Screening for anabolic steroids and related compounds in illegal cocktails by liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

Findings of illegal hormone preparations such as syringes, bottles, cocktails, and so on, are an important information source for the nature of the current abuse of anabolic steroids and related compounds as growth-promoting agents in cattle. A new screening method for steroids in cocktails is presented based on liquid chromatography (LC) with diode-array UV-absorbance detection and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS). Accurate mass measurements were performed at a mass resolution of 4000 using continuous introduction of a lock mass through a second (electro)sprayer. Similar experiments were carried out using dual-sprayer quadrupole time-of-flight mass spectrometry (ESI-QTOFMS/MS) at a mass resolution of 10 000 with data-dependent MS/MS acquisition; i.e. beyond an intensity threshold for the [M + H](+) ions, MS/MS spectra were automatically acquired at three different collision energies. Elemental compositions were calculated for precursor and product ions and it is shown that the combined information from LC retention behavior, UV spectra, elemental compositions, and accurate mass MS/MS spectra yield a fast impression of the steroids present in the complex mixture. Using a new software tool for structure elucidation of MS/MS spectra, an additional non-steroidal additive was identified as well.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Simultaneous determination of 24 sulfonamide residues in meat by ultra-performance liquid chromatography tandem mass spectrometry

The present study used the liquid extraction pretreatment method and developed an ultra-performance liquid chromatography triple quadrupole tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of 24 kinds of sulfonamide residues in meat. The meat samples were homogenized, extracted and deproteinized by acetonitrile, defatted by n-hexane, and further liquid-liquid extracted by ethyl acetate. All of 24 sulfonamide residues were simultaneously separated and determined by UPLC-MS/MS within 15 min. The sulfonamide residues were monitored via the ESI(+) ionization method and quantified by six-channel multiple reaction monitoring (MRM). The calibrations were performed in sample matrixes by the isotope dilution method and the interference effect of sample matrixes on the ionization was effectively eliminated. Good linear relationship (R(2)=0.991-0.999) was observed within the concentration range of 0.2-50 microg/kg. Satisfied recoveries (67.8-113.9%) of all the sulfonamides were demonstrated in different standard-spiked levels except sulfanitran (SNT). The analytical category, separation speed, selectivity, sensitivity and repeatability of sulfonamides using UPLC-MS/MS were significantly improved compared to other analytical methods. Quantitative results of 240 meat samples demonstrated that the present method has a convenient operation and good practicability, which can be applied to the quantitative analysis of a large number of samples.     

Presence of endogenous interferences in the urinary detection of selected anabolic steroids by liquid chromatography/electrospray tandem mass spectrometry

The use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) can give several benefits in the urinary detection of anabolic steroids (AS) such as avoiding the derivatization step or the possible direct detection of conjugated AS. However, the presence of endogenous compounds with similar structures can interfere with this detection, thus decreasing the reliability of the method. The circumvention of these interferences by the use of different MS/MS transitions has been studied. For 17alpha-trenbolone, an unusual odd-electron ion has been selected for detection. Using this approach, the interferences have been reduced drastically. The selection of a more specific precursor ion can also help in the circumvention of interferences. This has been shown for 1-testosterone, 5beta-androst-1-ene-17beta-ol-3-one and oxandrolone where the selection of the uncommon [M+H+MeOH](+) as the precursor ion increased the selectivity of the method. In addition, an approach to identify the endogenous interferences if they have corticosteroid or steroid structure has been evaluated. Precursor ion spectra can be used to define the molecular properties of the interferences and to predict whether it is an endogenous steroid or corticosteroid. The use of some restrictions for the structure can be helpful in outlining a feasible hypothesis. This data and the product ions from likely suspects can be used to identify AS. Using this approach, several endogenous interferences have been elucidated in this case study. The knowledge about the structures of these interferences has been found useful to circumvent their detection in the screening method for AS.     

