Leidenfrost Phenomenon-assisted Thermal Desorption (LPTD) and Its Application to Open Ion Sources at Atmospheric Pressure Mass Spectrometry

This work describes the development and application of a new thermal desorption technique that makes use of the Leidenfrost phenomenon in open ion sources at atmospheric pressure for direct mass spectrometric detection of ultratrace levels of illicit, therapeutic, and stimulant drugs, toxicants, and peptides (molecular weight above 1 kDa) in their unaltered state from complex real world samples without or with minor sample pretreatment. A low temperature dielectric barrier discharge ion source was used throughout the experiments and the analytical figures of merit of this technique were investigated. Further, this desorption technique coupled with other ionization sources such as electrospray ionization (ESI) and dc corona discharge atmospheric pressure chemical ionization (APCI) in open atmosphere was also investigated. The use of the high-resolution ‘Exactive Orbitrap’ mass spectrometer provided unambiguous identification of trace levels of the targeted compounds from complex mixtures and background noise; the limits of detection for various small organic molecules and peptides treated with this technique were at the level of parts per trillion and 10^–9?M, respectively. The high sensitivity of the present technique is attributed to the spontaneous enrichment of analyte molecules during the slow evaporation of the solvent, as well as to the sequential desorption of molecules from complex mixtures based on their volatilities. This newly developed desorption technique is simple and fast, while molecular ions are observed as the major ions.      

Desorption Mass Spectrometry for Nonvolatile Compounds Using an Ultrasonic Cutter

In this work, desorption of nonvolatile analytes induced by friction was studied. The nonvolatile compounds deposited on the perfluoroalkoxy substrate were gently touched by an ultrasonic cutter oscillating with a frequency of 40 kHz. The desorbed molecules were ionized by a dielectric barrier discharge (DBD) ion source. Efficient desorption of samples such as drugs, pharmaceuticals, amino acids, and explosives was observed. The limits of detection for these compounds were about 1 ng. Many compounds were detected in their protonated forms without undergoing significant fragmentation. When the DBD was off, no ions for the neutral samples could be detected, meaning that only desorption along with little ionization took place by the present technique.

Investigations into the feasibility of routine ultra high performance liquid chromatography–tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids,anabolic steroid esters and related compounds

The detection of the abuse of anabolic steroids in equine sport is complicated by the endogenous nature of some of the abused steroids, such as testosterone and nandrolone. These steroids are commonly administered as intramuscular injections of esterified forms of the steroid, which prolongs their effects and improves bioavailability over oral dosing. The successful detection of an intact anabolic steroid ester therefore provides unequivocal proof of an illegal administration, as esterified forms are not found endogenously. Detection of intact anabolic steroid esters is possible in plasma samples but not, to date, in the traditional doping control matrix of urine. The analysis of equine mane hair for the detection of anabolic steroid esters has the potential to greatly extend the time period over which detection of abuse can be monitored.     

A downscaled multi-residue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC-MS3)

Within the scope of the European Community member states' residue monitoring plan, illicit administration of anabolic steroids is monitored at slaughterhouse level as well as on living animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a procedure for which high performance liquid chromatographic (HPLC) fractionation prior to GC-MS(n) analysis was no longer required. Analytical results could be obtained within 1 day and only 5 mL urine was needed to carry out the screening procedure. Using the downscaled methodology, all validation criteria described in the European Commission document 2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs) established for anabolic steroids in urine, could be achieved. A higher GC-MS technique's specificity was achieved by detecting the steroids using GC-MS3. Nevertheless, it was decided to screen routinely sampled urine with GC-MS2 whereas GC-MS3 was applied to confirm the presence of anabolic steroid residues in suspected sample extracts.     

