     

Fit-for-purpose in veterinary drug residue analysis: development and validation of an LC-MS/MS method for the screening of thirty illicit drugs in bovine urine

A selective and sensitive method for screening 31 analytes (nine corticosteroids, eight β‐agonists, seven anabolic steroids, six promazines and zeranol) in bovine urine was validated according to 2002/657/EC guidelines. Upon optimization of sample treatment conditions, the extraction was performed by diethylether at pH 9, after deconjugation. Extraction yields (R%) proved higher than 70% for 19 analytes, 50<R%<70 for 5 analytes, lower than 50% but reproducible for the remaining six analytes. The analyses were carried out using HPLC‐ESI‐MS/MS. The method sensitivity proved high enough to largely exceed the CCβ requirements of the Italian residue detection plan, ranging from 1 to 3 ng/mL (20 ng/mL for promazines). The present method allowed the simultaneous analysis of most drugs for which the European legislation prescribes official controls. Its practical applicability was verified on 494 real samples as an alternative to the traditional screening protocols based on multiple immunometric analysis, demonstrating high efficiency and comprehensive investigation capacity, allowing epidemiological assessment of the current trends in cattle breeding drug abuse. Among non‐compliant results, nine borderline cases of growth‐promoters illegal treatments, making use of long‐term low‐dosage administrations and typically yielding urine residues below the cut‐off value for immunochemical methods, were detected by using the present LC‐MS/MS method.