High resolution orbitrap mass spectrometry in comparison with tandem mass spectrometry for confirmation of anabolic steroids in meat

A prominent trend which has been observed in recent years in the analysis of veterinary drugs and growth-promoting agents is the shift from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as time of flight (ToF) and Fourier Transform (FT) Orbitrap MS). In this study the applicability of high resolution single-stage-Orbitrap-MS for confirmatory analysis of growth-promoting agents in meat was compared to that of a QqQ-MS. Validation according to CD 2002/657/EC demonstrated that steroid analysis based on Orbitrap MS, operating at a resolution of 50,000 FWHM, is indeed capable to compete with QqQ-MS in terms of selectivity/specificity, while providing excellent linearity (for most compounds >0.99) but somewhat inferior sensitivity. Indeed, CC_αs reached from 0.04–0.88 μg kg⁻¹ for the 34 anabolic steroids upon MS/MS detection, while upon Orbitrap MS detection a range of 0.07–2.50 μg kg⁻¹ was observed. Using QqQ-MS adequate precision was obtained since relative standard deviations, associated with the repeatability and intra-laboratory reproducibility, were below 20%. In the case of Orbitrap MS, for some compounds (i.e. some estrogens) this threshold was exceeded and thus poor precision was observed, which is possibly caused by the lack in sensitivity. Overall, it may be concluded that Orbitrap-MS offers an adequate performance in terms of linearity and precision but lacks in sensitivity for some of the compounds.     

Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionisation tandem mass spectrometric (LC/ESI-MS/MS) method was developed for the analysis of acetylcholine and choline in microdialysis samples. A Ringer's solution that contains high (150 mM) concentrations of inorganic salts was used to extract acetylcholine and choline from a rat or mouse brain. The separation of acetylcholine, choline, an internal standard acetyl-beta-methylcholine, endogenous compounds and inorganic cations was achieved with hydrophilic interaction chromatography using a diol column. The eluent consisted of 20 mM ammonium formate (pH 3.3) and acetonitrile (20:80) which is favourable for the ESI process. Limits of detection (signal-to-noise (S/N) ratio = 3) of 0.02 nM (0.2 fmol) for acetylcholine and 1 nM (10 fmol) for choline were observed using standards diluted in Ringer's solution. A good linearity was obtained from the limit of quantitation: 0.1 nM (S/N ratio = 10) to 50 nM (r = 0.999) for acetylcholine and within the concentration range of 100-3500 nM (r = 0.998) for choline. The between-day repeatability of the method was good; RSD was 3.1% at 1 nM level of acetylcholine and 3.5% at 1000 nM level of choline. The recoveries for addition of 1 or 2.5 nM acetylcholine and 0.2 or 1 microM choline in microdialysis balancing samples were between 93 and 101% indicating that no suppressing endogenous compounds were co-eluting with acetylcholine or choline. The developed method was applied to the analysis of microdialysis balancing samples collected from rat and mouse brains.     

液相色谱串联质谱测定畜禽肉中黄霉素A的残留量

建立了高效液相色谱串联质谱法测定畜禽肉中黄霉素A残留量的方法,样品中残留的黄霉素A经提取,HLB柱净化后,以Agilent Ecillps C18色谱柱为分析柱,乙腈-10 mmol/L的乙酸铵为梯度洗脱液,经串联四极杆质谱多反应监测模式检测,黄霉素A得到了很好的分离。线性范围为10～1000μg/kg。黄霉素A的定量限为10μg/kg。添加浓度在10～50μg/kg时,回收率在70%～100%之间,变异系数在4.6%～8.8%之间。方法适合出口畜禽肉及组织中黄霉素A残留量的测定。     

Fast analysis of 19 anabolic steroids in bovine tissues by high performance liquid chromatography with tandem mass spectrometry

For the detection of 19 steroid hormones in bovine muscle, a fast and sensitive liquid chromatography with electrospray ionization tandem mass spectrometry method was developed using both positive and negative ionization mode. Chromatographic separation on Poroshell 120‐EC C18 column was achieved in less than 10 min using isocratic elution of mobile phase of acetonitrile/methanol/water. The compounds were extracted from muscle tissue using ethyl acetate and quick, easy, cheap, effective, rugged, and safe technique. The purification of the obtained extract was performed by dispersive solid‐phase extraction with sorbents C18, primary secondary amine and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. For all steroids tested good recoveries were obtained (from 51.2 to 121.4%) in the concentration range from decision limits until 5 µg/kg. The values of decision limits and the detection capabilities for individual compounds were in the range 0.10–0.48 and 0.17–0.95 µg/kg, respectively. The method was characterized by satisfactory linearity for most compounds (correlation coefficients  > 0.99) and the reproducibility was lower than 35%. The elaborated procedure has met the criteria for confirmatory methods and is currently used in the official control of hormones.     

