Comparison of Hair Fatty Alcohols by N‐Alkylpyridinium Isotope Quaternization and Matrix‐assisted Laser Desorption/ionization Mass Spectrometry for Drug Abuse Monitoring

Based on our previous report on N‐alkylpyridinium isotope quaternization (NAPIQ) for the analysis of alcoholic and α,β‐unsaturated ketone compounds, we have further applied NAPIQ method in the screening of hair lipids in drug abusers. Relative isotopic quantification was used for comparison of fatty alcohols between normal and drug abuse group. The NAPIQ strategy was proven to be a high‐throughput method in the metabolic comparison studies of different group samples. The attached N‐cationic pyridinium significantly improved the detection sensitivity for these fatty alcohols in matrix‐assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometric (MALDI‐FTMS) analysis. The experimental results showed that the levels of fatty alcohols in the hair of heroin abuse group decreased significantly compared with the normal groups, which may be the results of the inducing of peroxidation enzyme. NAPIQ was proven to be an effective and alternative method in the research of fatty alcoholic metabolism for drug abuse monitoring.     

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.     

Development and validation of a liquid chromatographic method for the analysis of positron emission tomography radiopharmaceuticals with Ru(bpy)(3)(2+)-KMnO(4) chemiluminescence detection.

A sensitive, selective and rapid high-performance liquid chromatographic (HPLC) method with chemiluminescence (CL) detection was developed and validated for the analysis of positron emission tomography (PET) radiopharmaceuticals. This method is based on the CL reaction of PET compounds with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)] and acidic potassium permanganate [KMnO(4)]. After optimization of the reaction conditions, 12 of the 14 PET compounds investigated could be successfully detected and showed good performance in terms of sensitivity, linearity and reproducibility. In particular, for compounds with a tertiary amine functional group, the limits of detection were ppb levels for a 20 microL injection volume. Finally, this method was used to determine PET compounds for calculating of specific radioactivity in pharmaceutical samples.     

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.     

An LC-MS screening method with library identification for the detection of steroids in dietary supplements

For many years anabolic-androgenic steroids (AAS) are by far the most frequently detected pharmacological substances in doping control. In order to improve their performances, professional sportsmen are often tempted to take dietary supplements. However, due to the frequent and widespread occurrence of contaminated supplements, the use of such products is not without risk for the athletes involved. In order to minimize the chances of an unattended positive doping test or serious health problems, fast and reliable screening methods for the detection of anabolic steroids in dietary supplements are needed. A general screening procedure requires the fast and unambiguous detection of a large range of steroids. Gas chromatography-mass spectrometry (GC-MS) has been used intensively in the detection of doping substances for the past 40 years. Over time, many laboratories have delivered spectra to be included in standard reference databases, one of which is maintained by the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). In recent years, however, liquid chromatography coupled to mass spectrometry (LC-MS) has gained popularity. Unfortunately, existing GC-MS libraries are not applicable to LC-MS analysis. In the present study, a new mass spectral library of 88 steroids was developed, along with a fast UPLC-MS method. For the construction of this mass spectral library, three different mass spectra were measured for each steroid, with a sample cone voltage of 30, 60 and 100 V, respectively. This method was then successfully tested on contaminated dietary supplements which had previously been tested by means of a targeted LC-MS/MS method. Overall, the library search was shown to identify the same compounds as the MRM method.     

Direct analysis of anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption-dielectric barrier discharge ionization mass spectrometry

Rapid detection of trace level anabolic steroids in urine is highly desirable to monitor the consumption of performance enhancing anabolic steroids by athletes. The present article describes a novel strategy for identifying the trace anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption (LPTD) coupled to dielectric barrier discharge (DBD) ionization mass spectrometry. Using this method the steroid molecules are enriched within a liquid droplet during the thermal desorption process and desorbed all-together at the last moment of droplet evaporation in a short time domain. The desorbed molecules were ionized using a dielectric barrier discharge ion-source in front of the mass spectrometer inlet at open atmosphere. This process facilitates the sensitivity enhancement with several orders of magnitude compared to the thermal desorption at a lower temperature. The limits of detection (LODs) of various steroid molecules were found to be in the range of 0.05–0.1 ng mL⁻¹ for standard solutions and around two orders of magnitude higher for synthetic urine samples. The detection limits of urinary anabolic steroids could be lowered by using a simple and rapid dichloromethane extraction technique. The analytical figures of merit of this technique were evaluated at open atmosphere using suitable internal standards. The technique is simple and rapid for high sensitivity and high throughput screening of anabolic steroids in urine.     

