Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis

Analysis of the broad range of trace chemical modifications of proteins in biological samples is a significant challenge for modern mass spectrometry. Modification at lysine and arginine residues, in particular, causes resistance to digestion by trypsin, producing large tryptic peptides that are not readily sequenced by mass spectrometry. In this work, we describe the analysis of ribonuclease (RNase) modified by methylglyoxal (MGO) under physiological conditions. For detection of modifications, we use comparative analysis of the single combined spectra extracted from the full-scan MS data of the tryptic digests from native and modified proteins. This approach revealed 11 ions unique to MGO-modified RNase, including a 32-amino acid peptide containing a modified Arg-85 residue. Sequential digestion of MGO-modified RNase by endoproteinase Glu-C and trypsin was required to obtain peptides that were amenable to sequencing analysis. Arg-39 was identified as the main site of modification (35% modification) on MGO-modified Rnase, and the dihydroxyimidazolidine and hydroimidazolone derivatives were the main adducts formed, with minor amounts of the tetrahydropyrimidine and argpyrimidine derivatives. For identification of these products, we used variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the tetrahydropyrimidine-containing peptides and dehydration of the dihydroxyimidazoline-containing peptides. The resultant spectra were dependent on the cone voltage and collision energy, and analysis of spectra at various settings permitted structural assignments. These studies illustrate the usefulness of single combined mass spectra extracted from full-scan data and variations in source and collision cell voltages for detection and structural characterization of chemical adducts on proteins.     

Fragmentation behavior of glycated peptides derived from D-glucose, D-fructose and D-ribose in tandem mass spectrometry

Nonenzymatic glycosylation (or glycation) is a common nonenzymatic side-chain specific sequence-independent posttranslational modification formed by the reaction of reducing carbohydrates with free amino groups. Thus, proteins can react with aldoses or ketoses to yield Amadori or Heynes compounds, respectively. Here, the fragmentation behavior of D-glucose and D-ribose-derived Amadori peptides as well as D-fructose-derived Heynes peptides were studied by collision-induced fragmentation (CID) after electrospray (ESI) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). All three sugar moieties displayed characteristic fragmentation patterns accompanying the parent and the fragment ions, which could be explained by consecutive losses of water and formaldehyde. Glucose-derived Amadori parent and fragment ions displayed losses of 18, 36, 54, 72, and 84 u at a characteristic intensity distribution compared with losses of 18, 36, 54, 72, 84, and 96 u for D-fructose-derived ions and losses of 18, 36, and 54 u for ribose-derived ions. Furthermore, each sugar moiety produced indicative lysine-derived immonium ions that were successfully used in a precursor ion scan analysis to identify Amadori peptides in a tryptic digest of bovine serum albumin (BSA) glycated with D-glucose. BSA was modified on lysine residues at positions 36, 160, 235, 256, 401, and 548.     

Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

Screening of methylenedioxyamphetamine‐ and piperazine‐derived designer drugs in urine by LC–MS/MS using neutral loss and precursor ion scan

This study describes a method for the screening of methylenedioxyamphetamine‐ and piperazine‐derived compounds in urine by liquid chromatography‐tandem mass spectrometry. These substances, characterized by possessing common moieties, are screened using precursor ion and neutral loss scan mode and then quantified in multiple reaction monitoring acquisition mode. Based on the product‐ion spectra of different known molecules, chosen as ‘model’, characteristic neutral losses and product ions were selected: piperazines were detected in precursor ion scan of m/z 44 and neutral loss of 43 and 86 while amphetamines in precursor ion scan of m/z 133, 135 and 163. The applicability of the screening approach was studied in blank urine spiked with selected analytes and processed by solid‐phase extraction. Linearity, matrix effect, precision, accuracy, limits of detection and limits of quantification were evaluated both for the screening and the quantification methods. The ability of the screening method to provide semi‐quantitative data was demonstrated. This method appears to be a useful tool for the identification of designer drugs derived from piperazines or methylenedioxyamphetamines and can be potentially applied to other drug classes. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of different scan methods for the urinary detection of corticosteroid metabolites by liquid chromatography tandem mass spectrometry