Screening for anabolic steroids in doping analysis by liquid chromatography/electrospray ion trap mass spectrometry

A fast and selective LC/MS/MS method for the screening of four anabolic steroids in human urine has been developed and validated. Liquid-liquid extraction with diethyl ether was applied after enzymatic hydrolysis. Analyses were performed on an ion trap mass spectrometer equipped with electrospray ionisation. MS/MS was applied for all compounds. The analytical run time was 11 min. The LOD for all compounds varied between 1 and 10 ng/mL. Left-over A samples, which were declared positive by GC/MS for the presence of 3'-hydroxystanozolol, were assessed using the described method.     

Ionization of anabolic steroids by adduct formation in liquid chromatography electrospray mass spectrometry

The ionization of 46 anabolic steroids has been studied. The absence of basic or acidic moieties in most of these analytes makes their direct ionization as [M + H]+ by atmospheric pressure interfaces difficult. The formation of adducts with different components of the mobile phase has been found to be an efficient way to ionize anabolic steroids by electrospray. Different mobile phases using methanol (MeOH) or acetonitrile as organic solvent and HCOOH, Na+ or NH4+ as additives have been tested to favor the adduct formation. A direct correlation between the chemical structure of the anabolic steroid and the possibility to ionize it in a particular chromatographic condition has been found. According to their ionization, anabolic steroids can be divided into seven different groups depending on both the nature and the relative position of their functional groups. The formation of different adducts such as [M + Na + MeOH]+ or [M + H + CH3 CN - H2O]+ is required in order to ionize some of these groups and the optimal mobile phase composition for each group of anabolic steroids is proposed. Despite the ionization limitations due to their chemical structure, most of tested anabolic steroids could be ionized using the adduct formation approach.     

Determination of steroids by liquid chromatography/mass spectrometry

On-line atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) were evaluated for the analysis of a variety of steroids. Steroids were classified into three major groups based on the spectra and the sensitivities observed: (I) those containing a 3-one, 4-ene functional group, (II) those containing at least one ketone group without conjugation, and (III) those containing hydroxy group(s) only. In the APCI mode, the best sensitivity and the lowest detection limit for all three groups were obtained by using a mobile phase consisting of methanol and 1%–2% acetic acid in water. The APCI spectra were characterized by MH⁺, MH⁺-H₂O, MH⁺-2H₂O, etc., with the degree of H₂O loss being compound dependent: group I steroids produced stable MH⁺ and group III steroids showed extensive water loss. In the electrospray mode the best sensitivity and the lowest detection limit for the first two groups were obtained when pure methanol and water were used as the mobile phase. This condition produced abundant stable MNa⁺ due to ubiquitous sodium. Detection limits in the 5–15 pg range can be easily achieved using ESI LC/MS. Addition of ammonium acetate or use of acetonitrile in the mobile phase, common in the LC/MS analysis of steroids, decreased the sensitivity for the group I and II steroids and thus should be avoided. For group III steroids, the detection limit can be improved by the addition of acetic acid to the mobile phase.     

Rapid determination of nitroimidazole residues in honey by liquid chromatography/tandem mass spectrometry