A new mixed micellar electrokinetic chromatography method for analysis of natural and synthetic anabolic steroids

A simple, rapid and low-costing new mixed surfactant MEKC method has been developed for the analysis of five neutral anabolic steroids in this paper. It was found that the bile salt coupling with Triton X-100 was a suitable bi-micellar surfactant for the separation of these anabolic steroids with similar structure. The separation conditions were optimized in detail. The five natural and synthetic anabolic steroids, such as androstenedione (AD), 19-norandrostenedione (NAD), 1,4-androstadiene-3,17-dione (ADD), methandrostenolone (MA) and methyltestosterone (MT) were separated and detected in an alkaline buffer system (pH 9.0) containing 15 mM Britton-Robinson (BR) buffer, 50 mM sodium cholate (SC) and 0.1% (v/v) Triton X-100 with detection wavelength at 241 nm and 18 kV of separation voltage. Under the optimal conditions, five coexistence neutral steroids were completely separated within 12 min with the detection limits ranged from 0.20 to 0.51 μg/mL. This method was successfully used for detection and confirmation of the anabolic steroid methandrostenolone in methandrostenolone tablets and in the real human urine, GC-MS method was applied to confirm the free methandrostenolone existence in the urine sample in order to validate the reliability of MEKC method.     

Testing for nandrolone metabolites in urine samples of professional athletes and sedentary subjects by GC/MS/MS analysis

The concentrations of nandrolone metabolites, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) were analysed in urine samples of professional athletes doing intense physical activity and sedentary subjects to verify if there was endogenous production of nandrolone and if there was any link between physical effort and the urinary metabolites of the steroid. We collected 18 urine samples from professional footballers age range 20-30 years, all from the same team, and 18 urine samples from males not doing any physical activity, age range 20-30 years. Neither group used nandrolone. Qualitative and quantitative analyses of urinary nandrolone metabolites were carried out by GC/MS followed by GC/MS/MS to confirm positive samples. This technique has been demonstrated to be an excellent analytical approach for the determination of anabolic steroids at very low detection limits in complex matrices such as urine. In five urine samples from professional footballers traces of 19-NA were detected. No trace of 19-NA was found in the group of sedentary subjects and no trace of 19-NE was found in any urine sample. The absence of nandrolone metabolites in sedentary subjects supports the hypothesis that the presence of 19-NA and 19-NE could be linked to physical effort even though the origin is not yet clear.     

Liquid chromatography/mass spectrometry in anabolic steroid analysis--optimization and comparison of three ionization techniques: electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

The applicability of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the detection of the free anabolic steroid fraction in human urine was examined. Electrospray ionization (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization methods were optimized regarding eluent composition, ion source parameters and fragmentation. The methods were compared with respect to specificity and detection limit. Although all methods proved suitable, LC/ESI-MS/MS with a methanol-water gradient including 5 mM ammonium acetate and 0.01% acetic acid was found best for the purpose. Multiple reaction monitoring allowed the determination of steroids in urine at low nanogram per milliliter levels. LC/MS/MS exhibited high sensitivity and specificity for the detection of free steroids and may be a suitable technique for screening for the abuse of anabolic steroids in sports.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

Flash Desorption/Mass Spectrometry for the Analysis of Less- and Nonvolatile Samples Using a Linearly Driven Heated Metal Filament

In this paper, the important issue of the desorption of less- and nonvolatile compounds with minimal sample decomposition in ambient mass spectrometry is approached using ambient flash desorption mass spectrometry. The preheated stainless steel filament was driven down and up along the vertical axis in 0.3 s. At the lowest position, it touched the surface of the sample with an invasion depth of 0.1 mm in 50 ms (flash heating) and was removed from the surface (fast cooling). The heating rate corresponds to ~10⁴ °C/s at the filament temperature of 500 °C. The desorbed gaseous molecules were ionized by using a dielectric barrier discharge ion source, and the produced ions were detected by a time-of-flight (TOF) mass spectrometer. Less-volatile samples, such as pharmaceutical tablets, narcotics, explosives, and C₆₀ gave molecular and protonated molecule ions as major ions with thermal decomposition minimally suppressed. For synthetic polymers (PMMA, PLA, and PS), the mass spectra reflected their backbone structures because of the suppression of the sequential thermal decompositions of the primary products. The present technique appears to be suitable for high-throughput qualitative analyses of many types of solid samples in the range from a few ng to 10 μg with minimal sample consumption. Some contribution from tribodesorption in addition to thermal desorption was suggested for the desorption processes.