Different approaches for the non‐target detection of corticosteroids in urine have been evaluated. As a result of previous studies about the ionization (positive/negative) and fragmentation of corticosteroids, several methods based on both precursor ion (PI) and neutral loss (NL) scans are proposed. The applicability of these methods was checked by the injection of a standard solution containing 19 model compounds. Five of the studied methods (NL of 76 Da; PI of 77, 91 and 105; PI of 237; PI of 121, 147 and 171; and NL of 38 Da) exhibited satisfactory results at the concentration level checked (corresponding to 20 ng/ml in sample). Some other methods in negative ionization mode such as the NL of 104 Da were found to lack sufficient sensitivity. Some of the applied methods were found to be specific for a concrete structure (NL of 38 Da for fluorine containing corticosteroids) while others showed a wide range applicability (PI of 77, 91 and 105 showed response in all model compounds). Interference by endogenous compounds was also tested by the analysis of negative urines and urines spiked with different corticosteroids. The suitability of these methods for the detection of corticosteroid metabolites was checked by the analysis of urine samples collected after the administration of methylprednisolone and triamcinolone. A combination of the reported methods seems to be the approach of choice in order to have a global overview about the excreted corticosteroid metabolites. Copyright © 2009 John Wiley & Sons, Ltd.     

Versatility of tandem mass spectrometry for focused analysis of oxylipids

Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) in the multiple reaction monitoring (MRM) scan mode has been the primary MS method applied for the target identification of specific and minor oxylipids in complex matrices, such as eicosanoids and docosanoids, which are potent lipid mediators derived from polyunsaturated fatty acid oxygenation. However, the high specificity of MRM can limit the detection of species with m/z MRM transitions not covered by the method. In addition to MRM, tandem‐quadrupole mass analyzers enable other experiments to be conducted, by fragmenting ions via collision‐induced dissociation process (CID). This paper presents the potential of tandem mass spectrometry for the focused analysis of oxylipids. We have successfully developed an LC‐MS/MS method for the identification of precursor ions of m/z 115, a diagnostic product ion of 5‐hydroxy‐ and 5‐epoxy‐fatty acids. As a proof of concept, the developed method was used to discover several oxylipids oxidized at C5 derived from arachidonic acid (C20 : 4) oxygenation in a hypothalamus rat extract that were not identified using the target MRM methodology. The proposed focused MS/MS‐based approach in a tandem mass analyzer has proven to be a powerful strategy to accelerate the identification of oxylipids with structural similarities and assist the field of lipidomic research. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of nifedipine in dog plasma by high‐performance liquid chromatography with tandem mass spectrometric detection

Nifedipine is a dihydropyridine calcium channel blocker used widely in the management of hypertension and other cardiovascular disorders. In this work, a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated to determine nifedipine in dog plasma using nimodipine as the internal standard. Chromatographic separation was carried out on a C₈ column. The mobile phase consisted of a mixture of acetonitrile, water and formic acid (60:40:0.2, v/v/v) at a flow rate of 0.5 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode via an atmospheric pressure chemical ionization source. The method has a lower limit of quantification of 0.20 ng/mL with consumption of plasma as low as 0.05 mL. The linear calibration curves were obtained in the concentration range of 0.20–50.0 ng/mL (r = 0.9948). The recoveries of the liquid extraction method were 74.5–84.1%. Intra‐day and inter‐day precisions were 4.1–8.8 and 6.7–7.4%, respectively. The quantification was not interfered with by other plasma components and the method was applied to determine nifedipine in plasma after a single oral administration of two controlled‐release nifedipine tablets to beagle dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid detection of cyanobacterial toxins in precursor ion mode by liquid chromatography tandem mass spectrometry