We developed a rapid and efficient means of determining residues of four nitroimidazoles-i.e., dimetridazole, ipronidazole, metronidazole, and ronidazole-and three hydrophilic metabolites- i.e., 2-hydroxymethyl-1-methyl-5-nitroimidazole, 1 -methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole, and 1-(2-hydroxyethyl)-2-hydroxymethyl-nitroimidazole--in honey. We applied a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure improved to suit a nitroimidazole analysis, which is fast (approximately 30 min) and uses less organic solvent. The procedure involves initial single-phase extraction of 5 g of honey with acetonitrile containing 1% acetic acid, followed by liquid-liquid partitioning involving the addition of 5 g sodium chloride, 1.5 g trisodium citrate dihydrate, and 4 g magnesium sulfate. Moreover, matrix from honey was reduced by an SPE method with an alumina-N cartridge. The samples were analyzed using LC/MS/MS. Chromatographic separation of these nitroimidazoles and metabolites was performed in the gradient mode on a pentafluorophenylpropyl-bonded silica column (150x2.0 mm, 3 pm particle size) at 40 degrees C. The mobile phase consisted of a 0.01% acetic acid solution and acetonitrile, and the flow rate was 0.2 mL/min. The method was validated using honey spiked with these nitroimidazoles from 0.1 to 0.5 microg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were <9.5 and <14.2%, respectively. The LOQ ranged from 0.1 to 0.5 microg/kg. LC/MS/MS coupled with the QuEChERS method showed good potential as a method for determining nitroimidazole residues in honey.     

Determination of nitroimidazole residues in porcine urine by liquid chromatography/tandem mass spectrometry

A reliable and sensitive method was developed for determination of nitroimidazoles in porcine urine. Sample preparation involves an extraction with ethyl acetate, followed by a solid-phase extraction cleanup step. The final extract is injected into the liquid chromatography/tandem mass spectrometry system in the positive-ion electrospray ionization mode. A C8 column with water and acetonitrile as the mobile phase is used for chromatographic separation under gradient conditions. Overall average recoveries ranged from 83 to 107%. The limits of detection ranged from 0.03 to 0.05 ng/mL, and the limits of quantitation, from 0.1 to 0.2 ng/mL.     

Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry

A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times.     

Determination of chloramphenicol residues in bee pollen by liquid chromatography/tandem mass spectrometry

A novel liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed for the trace residue determination of chloramphenicol (CAP) in bee pollen. CAP was extracted from bee pollen with a mixture of methanol and 1% metaphosphoric acid solution, followed by a 2-stage solid-phase extraction enrichment and cleanup. The first stage involved a polymeric cartridge, and the second stage involved an alumina neutral cartridge. The LC separation was performed on a C18 column with 10 mM ammonium formate-acetonitrile (7 + 3) as the mobile phase and MS detection with negative-ion electrospray ionization. CAP-d5 was used as the internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear between 0.1 and 5.0 ng/mL, and overall recoveries ranged from 98 to 113%. Decision limits (CCalpha) ranged from 0.05 to 0.07 microg/kg, and detection capabilities (CCbeta) ranged from 0.08 to 0.12 microg/kg. The developed method was applied to 11 samples.     

Feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization mass spectrometry in analyzing anabolic steroids in urine samples

We examined the feasibility of capillary liquid chromatography/microchip atmospheric pressure photoionization tandem mass spectrometry (capLC/µAPPI‐MS/MS) for the analysis of anabolic steroids in human urine. The urine samples were pretreated by enzymatic hydrolysis (with β‐glucuronidase from Helix pomatia), and the compounds were liquid‐liquid extracted with diethyl ether. After separation the compounds were vaporized by microchip APPI, photoionized by a 10 eV krypton discharge lamp, and detected by selected reaction monitoring. The capLC/µAPPI‐MS/MS method showed good sensitivity with detection limits at the level of 1.0 ng mL^?1, good linearity with correlation coefficients between 0.9954 and 0.9990, and good repeatability with relative standard deviations below 10%. These results demonstrate that microchip APPI combined with capLC/MS/MS provides a new potential method for analyzing non‐polar and neutral compounds in biological samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid determination of fosetyl-aluminum residues in lettuce by liquid chromatography/electrospray tandem mass spectrometry

This paper describes a new method for the sensitive and selective determination of fosetyl-aluminum (Al) residues in vegetable samples. The method involves extraction with water by using a high-speed blender and subsequent injection of the 5-fold diluted extract into the liquid chromatograph. Fosetyl-Al is determined by liquid chromatography with electrospray tandem mass spectrometry after the addition of tetrabutylammonium acetate as the ion-pairing reagent. The method has been used to assay lettuce samples spiked at 2 and 0.2 mg/kg. Recoveries were satisfactory, with mean values of 98 and 106%, respectively, and relative standard deviations were < 10%. The limit of quantitation was 0.2 mg/kg, and the limit of detection was as low as 0.05 mg/kg. Matrix-matched calibration was used for quantitation, and the addition of an internal standard improved repeatability. The developed method allows the accurate and rapid determination of low levels of fosetyl-Al residues in lettuce with very little sample handling and good sensitivity; it was shown to be robust by the analysis of almost 100 samples.     

