Agents In Human Urine Research Articles

Simultaneous analysis of degradation products of Novichok agents and conventional nerve agents in human urine by ion chromatography-tandem mass spectrometry using ammonium regeneration solution

An ion chromatography (IC)-tandem mass spectrometry (MS/MS) method to analyze nerve agent degradation products in human urine was developed. Six degradation products of conventional nerve agents and six Novichok agent degradation products were analyzed simultaneously despite their differences in hydrophilicity and acidity. Using ammonium regeneration solution improved the peak shapes greatly compared with the results obtained with the ordinary IC-MS/MS configuration. For urine samples, a simple pretreatment method of dilution with water and ultrafiltration was used. The detection limits of the nerve agent degradation products were sufficiently low (10–250 ng/mL) and the calibration curves showed acceptable linearity. Due to the absence of a derivatization step, throughput was higher than for our previous derivatization-liquid chromatography-MS/MS method.

Analysis of degradation products of Novichok agents in human urine by hydrophilic interaction liquid chromatography–tandem mass spectrometry

PurposeThe detection of hydrolysis products of Novichok agents in biological samples from victims is important for confirming exposure to these agents. However, Novichok agents are new class of nerve agent and there have been only few reports on analyses of Novichok agent degradation products. Here, we developed hydrophilic interaction liquid chromatography (HILIC)–tandem mass spectrometry (MS/MS) methods to detect Novichok agent degradation products in human urine with simple pretreatment and high sensitivity.MethodsA Poroshell 120 HILIC-Z column was used to analyze six Novichok agent degradation products. For urine samples, we used a simple pretreatment method, which consisted of deproteinization with acetonitrile and microfiltration. We calculated the pKa values of the OH groups, the log P values, and the molecular weights to investigate the difference in chromatographic behaviors of the Novichok agent degradation products and the degradation products of conventional nerve agents.ResultsSix Novichok agent degradation products, including N-(bis-(diethylamino)methylidene)-methylphosphonamidic acid (MPGA), which could not be detected by our previous method, could be analyzed with sufficient peak shape and mutual separation. The detection limits of six Novichok agent degradation products were sufficiently low (1–50 ng/mL) and the calibration curves showed sufficient linearity. The physicochemical parameters of Novichok agent degradation products were different from those of conventional nerve agent degradation products, and this explains the difference in chromatographic behaviors.ConclusionSix Novichok agent degradation products were successfully analyzed by HILIC–MS/MS. Due to the absence of a derivatization step, throughput performance was higher than our previous derivatization-liquid chromatography–MS/MS method.

A Smartphone-Based Fluorescent Electronic Tongue for Tracing Dopaminergic Agents in Human Urine.

The importance of tracing dopaminergic agents in the progression assessment of Parkinson's disease has boosted the demand for fast, sensitive, and real-time multi-analyte detection. Herein, visual and fingerprint fluorimetric patterns have been created by an optical sensor array to simultaneously detect and discriminate among levodopa, carbidopa, benserazide, and entacapone, as important dopaminergic agents. A dual emissive nanoprobe consisting of red quantum dots and blue carbon dots with an overall pink emission has been fabricated to provide unique emission patterns in the presence of the target analytes. The sensor elements in the array come from it's differential response in the absence and presence of cetyltrimethylammonium bromide under alkaline conditions. A smartphone camera was used to take photos from the solutions in the wells. Distinct changes in the spectral profiles along with vivid and concentration-dependent color variations led to visual discrimination of dopaminergic agents in a broad concentration range. The results of linear discriminant analysis revealed great discrimination accuracies. Different concentrations of the target analytes were excellently recognized in human urine. The high sensitivity of the array, which is a bonus to rapid, on-site, and visual discrimination of dopaminergic agents, holds great promise for routine analysis of real-world clinical samples.