We established an analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in the precursor ion mode for simultaneous qualitative monitoring of various groups of cyanobacterial toxins. The toxin groups investigated were paralytic shellfish poisoning (PSP) toxins, anatoxins (ANAs), cylindrospermopsins (CYNs), microcystins (MCs), and nodularins (NODs), including rare and uncharacterized derivatives found in plankton and water matrices. Alternative analytical methods based on tandem mass spectrometry commonly operate in multiple reaction monitoring (MRM) mode and depend on prior knowledge of putative toxigenicity of the cyanobacterium species and strain, and the expected toxin variants. In contrast, the precursor ion mode yields diagnostic mass fragments for the detection of characteristic compounds of the different toxin classes and thus allows monitoring of a large set of unspecified cyanotoxins of various groups, even when the species composition is undetermined or uncertain. This rapid method enables screening for a wide spectrum of toxic cyanobacterial metabolites and degradation products in a single chromatographic separation with detection limits at nanogram levels. The precursor ion technique is a valuable adjunct to existing mass spectrometric methods for cyanotoxins, although it is not a complete replacement for detailed quantitative analysis requiring comprehensive sample cleanup.     

High‐throughput determination of Sudan Azo‐dyes within powdered chili pepper by paper spray mass spectrometry

A high‐throughput mass spectrometric method is presented for the simultaneous detection of Sudan I, II, III, IV and Para‐Red azo‐dyes in foodstuff. The method is based on the use of paper spray mass spectrometry (MS) and deuterium‐labeled internal standards on a triple‐quadrupole instrument. A detailed assay of each azo‐dye was performed by the isotope dilution method, through the precursor ion scan approach, using deuterium‐labeled internal standards. The gas‐phase breakdown pattern of each labeled and unlabeled analogue displays the naphthoic moiety as a common fragment. Sudan dyes can be determined above the threshold of 1 ppm. Paper spray allows for a wide range of analytes and samples to be investigated by MS in the open air and without sample preparation and bypassing chromatography. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of isomeric 5‐hydroxytryptophan‐ and oxindolylalanine‐containing peptides by mass spectrometry

Cells continuously produce reactive oxidative species that can modify all cellular components. In proteins, for example, cysteine, methionine, tryptophan (Trp), and tyrosine residues are particularly prone to oxidation. Here, we report two new approaches to distinguish two isomeric oxidation products of Trp residues, i.e. 5‐hydroxytryptophan (5‐HTP) and oxindolylalanine (Oia) residues, in peptides. First, 2‐nitrobenzenesulfenyl chloride, known to derivatize Trp residues in position 2 of the indole ring, was used to label 5‐HTP residues. The mass shift of 152.98 m/z units allowed identifying 5‐HTP‐ besides Trp‐containing peptides by mass spectrometry, whereas Oia residues were not labeled. Second, fragmentation of the Oia‐ and 5‐HTP‐derived immonium ions at m/z 175.08 produced ions characteristic for each residue that allowed their identification even in the presence of y₁ ions at m/z 175.12 derived from peptides with C‐terminal arginine residues. The pseudo MS³ spectra acquired on a quadrupole time‐of‐flight hybrid mass spectrometer displayed two signals at m/z 130.05 and m/z 132.05 characteristic for Oia‐containing peptides and a group of six signals (m/z 103.04, 120.04, 130.04, 133.03, 146.04, and 148.04) for 5‐HTP‐cointaining peptides. In both cases, the relative signal intensities appeared to be independent of the sequence providing a specific fingerprint of each oxidative modification. Copyright © 2012 John Wiley & Sons, Ltd.     