Direct detection of explosives on solid surfaces by mass spectrometry with an ambient ion source based on dielectric barrier discharge

Trace amounts of explosives on solid surfaces were detected by mass spectrometry at ambient conditions with a new technique termed dielectric barrier discharge ionization (DBDI). By the needle-plate discharge mode, a plasma discharge with energetic electrons was generated, which could launch the desorption and ionization of the explosives from solid surfaces. Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) were desorbed directly from the explosives-contaminated surface by DBDI, forming the typical anions of [TNT](-), [TNT - H](-), [RDX + NO(2)](-), [PETN + ONO(2)](-), and [RDX + ONO(2)](-). The ions were transferred into the MS instrument for analysis in the negative ion mode. The detection limit of present method was 10 pg for TNT (m/z 197, S/N 8 : 1), 0.1 ng for RDX (m/z 284, S/N 10 : 1), and 1 ng for PETN (m/z 260, S/N 12 : 1). The present method allowed the detection of trace explosives on various matrices, including paper, cloth, chemical fiber, glass, paints, and soil. A relative standard deviation of 5.57% was achieved by depositing 100 pg of TNT on these matrices. The analysis of A-5, a mixture of RDX and additives, has been carried out and the results were consistent with the reference values. The DBDI-MS method represents a simple and rapid way for the detection of explosives with high sensitivity and specificity, which is especially useful when they are present in trace amounts on ordinary environmental surfaces.     

Norchlorotestosterone Acetate: An Alternative Metabolism Study and GC–MS<Superscript>2</Superscript> Analysis in Kidney Fat,Urine, and Faeces

Norchlorotestosterone acetate (NClTA) is an anabolic steroid which resembles chlorotestosterone acetate. It cannot yet be detected by routine methods used for anabolic steroids, because there is no knowledge of its metabolic pathway. The invertebrate Neomysis integer has been used as an alternative model to study the metabolism of NClTA. The experimental results indicated the presence of 4-norchloroandrost-4-ene-17-ol-3-one (NClT) and 4-norchloroandrost-4-ene-3,17-dione (NorClAD) as possible metabolites of NClTA. Subsequently NClTA and the synthesised metabolites NClT and NorClAD were incorporated into the routine multi-residue method for detection of anabolic steroids in kidney fat, urine, and faeces.     

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.     

Determination of nitroaromatic and nitramine explosives from a PTFE wipe using thermal desorption-gas chromatography with electron-capture detection

A method for the detection of nitroaromatic and nitramine explosives from a PTFE wipe has been developed using thermal desorption andgas chromatography with electron-capture detection (TD-GC-ECD). For method development a standard mixture containing eight nitroaromatic and two nitramine (HMX and RDX) explosive compounds was spiked onto a PTFE wipe. Explosives were desorbed from the wipe in a commercial thermal desorption system and trapped onto a cooled injection system, which was incorporated into the injection port of the GC. A dual column, dual ECD configuration was adopted to enable simultaneous confirmation analysis of the explosives desorbed. For the desorption of 50 ng of each explosive, desorption efficiencies ranged between 80.0 and 117%, for both columns. Linearity over the range 2.5-50 ng was demonstrated for each explosive on both columns with r2 values ranging from 0.979 to 0.991 and limits of detection less than 4 ng. Desorption of HMX from a PTFE wipe has also been demonstrated for the first time, albeit at relatively high loadings (100 ng).     

Analytical methods for anti-doping control in sport: anabolic steroids with 4,9,11-triene structure in urine

Doping control in sport is mandatory to detect and to control the use of prohibited substances. Due to the growing number of targets, the analysis of doping compounds and their metabolites is carried out using established screening methods. However, detection of anabolic steroids with 4,9,11-triene structure in urine is problematic, so it is necessary to improve the methods.We review the state of the art in doping-control analysis of 4,9,11-trien-3-one steroids, providing an overview of the screening and confirmatory methods developed for these analytes in human urine. First, we review chromatographic techniques. We discuss difficulties in the derivatization of those compounds prior to gas chromatography analyses. In recent years, liquid chromatography has been the preferred technique in drug testing in sport, due to the reduced sample pre-treatment, improved limits of detection and comprehensiveness. We also report on advances and limitations of immunochemical techniques for the analysis of this group of substances.     