An automatic online solid-phase dehydrate extraction-ultra-high performance supercritical fluid chromatography-tandem mass spectrometry system using a dilution strategy for the screening of doping agents in human urine

An automatic online solid-phase dehydrate extraction (SPDE)-ultra-high performance supercritical fluid chromatography (UHPSFC)-MS/MS system was developed in this study, in which the automatic SPDE procedure was coupled with UHPSFC to allow UHPSFC to analyze aqueous samples directly. Moreover, a pre-column dilution strategy was employed, which focused the analytes in strong desorption solvent on the column head and helped to obtain narrow and symmetric peaks. The online SPDE-UHPSFC-MS/MS system was firstly applied to the screening of 45 prohibited substances in human urine for doping control, during which all the mechanisms and features of the online system were fully studied. The majority (91%) of the target compounds achieved weak matrix effects (80–120%), indicating that the online method was accurate and reliable thanks to the SPDE procedure and efficient UHPSFC separation. Owing to the reduction of the matrix effects, large volume injection and the pre-column dilution, the online system could achieve high sensitivity with the LODs ranging from 0.0380 ng L−1 to 1.24 μg L−1. Under the optimized conditions, the extraction recoveries of 66% target analytes were more than 50%. All the target compounds showed good linearity with linear correlation coefficients higher than 0.9928. The accuracy values of all the spiked prohibited substances were within 80.8–119.7%, while the RSDs% for the intra-/inter-day precision were within 10.8% and 15.4%. Compared with the dilute-and-shoot-ultra-high performance liquid chromatography-MS/MS method, in which the urine samples were simply diluted before analyzing, this online method was superior in sensitivity and reducing matrix effects, which demonstrated its utility in doping control. Compared with the previously reported online SPE-SFC system, the online SPDE-UHPSFC-MS/MS system showed advantages in automation, efficiency, sensitivity and chromatographic performance. In summary, the online SPDE-UHPSFC-MS/MS system is capable of analyzing complex aqueous samples.

Liquid chromatography and supercritical fluid chromatography as alternative techniques to gas chromatography for the rapid screening of anabolic agents in urine

This work describes the development of two methods involving supported liquid extraction (SLE) sample treatment followed by ultra-high performance liquid chromatography or ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS and UHPSFC–MS/MS) for the screening of 43 anabolic agents in human urine. After evaluating different stationary phases, a polar-embedded C18 and a diol columns were selected for UHPLC–MS/MS and UHPSFC-MS/MS, respectively. Sample preparation, mobile phases and MS conditions were also finely tuned to achieve highest selectivity, chromatographic resolution and sensitivity.Then, the performance of these two methods was compared to the reference routine procedure for steroid analyses in anti-doping laboratories, which combines liquid–liquid extraction (LLE) followed by gas chromatography coupled to tandem mass spectrometry (GC–MS/MS). For this purpose, urine samples spiked with the compounds of interest at five different concentrations were analyzed using the three analytical platforms. The retention and selectivity of the three techniques were very different, ensuring a good complementarity. However, the two new methods displayed numerous advantages. The overall procedure was much faster thanks to high throughput SLE sample treatment using 48-well plates and faster chromatographic analysis. Moreover, the highest sensitivity was attained using UHPLC–MS/MS with 98% of the doping agents detected at the lowest concentration level (0.1ng/mL), against 76% for UHPSFC–MS/MS and only 14% for GC–MS/MS. Finally, the weakest matrix effects were obtained with UHPSFC–MS/MS with 76% of the analytes displaying relative matrix effect between −20 and 20%, while the GC–MS/MS reference method displayed very strong matrix effects (over 100%) for all of the anabolic agents.

TiO₂ nanoparticles functionalized monolithic capillary microextraction online coupled with inductively coupled plasma mass spectrometry for the analysis of Gd ion and Gd-based contrast agents in human urine.