Electrospray ionization mass spectrometric study of end‐groups in peroxydicarbonate‐initiated radical polymerization

Initiation by diethyl peroxydicarbonate (E‐PDC), di‐n‐tetradecyl peroxydicarbonate (nTD‐PDC), di‐n‐hexadecyl peroxydicarbonate (nHD‐PDC), and di‐2‐ethylhexyl peroxydicarbonate (2EH‐PDC) of free‐radical polymerizations of methyl methacrylate in benzene solution was studied by end‐group analysis via electrospray ionization mass spectrometry (ESI‐MS). Unambiguous assignment of ESI‐MS peaks allows for identification of the type of radical that starts chain growth. In case of initiation by dialkyl peroxydicarbonates with linear alkyl groups, almost exclusively alkoxy carbonyloxyl species, which are the primary fragments from initiator decomposition, occur as end‐groups. With 2EH‐PDC, however, both the primary 2‐ethylhexoxy carbonyloxyl fragment and a second moiety, which is formed by decarboxylation of the 2‐ethylhexoxy carbonyloxyl radical, are clearly observed as end‐groups. The decarboxylation process is described by a concerted mechanism which involves a 1,5‐hydrogen shift reaction. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6071–6081, 2008     

Structural identification of hindered amine light stabilisers in coil coatings using electrospray ionisation tandem mass spectrometry

Hindered amine light stabilisers (HALS) are the most effective antioxidants currently available for polymer systems in post‐production, in‐service applications, yet the mechanism of their action is still not fully understood. Structural characterisation of HALS in polymer matrices, particularly the identification of structural modifications brought about by oxidative conditions, is critical to aid mechanistic understanding of the prophylactic effects of these molecules. In this work, electrospray ionisation tandem mass spectrometry (ESI‐MS/MS) was applied to the analysis of a suite of commercially available 2,2,6,6‐tetramethylpiperidine‐based HALS. Fragmentation mechanisms for the [M + H]⁺ ions are proposed, which provide a rationale for the product ions observed in the MS/MS and MS³ mass spectra of N‐H, N‐CH₃, N‐C(O)CH₃ and N‐OR containing HALS (where R is an alkyl substituent). A common product ion at m/z 123 was identified for the group of antioxidants containing N‐H, N‐CH₃ or N‐C(O)CH₃ functionality, and this product ion was employed in precursor ion scans on a triple quadrupole mass spectrometer to identify the HALS species present in a crude extract from of a polyester‐based coil coating. Using MS/MS, two degradation products were unambiguously identified. This technique provides a simple and selective approach to monitoring HALS structures within complex matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

A systematic tandem mass spectrometric study of anion attachment for improved detection and acidity evaluation of nitrogen‐rich energetic compounds

The development of rapid, efficient, and reliable detection methods for the characterization of energetic compounds is of high importance to security forces concerned with terrorist threats. With a mass spectrometric approach, characteristic ions can be produced by attaching anions to analyte molecules in the negative ion mode of electrospray ionization mass spectrometry (ESI‐MS). Under optimized conditions, formed anionic adducts can be detected with higher sensitivities as compared with the deprotonated molecules. Fundamental aspects pertaining to the formation of anionic adducts of 1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocane (HMX), 1,3,5‐trinitro‐1,3,5‐triazinane (RDX), pentaerythritol tetranitrate (PETN), nitroglycerin (NG), and 1,3,5‐trinitroso‐1,3,5‐triazinane energetic (R‐salt) compounds using various anions have been systematically studied by ESI‐MS and ESI tandem mass spectrometry (collision‐induced dissociation) experiments. Bracketing method results show that the gas‐phase acidities of PETN, RDX, and HMX fall between those of HF and acetic acid. Moreover, PETN and RDX are each less acidic than HMX in the gas phase. Nitroglycerin was found to be the most acidic among the nitrogen‐rich explosives studied. The ensemble of bracketing results allows the construction of the following ranking of gas‐phase acidities: PETN (1530‐1458 kJ/mol) > RDX (approximately 1458 kJ/mol) > HMX (approximately 1433 kJ/mol) > nitroglycerin (1427‐1327.8 kJ/mol).     