0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride as a sensitive derivatizing agent for the electron capture gas liquid chromatographic analysis of keto steroids.

0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride was used to prepare oximes of steroids with keto groups in selected positions; 3,17 and 20-monoketo; 3,17 and 3,20-diketo. Some of the 3-keto steroids had hindered 17-hydroxyl groups which were not readily amenable to esterification with perfluoroanhydrides, the most commonly used derivatizing agents for electron capture gas chromatographic analysis of hydroxy steroids. The oximes were readily prepared from 5 ng of each of the compounds tested, and with testosterone it was demonstrated that the derivative could be prepared from as little as 0.1 ng. The derivatives were stable to gas chromatography and extremely sensitive to electron capture detection. The sensitivity ranged from 1.5 X 10(4) coulombs per mole of progesterone. Because of the ease of preparation of the derivatives, their stability in common solvents and analytical manipulative techniques, the reagent would be suitable for the micro analysis of biologically significant keto steroids by electron capture gas chromatography.     

A new approach for generic screening and quantitation of potential genotoxic alkylation compounds by pre-column derivatization and LC-MS/MS analysis

A generic LC-MS/MS method was developed for the analysis of potentially genotoxic alkyl halides. A broad selection of alkyl halides were derivatized using 4-dimethylaminopyridine in acetonitrile. The reaction conditions for derivatization, i.e., solvent, reaction time, temperature and concentration of alkyl halide, active pharmaceutical ingredient (API), and reagent, were optimized for sensitivity and robustness. The interference of the matrix and the API and the presence of water on the derivatization reaction were investigated for a model drug product (paracetamol/caffeine tablets). Hydrophilic interaction liquid chromatography was used to allow a quantitative determination of the derivatives by tandem mass spectrometry. The derivatization reaction was shown to be selective for alkyl halides, although some reactivity was also observed for an aromatic sulfonate, which is also genotoxic. Even though differences in reaction efficiencies have been observed, the enhanced sensitivity obtained by the derivatization allows the majority of the alkyl halides to be detected by MS/MS at relevant levels for genotoxic impurity evaluation, i.e., 10 mg kg(-1). Another key advantage is that for the majority of derivatives, reagent-related fragments are produced, which allows low-level screening for alkyl halides. Highly specific MS detection can be performed using neutral loss and precursor ion scan experiments. The applicability of a generic screening method will make the genotox evaluation less dependent on the quality of assessments based on predictions only, and it will provide essential information during the development of new chemical entities. In addition to screening, target analysis in the low milligrams per kilogram range can be performed. A similar response of the derivatized compounds was obtained in the range of 1-100 mg kg(-1) with a reproducibility better than 10%, which is sufficient for the determination of alkyl halides in APIs and drug products.     

Quantum dots assisted laser desorption/ionization mass spectrometric detection of carbohydrates: qualitative and quantitative analysis

A quantum dots (QDs) assisted laser desorption/ionization mass spectrometric (QDA‐LDI‐MS) strategy was proposed for qualitative and quantitative analysis of a series of carbohydrates. The adsorption of carbohydrates on the modified surface of different QDs as the matrices depended mainly on the formation of hydrogen bonding, which led to higher MS intensity than those with conventional organic matrix. The effects of QDs concentration and sample preparation method were explored for improving the selective ionization process and the detection sensitivity. The proposed approach offered a new dimension to the application of QDs as matrices for MALDI‐MS research of carbohydrates. It could be used for quantitative measurement of glucose concentration in human serum with good performance. The QDs served as a matrix showed the advantages of low background, higher sensitivity, convenient sample preparation and excellent stability under vacuum. The QDs assisted LDI‐MS approach has promising application to the analysis of carbohydrates in complex biological samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Development of an on-line automated sample clean-up method and liquid chromatography–tandem mass spectrometry analysis: application in an in vitro proteolytic assay