In this work, a novel method of TiO2 nanoparticles (NPs) functionalized monolithic capillary microextraction (CME) online coupling with inductively coupled plasma mass spectrometry (ICPMS) was developed for the sequential determination of Gd(3+) and Gd-based contrast agents in human urine samples. The monolithic capillary was prepared by embedding anatase TiO2 NPS in the poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EDMA) framework. The Gd(3+) and Gd-based contrast agents (such as gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) and Gd-DTPA-bismethylamide (Gd-DTPA-BMA)) display different adsorption behaviors on the prepared monolithic capillary which possesses the adsorption properties of both anatase TiO2 NPS and poly(MAA-EDMA) monolith. Under the optimized conditions, the limits of detection (LODs) were found to be 3.6, 3.2, and 4.5 ng L(-1) for Gd(3+), Gd-DTPA, and Gd-DTPA-BMA, respectively, which are the lowest up to date. The enrichment factor was 25-fold with the sample throughput of 5 h(-1). The proposed method was validated by the analysis of Gd(3+) and Gd-DTPA in the healthy human urine samples as well as Gd(3+) and Gd-DTPA-BMA in patient urine samples. It was found that only a small amount of the free Gd(3+) was released from Gd-DTPA-BMA, and accurate results could be obtained since no oxidation/reduction or subtraction is involved in this method. This method is simple, sensitive, and rapid and provides a very attractive nonchromatography strategy for the speciation of Gd(3+) and Gd-based contrast agents in urine samples.

Antibiotic exposure in a low-income country: screening urine samples for presence of antibiotics and antibiotic resistance in coagulase negative staphylococcal contaminants.

Development of antimicrobial resistance has been assigned to excess and misuse of antimicrobial agents. Staphylococci are part of the normal flora but are also potential pathogens that have become essentially resistant to many known antibiotics. Resistances in coagulase negative staphylococci (CoNS) are suggested to evolve due to positive selective pressure following antibiotic treatment. This study investigated the presence of the nine most commonly used antimicrobial agents in human urine from outpatients in two hospitals in Ghana in relation to CoNS resistance. Urine and CoNS were sampled (n = 246 and n = 96 respectively) from patients in two hospitals in Ghana. CoNS were identified using Gram staining, coagulase test, and MALDI-TOF/MS, and the antimicrobial susceptibility to 12 commonly used antimicrobials was determined by disk diffusion. Moreover an analytical method was developed for the determination of the nine most commonly used antimicrobial agents in Ghana by using solid-phase extraction in combination with HPLC-MS/MS using electron spray ionization. The highest frequency of resistance to CoNS was observed for penicillin V (98%), trimethoprim (67%), and tetracycline (63%). S. haemolyticus was the most common isolate (75%), followed by S. epidermidis (13%) and S. hominis (6%). S. haemolyticus was also the species displaying the highest resistance prevalence (82%). 69% of the isolated CoNS were multiple drug resistant (≧4 antibiotics) and 45% of the CoNS were methicillin resistant. Antimicrobial agents were detected in 64% of the analysed urine samples (n = 121) where the most frequently detected antimicrobials were ciprofloxacin (30%), trimethoprim (27%), and metronidazole (17%). The major findings of this study was that the prevalence of detected antimicrobials in urine was more frequent than the use reported by the patients and the prevalence of resistant S. haemolyticus was more frequent than other resistant CoNS species when antimicrobial agents were detected in the urine.

Sensitive and robust method for anabolic agents in human urine by gas chromatography–triple quadrupole mass spectrometry

A rapid, sensitive and robust gas chromatography–triple quadrupole mass spectrometry method was developed for the determination of seven anabolic agents in human urine. The selection of analytes includes the main metabolites of all anabolics with higher sensitivity requirements. After optimizing the fragmentation conditions for each compound, 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 compound. Adequate intermediate precision, below 14%, was observed for all of the compounds at the lower concentration tested. The concentrations assayed were in accordance with the performance limits required by the World Anti-Doping Agency (WADA). Unlike a previously published GC/QqQ method, detection of 17α-methyl-5β-androstane-3α,17β-diol (the main metabolites of methyltestosterone) at 2ng/mL was accomplished under routine conditions. The qualitative method was applied to the analysis of 1367 samples in the span of 2 weeks, as part of the doping control of the XVI Pan American Games which took place in Mexico (14th–30th October, 2011). The high sensitivity was maintained during the analysis of all analytical batches, proving for the first time the excellent ruggedness of GC/QqQ methods.

Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry

The chromatographic behaviour of 44 polar compounds (23 beta-adrenergic agents, 11 stimulants, 4 narcotics and 6 phenolalkylamines) included in the list of prohibited substances and methods of the World Anti-Doping Agency, has been investigated under hydrophilic interaction liquid chromatography conditions by application of different mobile phase compositions (percentage of the organic solvent, type and amount of mobile phase additive and ionic strength) and column temperatures. Detection of analytes was performed by a triple quadrupole mass spectrometer in positive ionization mode and selected reaction monitoring acquisition mode after liquid/liquid extraction. Data collected using as stationary phase type-B silica materials from different producers, showed that the best chromatographic conditions in terms of peak shape, selectivity and chromatographic retention were obtained using an initial percentage of acetonitrile of 90%, a column temperature of 35 °C, a mobile phase pH of 4.5 and ammonium acetate (5 mM) and acetic acid (0.1%) as mobile phase additives. The selected chromatographic conditions were used to develop screening and confirmation analytical procedures to detect polar compounds in human urine for antidoping purpose. The developed methods were validated in terms of specificity, matrix effect, linearity, precision, accuracy, sensitivity, robustness and repeatability of retention times and relative ion abundances. Such methods offer attractive alternatives and considerable advantages over traditional approaches especially for the analysis of the phenolalkylamines.

Qualitative detection of diuretics and acidic metabolites of other doping agents in human urine by high-performance liquid chromatography–tandem mass spectrometry: Comparison between liquid–liquid extraction and direct injection

Direct injection of urine has gained interest in the field of analytical toxicology, including doping control analysis. However, implementation of a direct urinalysis method for the LC–MS/MS detection of 34 diuretics and 9 other doping agents yielded several analytical problems, which were not observed using a traditional liquid–liquid extraction. Therefore a comparative study was made between liquid–liquid extraction and direct injection. Comparison of validation results showed that the liquid–liquid extraction at pH 7 allows to analyze samples without major drawbacks regarding matrix effects. Hence, good sensitivity was observed and detection limits ranged between 1 and 250 ng/mL for all compounds. In the direct injection approach shifted retention times were observed for several acidic and basic compounds due to unwanted matrix effects. This shift was reduced by a 25-fold dilution of the urine samples. Besides the improved retention time stability the diluted samples also exhibited lower ion suppression than the undiluted ones. After 25-fold dilution, detection limits ranged between 10 and 250 ng/mL for all compounds. Since these detection limits are at or below the minimum required performance level, imposed by the World Anti-Doping Agency, the method could be applied to routine anti-doping analysis. Samples, previously declared positive, were reanalysed using both the liquid–liquid extraction and direct injection. With both techniques all 26 samples were found to be positive, showing the applicability of direct injection for the analysis of diuretics.

Generic sample preparation and dual polarity liquid chromatography—time‐of‐flight mass spectrometry for high‐throughput screening in doping analysis

The requirements on initial testing in doping control are getting tighter regarding efficiency and speed while the scope of analytes is getting more diverse and, consequently, the need for high-throughput methods is apparent. In this study, a comprehensive screening method for doping agents in human urine is presented, based on solid phase extraction (SPE) and liquid chromatography-time-of-flight mass spectrometry (LCTOFMS). The method covers most of the compound groups in the list of prohibited substances by World Anti-Doping Agency (WADA). Mixed-mode SPE on two types of sorbent and the use of negative ionization mode besides the commonly used positive mode in electrospray ionization (ESI) allowed detection of acidic compounds, such as sulpho-conjugated metabolites. A run time of 8 minutes for each of the two ESI polarities was achieved. The method was validated regarding relative ionization efficiency, selectivity and signal to noise at the WADA's minimum required performance limit (MRPL) level, resulting in the acceptance of 197 compounds. A selection of 20 compounds was submitted for a more thorough validation, including extraction recovery, repeatability and linearity. Recovery and linearity (R(2)) varied mainly between 83-115% and 0.78-0.99, respectively. Median values for repeatability at the MRPL and 10 x MRPL levels were below 20%. A mean and median mass accuracy of 1.2 and 0.80 mDa, respectively, was achieved. The present method represents at the moment the widest coverage of low molecular weight prohibited substances for the screening in sports, providing an approach for further rationalisation of the analytical work-flow in the doping control laboratories.