Determination of sirolimus in rabbit arteries using liquid chromatography separation and tandem mass spectrometric detection

Sirolimus, an effective immunosuppressive agent, is used for drug eluting stents. During stent development, an analytical method for the determination of sirolimus in tissue needs to be established. Normally, tissue samples are homogenized and then analyzed against the calibration standards prepared in a tissue homogenate. This approach provides insufficient control of the homogenization process. In this paper, tissue quality control samples were introduced for the optimization of the homogenization process during method development, but also allowance for the performance evaluation of the entire analytical process. In addition, a new approach using rabbit blood as a homogenization medium was developed to stabilize sirolimus in rabbit tissue homogenates. Calibration standards and quality controls were prepared by spiking different sirolimus working solutions into rabbit blood. Homogenization quality control samples were prepared by injecting other sirolimus working solutions into empty test tubes and pre-cut arteries within pre-defined masses. A high-throughput homogenization procedure was optimized based on the specific chemical properties of sirolimus. The linear dynamic range was between 49.9 pg/mL and 31.9 ng/mL to accommodate the expected artery homogenate concentrations. Additionally, quality controls in rabbit blood were also used in the extraction to support the calibration standards. The accuracy and precision of the quality controls in rabbit blood reflect the extraction performance and the accuracy and precision of the homogenization tissue quality controls reflect the overall performance of the method. The mean bias was between -4.5 and 0.2% for all levels of quality controls in the blood and between 4.8 and 14.9% for all levels of the homogenization tissue quality controls. The CVs of all concentration levels were < or =5.3% for the quality controls in blood and < or =9.2% for the homogenization tissue quality controls. The method was successfully applied to determine the concentration of sirolimus in the rabbit arteries.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

Fast liquid chromatography separation and multiple‐reaction monitoring mass spectrometric detection of neurotransmitters

We describe here the fast LC‐MS/MS separation of a mixture of neurotransmitters consisting of dopamine, epinephrine, norepinephrine, 3,4‐dihydroxybenzylamine (DHBA), salsolinol, serotonin, and γ‐aminobutyric acid (GABA). The new UltiMate® 3000 Rapid Separation system (RSLC) was successfully coupled to the 4000 QTRAP mass spectrometer operating in multiple‐reaction monitoring (MRM) mode. The separation was attained using a 100 mm length, 2.2 μm particle size Acclaim column at a flow rate of 0.5 mL/min. The column back pressure was 350 bar, while the total run time including column re‐equilibration was 5.2 min. The peak resolution was minimally affected by the fast separation. The RSLC‐MRM separation was found to have a precision range based on peak area for 50 replicate runs of 2–5% CV for all analytes, and the reproducibility of the retention time for all analytes was found to range from 0–2% CV. The described method represents an almost seven times shorter analysis time of neurotransmitters using LC/MRM which is very useful in screening large quantities of biological samples for various neurotransmitters.     

Quantitation and pharmacokinetics of 1,4‐diamino‐2,3‐dicyano‐1,4‐bis (2‐aminophenylthio) butadiene (U0126) in rat plasma by liquid chromatography‐tandem mass spectrometry

A simple, robust, and rapid LC‐MS/MS method was developed for the quantitation of U0126 and validated in rat plasma. Plasma samples (20 μL) were deproteinized using 200 μL ACN containing 30 ng/mL of chlorpropamide, internal standard. Chromatographic separation performed on an Agilent Poroshell 120 EC‐C₁₈ column (4.6 × 50 mm, 2.7 μm particle size) with an isocratic mobile phase consisting of a 70:30 v/v mixture of ACN and 0.1% aqueous formic acid. Each sample was run at 0.6 mL/min for a total run time of 2 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with positive ESI at m/z 381 → 123.9 for U0126 and m/z 277 → 175 for the internal standard. The standard curve was linear over a concentration range of 20–5000 ng/mL with correlation coefficients greater than 0.9965. Precision, both intra‐ and interday, was less than 10.1% with an accuracy of 90.7–99.4%. No matrix effects were observed. U0126 in rat plasma degraded approximately 41.3% after 3‐h storage at room temperature. To prevent degradation, sample handling should be on an ice bath and all solutions kept at 4°C. This method was successfully applied to a pharmacokinetic study of U0126 at various doses in rats.     