Direct determination of anabolic steroids in pig urine by a new SPME-GC-MS method

A new solid phase microextraction (SPME) method coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of four anabolic steroids such as 3α-hydroxy-5α-androstane-17-one (HA), dihydrotestosterone (DHT), androstenedione (AD) and methyltestosterone (MT) in pig urine. SPME was used to extract the four anabolic compounds directly without derivatization. The optimum SPME sampling conditions were based on the home-made carbowax-divinylbenzene (CW-DVB) fiber coating during extraction at 40 °C for 50 min with 0.18 g/mL NaCl solution and 750 rpm stirring speed. The linear ranges of the proposed method were in the range of 8-640 pg/mL for HA and DHT and 16-510 pg/mL for AD and MT, respectively. The detection limits (S/N = 3) were from 2 to 8 pg/mL for the four anabolic steroids. This SPME method provided very high enrichment factors for the four anabolic steroids, which were 1063-fold and 965-fold for HA and DHT at the concentration of 8 pg/mL and 207-fold and 451-fold for AD and MT at the concentration of 16 pg/mL, respectively. The recoveries ranged from 71.3 to 121%, and the RSDs were lower than 12.9%. The method was sensitive and reliable for determination of trace anabolic steroids in biological samples.     

Excretion Study of Stanozolol in Bovine by HPLC-Tandem Mass Spectrometry

Stanozolol is an anabolic steroid illicitly used for growth promoting purposes in animal production. For reasons of public health the use of anabolic steroids as growth promoters is officially banned in Europe in animals intended for consumption. The aim of this work is the investigation of stanozolol metabolites, 3^′-hydroxystanozolol, 4β- hydroxystanozolol and 16β- hydroxystanozolol in urine samples after stanozolol administration to a young calf. After solid phase extraction of urine samples, detection is carried out by HPLC-MS-MS multiple reaction monitoring. Average recovery for the three metabolites is 80%. The method is highly specific and has been validated in terms of linearity, inter and intra day precision. In addition the decision limit CCα and the detection capability CCβ have been determined.     

Development of a dielectric barrier discharge ion source for ambient mass spectrometry

A new ion source based on dielectric barrier discharge was developed as an alternative ionization source for ambient mass spectrometry. The dielectric barrier discharge ionization source, termed as DBDI herein, was composed of a copper sheet electrode, a discharge electrode, and a piece of glass slide in between as dielectric barrier as well as sample plate. Stable low-temperature plasma was formed between the tip of the discharge electrode and the surface of glass slide when an alternating voltage was applied between the electrodes. Analytes deposited on the surface of the glass slide were desorbed and ionized by the plasma and the ions were introduced to the mass spectrometer for mass analysis. The capability of this new ambient ion source was demonstrated with the analysis of 20 amino acids, which were deposited on the glass slide separately. Protonated molecular ions of [M + H](+) were observed for all the amino acids except for L-arginine. This ion source was also used for a rapid discrimination of L-valine, L-proline, L-serine and L-alanine from their mixture. The limit of detection was 3.5 pmol for L-alanine using single-ion-monitoring (SIM). Relative standard deviation (RSD) was 5.78% for 17.5 nmol of L-alanine (n = 5). With the advantages of small size, simple configuration and ease operation at ambient conditions, the dielectric barrier discharge ion source would potentially be coupled to portable mass spectrometers.     

Laser desorption lamp ionization source for ion trap mass spectrometry

A two‐step laser desorption lamp ionization source coupled to an ion trap mass spectrometer (LDLI‐ITMS) has been constructed and characterized. The pulsed infrared (IR) output of an Nd:YAG laser (1064 nm) is directed to a target inside a chamber evacuated to ~15 Pa causing desorption of molecules from the target's surface. The desorbed molecules are ionized by a vacuum ultraviolet (VUV) lamp (filled with xenon, major wavelength at 148 nm). The resulting ions are stored and detected in a three‐dimensional quadrupole ion trap modified from a Finnigan Mat LCQ mass spectrometer operated at a pressure of ≥ 0.004 Pa. The limit of detection for desorbed coronene molecules is 1.5 pmol, which is about two orders of magnitude more sensitive than laser desorption laser ionization mass spectrometry using a fluorine excimer laser (157 nm) as the ionization source. The mass spectrum of four standard aromatic compounds (pyrene, coronene, rubrene and 1,4,8,11,15,18,22,25‐octabutoxy‐29H,31H‐phthalocyanine (OPC)) shows that parent ions dominate. By increasing the infrared laser power, this instrument is capable of detecting inorganic compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

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.