Introduction of HPLC/orbitrap mass spectrometry as screening method for doping control

A new doping control screening method has been developed, for the analysis of doping agents in human urine, using HPLC/orbitrap with in-source collision-induced dissociation and atmospheric pressure chemical ionization. The developed method allows the detection of 29 compounds, including agents with antiestrogenic activity, beta(2) agonists, exogenous anabolic steroids, and other anabolic agents. The mass accuracy of this method is better at 2 ppm using an external reference. The detection limit for all compounds tested was better than 100 pg/ml. The recoveries of most analytes were above 70%. The measured median repeatability values for doping agents included in the method at concentrations of 1 and 10 ng/ml were 21 and 17%, respectively. The relative standard deviation (RSD) of the intraday precision (n = 6) ranged from RSD = 16-22%, whereas the interday precision (n = 18), ranged from RSD = 17-26%, depending on the solute concentration investigated.

Rapid Screening of Doping Agents in Human Urine by Vacuum MALDI-Linear Ion Trap Mass Spectrometry

Rapid Screening of Doping Agents in Human Urine by Vacuum MALDI-Linear Ion Trap Mass Spectrometry

Rapid Screening of Doping Agents in Human Urine by Vacuum MALDI-Linear Ion Trap Mass Spectrometry

Detection of doping agents in urine frequently requires extensive separation prior to chemical analyses. Gas or liquid chromatography coupled to mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time and, sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique. Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range (m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit of detection and lower limit of quantification for all five analytes have been determined to be 0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal standard, and the quantitative measurements were achieved by the selective reaction monitoring (SRM) mode. The method was validated and showed consistency in the results. Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and quantification of doping agents in urine.

A general screening method for doping agents in human urine by solid phase extraction and liquid chromatography/time-of-flight mass spectrometry

A general screening method based on solid phase extraction (SPE) and liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) was developed and investigated with 124 different doping agents, including stimulants, β -blockers, narcotics, β 2 -adrenergic agonists, agents with anti-estrogenic activity, diuretics and cannabinoids. Mixed mode cation exchange/C8 cartridges were applied to SPE, and chromatography was based on gradient elution on a C18 column. Ionization of the analytes was achieved with electrospray ionization in the positive mode. Identification by LC/TOFMS was based on retention time, accurate mass and isotopic pattern. Validation of the method consisted of analysis of specificity, analytical recovery, limit of detection and repeatability. The minimum required performance limit (MRPL), established by World Anti-Doping Agency (WADA), was attained to 97 doping agents. The extraction recoveries varied between 33 and 98% and the median was 58%. Mass accuracy was always better than 5 ppm, corresponding to a maximum mass error of 0.7 mDa. The repeatability of the method for spiked urine samples, expressed as median of relative standard deviations (RSD%) at concentrations of MRPL and 10 times MRPL, were 14% and 9%, respectively. The suitability of the LC/TOFMS method for doping control was demonstrated with authentic urine samples.

Simultaneous determination of beta‐blocking agents and diuretics in doping analysis by liquid chromatography/mass spectrometry with scan‐to‐scan polarity switching

A previously described method for the screening of 18 diuretics and probenecid was substantially extended with 21 beta-blockers and 8 other diuretics allowing simultaneous determination of diuretics and beta-adrenergic blocking agents in human urine. Analysis was performed using an ion trap instrument with an electrospray ionisation (ESI) interface after liquid/liquid extraction with ethyl acetate. Full-scan MS and full-scan MS2 were applied in combination with scan-to-scan polarity switching. All compounds were separated in less than 22 min. The detection limits for the diuretics were between 5 and 100 ng/mL and for the beta-adrenergic blocking agents were between 5 and 500 ng/mL. The excretion of carvedilol was followed after intake of one tablet of Dimitone. Other doping agents including strychnine, norbuprenorphine and mesocarb hydroxysulfate could also be detected with this method.