Validation and application of a liquid chromatography‐tandem mass spectrometric method for the determination of GDC‐0152 in human plasma using solid‐phase extraction

A liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of GDC‐0152 in human plasma to support clinical development. The method consisted of a solid‐phase extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₇‐GDC‐0152 was used as the internal standard. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 0.02–10.0 ng/mL for GDC‐0152. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 99.3% with a precision (%CV) of 13.9%. For quality control samples at 0.0600, 2.00 and 8.00 ng/mL, the between‐run %CV was ≤8.64. Between‐run percentage accuracy ranged from 98.2 to 99.6%. GDC‐0152 was stable in human plasma for 363 days at ?20°C and for 659 days at ?70°C storage. GDC‐0152 was stable in human plasma at room temperature for up to 25 h and through three freeze–thaw cycles. In whole blood, GDC‐0152 was stable for 12 h at 4°C and at ambient temperature. This validated LC‐MS/MS method for determination of GDC‐0152 was used to support clinical studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Confirmation of anabolic hormone residues in bovine blood by micro-HPLC–ion spray–tandem mass spectrometry

Sensitive, specific analytical methods for the determination of an-abolics in biological matrices are essential to control the illegal use of these substances in food-producing animals. Programs of residue control are performed annually in Italy for the determination of endogenous sex hormones (17β-estradiol, progesterone, testoster-one) for which maximum physiological levels have been established. At present the methods used in the Italian programs to determine natural hormones in bovine blood are based on the sensitive radio-immunoassay (RIA), due to relatively low levels of these substances in plasma/serum. In this study, we report a new method based on tandem mass spectrometry with on-line micro-high performance liquid chromatography (micro-HPLC-MS-MS) using an atmos-pheric pressure ionization (API) source and an ion spray (IS) interface for the specific direct detection of natural (progesterone and testosterone), and synthetic (17β19-nortestosterone and me-droxyprogesterone) hormone residues in bovine serum. 17-Methyl-testosterone was used as the internal standard. Analytes were extracted with acetate buffer, purified on C18 Solid Phase Extrac-tion (SPE) cartridge and separated on a reverse phase C18 micro-HPLC column (300 mm × 1 mm, 5 μm), using acetonitrile-water, 80:20 (v/v) containing 2mM ammonium acetate as the mobile phase, at a flow rate of 10 μl/min. When anabolic hormones were ionized in the IS interface operating in the positive ion mode, only the protonated molecules, [M+H]⁺, were generated, without evidence of any fragmentation. These served as precursor ions for collision induced dissociation (CID) and Diagnostic daughter ions for each analyte were identified in order to carry out analysis by micro-HPLC-MS-MS in the selected reaction monitoring (SRM) mode. For the analytes in question, the response of the mass detector was related linearly to the quantity of each analyte injected between 10 and 300 pg, in the SRM mode. The limit of detection, based on a 3:1 signal-to-noise ratio, is 6–7 pg for the hormones. Recoveries were higher than 83% for 17β-19-nortestosterone, testosterone, and 17-methyltestosterone, and 72% for the medroxyprogesterone, and progesterone. The micro-HPLC-MS-MS method for the determi-nation of anabolic hormones in bovine blood requires no sample derivatization, minimal sample preparation, and provides a sensi-tive, selective, rapid alternative to the existing purification, separa-tion, and detection techniques. At present this very sensitive method is being successfully applied to measure bovine serum concentra-tions of natural hormones, such as testosterone and progesterone, in order to then confirm any illegal administration of these sub-stances.