Fluorescence detection has been a method of choice in industry for screening assays, including identification of enzyme inhibitors, owing to its high-throughput capabilities, excellent reproducibility, and sensitivity. Occasionally, inhibitors are identified that challenge the fluorescence assay limit, necessitating the development of more sensitive detection methods to assess these compounds. For data mining purposes, however, original assay conditions may be required. A direct method transfer to highly sensitive and specific LC-MS-based methods has not always been possible due to the presence of MS-incompatible neutral detergents and non-volatile salts in the assay matrix. Utilizing an in vitro proteolytic screening assay for the serine protease hepatitis C virus (HCV) nonstructural (NS) 3 protease as a test case, we report the development of an automated sample clean-up procedure implemented on-line with liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis to complement fluorescence detection. Ion exchange and peptide microtraps were employed to remove MS-incompatible assay matrix components. Three protease inhibitors were used to validate the MS/MS method. Comparable potencies were achieved for these compounds when assessed by fluorescence and MS/MS detection. Furthermore, four-fold less enzyme could be utilized when employing the MS/MS method compared to fluorescence detection. The longer analysis time, however, resulted in reduced sample capacity. The potency of our designed HCV NS3 protease inhibitors are thus routinely evaluated using a continuous fluorescence-based assay. Only pertinent inhibitors approaching the fluorescence assay sensitivity limit are subsequently analyzed further by LC-MS/MS. This methodology allows us to maintain a database and to compare results independent of the detection method. Despite the relatively slow sample turnaround time of this LC-MS approach, the versatility of the automated on-line clean-up procedure and sample analysis can be applied to assays containing reagents which were historically considered to be MS incompatible.     

The use of library identification and common fragments for the identification of corticosteroids in forensic samples

The detection of corticosteroids and sex steroids in samples with no content indication, which are confiscated for forensic investigation, is a challenge in doping analysis. A screening method based on the identification of androgens, estrogens, gestagens, and their esters by means of a mass spectral library, along with a fast ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) method, was recently developed in our lab for the analysis of dietary supplements. However, for forensic investigations, it is important to extend the scope of the method to corticosteroids in various matrices. Therefore, 36 corticosteroids were added to the mass spectral library, and the sample preparation step was modified so that androgens, gestagens, corticosteroids, and their esters could be analyzed with only one injection with the UPLC-MS method. A complementary tool to the existing library identification was found in the extraction of common fragment ions out of the full scan data obtained for the library search. The fragment ion with m/z 147 was found to be a good marker for the detection of steroids. Extra confirmation was obtained from the fragment ions with m/z 135 (for all steroids) and 237 (specific for corticosteroids) or from the fragment ions with m/z 77, 91, and 105. The effectiveness of this approach was evaluated on some samples previously screened for forensic investigation with thin-layer chromatography and confirmed with a targeted gas chromatography–mass spectrometry method. This study shows that the combination of the library identification and the common fragment ions approach can be a valuable tool in the detection of steroids without defining any target at the start of the analysis.     

Analysis of meat samples for anabolic steroids residues by liquid chromatography/tandem mass spectrometry

A rapid, specific and highly sensitive multi-residue method for the determination of anabolic steroid residues in bovine, pork and poultry muscle tissues was developed. The sample preparation involves enzymatic digestion followed by extraction with methanol. The crude extract was cleaned up by solid-phase extraction (SPE) combining C18 and NH2 columns. The detection was carried out by a highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using both positive and negative ionization modes. Natural and synthetic steroids covering different polarities could be extracted, concentrated and purified using one single method. Mobile phase composition and additives were optimized to achieve the highest sensitivity. The linearity was not good enough for quantitative analysis but the method was well-suited for qualitative confirmation. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.5 ng g(-1) for all the compounds in the three types of meat studied. The developed method is suitable for routine analysis in our laboratories.     

Sample preparation for the determination of steroids (corticoids and anabolics) in feed using LC

An improved sample preparation procedure for the determination of 17 steroids (corticoids (CC) and androgenic anabolic steroids (AAS)), used potentially as growth promoters, in feed samples has been developed. This procedure is based on two reported LC-UV methods. The improved procedure includes a leaching process using ACN, saponification, and SPE using polymeric cartridges. The proposed method was validated according to the EU criteria established for quantitative screening methods in PFS. The extraction efficiencies, decision limits (CCalpha) and detection capabilities (CCbeta), for these compounds were in the ranges of 82-100%, 19-40, and 24-53 microg/kg, respectively. The repeatability and the within-laboratory reproducibility at 1.0, 1.5, and 2.0 CCbeta levels were smaller than 10%. Accuracy was in the 97-101% range. The robustness was evaluated using the Youden robustness test. This method was applied to the analysis of steroids in different kinds of FS with satisfactory results.     

Electron-capturing derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry.

Derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) has been examined. Under APCI conditions, gas-phase electrons are provided by the corona discharge and captured by electron-affinitive compounds. In negative APCI-MS, therefore, ultrahigh sensitivity can be obtained by tagging neutral steroids, whose ionization efficiencies are low in the conventional APCI-MS, with electron-capturing moieties, such as a nitro group. We synthesized various boronic acid and hydrazine derivatives having electron-capturing moieties as derivatization reagents for 1,2-diol compounds and oxosteroids, respectively. Among reagents examined, those having the 2-nitro-4-trifluoromethylphenyl moiety were most effective in increasing sensitivity. That is, the detection responses of the derivatives with these reagents were increased by several to more than 200-fold over intact steroids, where limits of detection were some picograms. The developed derivatization procedures were applied to analyses of small amounts of steroids in human plasma and gave satisfactory results.     

The analysis of café espresso using two-dimensional reversed phase-reversed phase high performance liquid chromatography with UV-absorbance and chemiluminescence detection

In this study, an activity based screening technique combining two-dimensional liquid chromatography (2DHPLC) with UV-absorbance and chemiluminescence detection was applied to study “Ristretto”, “Decaffeinatto” and “Volluto” espresso coffees. This technique, which coupled the separation power of 2DHPLC with the sensitivity and selectivity of the chemiluminescence detection, offers great potential for screening complex samples for antioxidant compounds. Detailed information regarding the complexity of the sample, and the variation between these three coffees could be obtained using this multidimensional-hyphenated method of analysis.     

Relationships between structure,ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I–IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H]⁺ or [M + H–nH₂O]⁺ in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05–20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05–1, 2–5 and 10–20 ng/mL, respectively. Steroids including the conjugated keto‐functional group at C3 showed good proton affinity and stability, and generated the [M + H]⁺ ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H]⁺ ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H ? H₂O]⁺ or [M + H ? 2H₂O]⁺ ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC‐MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I–V) in human urine. Copyright © 2015 John Wiley & Sons, Ltd.     

End-column chemiluminescence detection for pressurized capillary electrochromatographic analysis of norepinephrine and epinephrine

A simple and convenient end-column chemiluminescence (CL) detection coupled to pressurized capillary electrochromatography (pCEC) was described. Luminol and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) were adopted as mode compounds to evaluate the feasibility of end-column reactor. Detailed analysis of ABEI revealed that the high sensitivity could be obtained with the reactor. Furthermore, determination of norepinephrine (NE) and epinephrine (EP), which were labeled with ABEI, was accomplished by using the end-column pCEC-CL detection based on ABEI-potassium ferricyanide-alkaline medium CL reaction system. Under the optimum conditions, the detection limit (S/N=3) of NE and EP was 0.08 microM and 0.06 microM, respectively. The proposed method has also been successfully applied to the analysis of adrenaline hydrochloride injection sample.     

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.     

Multi-residue screening of a minimum package of anabolic steroids in urine with GC-MS

The method comprises the screening of two groups of anabolic compounds, the stilbenes and several steroids. All compounds, inclusive their metabolites when possible, for which gas chromatography-mass spectrometry (GC-MS) currently is the preferred analytical technique, are included. Two different derivatives are prepared. One group, including the stilbenes, is detected as HFB derivative (Method 1), the second group is detected as TMS derivative (Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a minimum package of anabolic steroids to be included in National Residue Control Plans based on Council Directive 96/23 and complies with the current Minimum Required Performance Limits. The method has been validated according to Commission Decision 2002/657/EC. The CCalpha and CCbeta values are based on the detection of the most abundant ion. Results of validation experiments are presented. The method is flexible and due to the non-specific sample clean-up more and new anabolic compounds can be easily added in order to new monitoring requirements.     

Fast screening of anabolic steroids and other banned doping substances in human urine by gas chromatography/tandem mass spectrometry

A fast and sensitive method for the comprehensive screening of anabolic agents and other banned doping substances using gas chromatography/tandem mass spectrometry (GC/MS/MS) with an external ionization ion trap mass spectrometer is presented. The method takes advantage of the resolving power of MS/MS to eliminate background interferences, thus speeding up the chromatographic analysis. For each compound, different fragmentation reactions were studied and their collision energies optimized to obtain the best sensitivity in terms of their signal-to-noise ratio (S/N). A dramatic reduction in overall analysis time was achieved compared with other common approaches. More than 50 substances could finally be monitored in less than 7.4 min with detection limits (S/N >3) lower than 0.5 ng ml(-1) for most of the compounds with special sensitivity requirements according to the International Olympic Committee (IOC). A validation procedure for qualitative analysis was performed. The selectivity of the method showed that no interfering peaks were observed at the retention time of the analytes. Good intermediate precision, below 25% for most of the compounds, and robustness were observed. The optimized method was successfully applied to analyse more than 100 real human urine samples with optimum sensitivity and specificity rates.