Liquid chromatography clean-up method to improve identification of anabolic agents in human urine by gas chromatography–mass spectrometry

A confirmatory procedure to allow the unequivocal identification of the presence of urinary anabolic steroids and metabolites banned for use in sport by athletes is presented. A single liquid chromatography (LC) procedure was developed to selectively isolate the metabolites of the most abused anabolic agents. Five millilitre urine samples, with trenbolone added as a retention time marker, were concentrated by solid-phase extraction, enzymatically hydrolysed and directly injected onto the LC system. Chromatographic separation was carried out on a 150 mm×4.6 mm I.D. column, packed with 3 μm Hypersil BDS-C 18 with gradient elution and UV absorbance detection (340 nm). Fractions containing compounds of interest were collected and evaporated. After solvent evaporation and derivatisation to form trimethylsilyl (TMS) derivatives, fractions were analysed by GC/MS in full scan mode with an external ionisation ion trap mass spectrometer. A validation procedure for qualitative analysis was performed for the anabolic agents with special sensitivity requirements according to the International Olympic Committee (IOC) and the World Anti-Doping Agency (WADA). Appropriate precision, accuracy and recoveries were found. The selectivity of the method showed that no interfering peaks were observed at the retention time of the analytes, allowing obtaining full scan spectra even at the low concentrations required (2 ng/ml).

Identification of isopropyl substituted β-blocking agents in human urine by gas chromatography and tandem mass spectrometry

A method for the sensitive detection of isopropyl substituted β-blocking agents in human urine is presented. The sample preparation step involves enzymatic hydrolysis, solid phase extraction and derivatisation withN-methyl-N-trimethylsilyl-trifluoroacetamide. The instrumental analysis was performed using gas chromatography-mass spectrometry with an ion trap mass spectromeler. The mass spectrometer was operated in the scan as well as in the MS-MS mode.

Screening and confirmation analysis of anabolic agents in human urine by gas chromatography — hybrid mass spectrometry (high resolution — time of flight)

This work presents a novel method for the detection of anabolic agents administration by the analysis of human urine. The method here presented allows both the screening and the confirmation of five anabolic agents at concentrations below 1μgl−1, i.e. whenever it is not possible to obtain a full spectrum by gas chromatography technique with mass spectrometry detection unit (GC–MS) (single quadrupole mass detector), neither in electron impact (EI) nor in chemical ionization (CI) mode.The instrumental apparatus here used is constituted by a gas chromatograph (GC) coupled to a hybrid mass spectrometry detection (MS) unit, formed by a high resolution magnetic mass spectrometer (HRMS) and by a time of flight (TOF) orthogonal mass spectrometer.The proposed approach has been revealed very useful in the long term retrospective analysis of low concentration of anabolic agents and/or their metabolites, and especially of the five major anabolic agents (unchanged clenbuterol, and the main metabolites of methandienone, methyltestosterone, 19-nortestosterone and stanozolol), whose detection and quantitative confirmation, at levels of 2μgl−1, is presently imposed by the International Olympic Committee Medical Commission for the antidoping analysis of athletes urine.

High speed determination of beta-receptor blocking agents in human urine by liquid chromatography/tandem mass spectrometry.

Beta-receptor blocking agents are present on the international market in a huge variety. The International Olympic Committee prohibits the use of these drugs in several sport sections and doping control laboratories analyse urine samples of high-performance athletes with different techniques. Therefore, fast and reliable methods are required to enable a sensitive detection of many drugs and a high throughput of samples. In the present study a screening procedure is described using high speed liquid chromatography and multiple reaction monitoring to identify 32 beta-receptor blocking agents extracted from human urine. Urine specimens (blank urine samples, spiked urine samples and specimens of excretion studies) were hydrolysed, extracted and analysed within 7 min. Quasi-molecular ions (M(+) + H) of the beta-blockers are generated by means of an atmospheric pressure chemical ionization interface followed by collision-induced dissociation in a triple quadrupole mass spectrometer and subsequent detection of daughter ions. Proposals for the origin of common and individual secondary ions are presented.