Determination of lincomycin and tylosin residues in honey by liquid chromatography/tandem mass spectrometry

A simple and rapid analytical method was developed for the determination of lincomycin and tylosin residues in honey as part of field studies examining the efficacy and target animal safety of these antibiotics to control American foulbrood disease in honey bees. Residues of the antibiotics were determined using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Honey samples were diluted and injected directly into the LC/MS/MS system without additional cleanup by solid-phase extraction or liquid-liquid partitioning. A six-port valve system was utilized to selectively route eluant from the LC column into the mass spectrometer only during a relatively short portion of the chromatographic run corresponding to the elution of the analytes of interest. Minimal contamination of the MS source chamber was observed despite the analysis of large numbers of samples. Using internal standard quantitation, excellent accuracy and precision were obtained with no apparent matrix-to-matrix variation. Based on the analysis of fortified replicates, the mean percent deviation from the theoretical concentration and the percent relative standard deviation were both less than 10% for tylosin over an analytical range of 10-1000 microg/kg. Slightly higher mean percent deviations and relative standard deviations were observed for the analysis of lincomycin in fortified replicate samples. The method detection limits were determined to be 5 and 2 microg/kg for lincomycin and tylosin, respectively.     

Determination of anabolic steroids by gas chromatography/negative-ion chemical ionization mass spectrometry and gas chromatography/negative-ion chemical ionization tandem mass spectrometry with heptafluorobutyric anhydride derivatization

A gas chromatography/mass spectrometry (GC/MS) method is described which uses negative ion chemical ionization (NCI) and tandem mass spectrometry (MS/MS) for the determination of eight anabolic steroids in human urine. Eight anabolic steroids were derivatized by heptafluorobutyric anhydride (HFBA), and were determined using GC/NCI-MS and GC/NCI-MS/MS. The linear correlation coefficients for calibration in NCI-MS/MS were in the range 0.9880-0.9988. This method of derivatization with HFBA for use with GC/NCI was useful in determinations of 19-norandrosterone, boldenone, 19-noretiocholanolone, 2-methylandrosterone, nandrolone, 1-methyleneandrosterone, 1-methylandrosterone, 4-dihydroboldenone and mesterolone. The detection limits of this procedure were 5-20 ppb at a signal-to-noise (S/N) ratio of 3.     

Determination of ethyl glucuronide in hair samples by liquid chromatography/electrospray tandem mass spectrometry

A method for the determination of ethyl glucuronide (EtG) in hair samples, using liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS), was developed and validated. The treatment of hair samples was as follows: to 100 mg of washed (dichloromethane followed by methanol, 1 ml each) and cut (1-2 mm) material, 700 microl of water, 20 microl of internal standard solution (pentadeuterated EtG, D(5)-EtG, 500 microg/l) and 20 microl of methanol were added. Samples were incubated at 25 degrees C overnight and then ultrasonicated for 2 h. Finally, 8 microl of the centrifuged solution (13,000 rpm) were analyzed by LC/ESI-MS/MS in negative ion mode. The surviving ions of EtG and D(5)-EtG were monitored together with the following MRM transitions: m/z 221 --> 75, m/z 221 --> 85 (EtG) and m/z 226 --> 75, m/z 226 --> 85 (D(5)-EtG). The method exhibited a mean correlation coefficient better than 0.9998 over the dynamic range (3-2000 pg/mg). The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 3 and 2 pg/mg respectively. The intra- and interday precision and accuracy were studied at four different concentration levels (3, 5, 56 and 160 pg/mg) and were always better than 7% (n = 5). Matrix effects did not exceed 20%. The method was applied to several hair samples taken from autopsies of known alcoholics, from patients in withdrawal treatment, from social drinkers, from adult teetotalers and from children not exposed to ethanol, with EtG concentrations globally ranging from < or =2 to 4180 pg/mg.