Study of the non-covalent interaction between amyloid-beta-peptide and melatonin using electrospray ionization mass spectrometry

Oxidative stress and unregulated immune response are believed to play a key role in the processes inherent to Alzheimer's disease (AD). The fact that free radicals can result in neurodegeneration suggests that actions against reactive oxygen species may be beneficial in treating and preventing AD. In the light of the suggested link between oxidative stress and AD, it is proposed that antioxidants and, even more, endogenous antioxidants may offer a therapeutic regime for protection against the risk of this disease. For this reason, the formation of non-covalent complexes between amyloid-beta-peptide (A beta) or its oxidized forms and melatonin was studied by quadrupole and Fourier transform ion cyclotron resonance electrospray ionization mass spectrometry. The stability of the non-covalent complex was examined under several experimental conditions, such as orifice voltage, pH, presence of organic modifier, concentration and time. Two different digestion protocols combined with mass spectrometric analysis of the resulting peptide fragments were employed in order to locate the binding site of melatonin in A beta.     

Probing the interaction of kojic acid antibiotics with iron(III) chloride by using electrospray tandem mass spectrometry

Kojic acid, 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, has been used extensively as a clinical iron-chelating drug although the nature of the complexes of iron and kojic acid has not been established. In this article we demonstrate the complexation of kojic acid with iron(III) chloride by using electrospray ionization mass spectrometry (ESI-MS). The ESI-MS analysis revealed different reactions between iron(III) chloride and kojic acid (M), and the mass spectrum exhibited four complexes: [Fe+2(M-H)]+, [Fe+3(M-H)+H]+, [Fe2+4(M-H)+Cl]+, and [Fe2+5(M-H)]+. All these proposed complexes and the presence of chloride ion in one of the dinuclear complexes have been confirmed by isotopic patterns and fragmentation studies by means of tandem mass spectrometry (MSn).     

Studies on the non-covalent complexes between oleanolic acid and cyclodextrins using electrospray ionization tandem mass spectrometry

Non-covalent inclusion complexes formed between an anti-inflammatory drug, oleanolic acid (OA), and alpha-, beta- and gamma-cyclodextrins (CDs) were investigated by means of solubility studies and electrospray ionization tandem mass spectrometry (ESI-MS(n)). The order of calculated association constants (K(1 : 1)) of complexes between OA and different CDs in solution is in good agreement with the order of their relative peak intensities and the relative CID energies of the complexes under the same ESI-MS(n) conditions. These results indicate a direct correlation between the behaviors of solution- and gas-phase complexes. ESI-MS can thus be used to evaluate solution-phase non-covalent complexes successfully. The experimental results show that the most stable 1 : 1 inclusion complexes between three CDs and OA can be formed, but 2 : 1 CD-OA complexes can be formed with beta- and gamma-CDs. Multi-component complexes of alpha-CD-OA-beta-CD (1 : 1 : 1), alpha-CD-OA-gamma-CD (1 : 1 : 1) and beta-CD-OA-gamma-CD (1 : 1 : 1) were found in equimolar CD mixtures with excess OA. The formation of 2 : 1 and multi-component 1 : 1 : 1 non-covalent CD-OA complexes indicates that beta- and gamma-CD are able to form sandwich-type inclusion non-covalent complexes with OA. The above results can be partly supported by the relative sizes of OA and CD cavities by molecular modeling calculations. All the complexes allow the detection of gaseous deprotonated CD-OA complexes in the negative ion mode at high abundances. The relative stabilities of the CDs-OA inclusion complexes in the gas phase can be evaluated from the relative CID energies in the ion trap (alpha-CD-OA < beta-CD-OA < gamma-CD-OA) in the negative ion mode.     

Structural characterization and identification of dibenzocyclooctadiene lignans in Fructus Schisandrae using electrospray ionization ion trap multiple-stage tandem mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry

The electrospray ionization ion trap multiple-stage tandem mass spectrometry (ESI-MSⁿ) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MSⁿ) have been applied successfully to the direct investigation of a number of dibenzocyclooctadiene lignan constituents from the methanol extracts of the Fructus Schisandrae in the positive ion mode. The detailed structural characterization of the same skeleton and different peripheral substituents had been studied and the precise elemental compositions of ions at high mass resolution had been obtained. So the fragmentation mechanisms could be clarified. And the lignan components in Schisandra chinensis (Turcz.) Baill. fruits (SCF) and Schisandra sphenanthera Rehd. et Wils. fruits (SSF) were identified by comparing the structural information and fragmentation mechanisms. Then a pair of isobaric compounds was differentiated. Meanwhile these two similar fruits were distinguished. The research results demonstrated that ESI-MSⁿ technique is a sensitive, selective and effective tool for the direct analysis and rapid determination of constituents in complex mixtures from nature products. And these should be useful for the identification of similar compounds and differentiation of similar species from Chinese herbs.     

Determination of binding selectivities in host-guest complexation by electrospray/quadrupole ion trap mass spectrometry

The quantifiable relationship between the equilibrium solution composition and electrospray (ESI) mass spectral peak intensities of simple host-guest complexes was investigated. Specifically, host-guest complexes of simple crown ethers or glymes with alkali metals and ammonium ions were studied. Comparisons were made between the theoretical concentrations of host-guest complexes derived in solution from known stability constants and the peak intensities for the complexes observed by ESI mass spectrometry (ESI-MS). Two types of complexation experiments were undertaken. First, complexation of a single guest ion, such as an alkali metal, and two crown ethers was studied to evaluate the determination of binding selectivities. Second, complexation of two different guest ions by a single polyether host was also examined. In general, solvation was found to play an integral part in the ability to quantify binding selectivities by ESI-MS. The more similar the solvation energies of the two complexes in the mixture, the more quantifiable their binding selectivities by ESI-MS. In some cases, excellent correlation was obtained between the theoretically predicted selectivity ratios and the ESI mass spectral ratios, in particular when the ESI ratios were adjusted based on evaluation of ESI response factors for the various host-guest complexes.     

Study on the formation of an amoxicillin adduct with methanol using electrospray ion trap tandem mass spectrometry

The possibility of using the protonated methanol-adduct of antimicrobial amoxicillin for its identification and quantification at residue levels has been investigated, since it is impossible to completely suppress the formation of these adducts when methanol is present in the solvent system. This process has been monitored over time and as a function of concentration. It was determined that adducts were instantly formed and that the abundance of the protonated methanol-adduct at m/z 398 increased at the expense of the protonated molecule m/z 366 with storage time. The effect of several common solvents and mobile-phase additives on the ionization efficiency of amoxicillin and the formation of the methanol adduct has also been investigated. It was shown that the mass spectra of amoxicillin were strongly influenced by the solvent in which the analyte is dissolved and by the analyte concentration, as well as by the composition of mobile phase. Methanol was determined to be the best spray solvent, as it provided spectra with the lowest abundance of dimer ions. It was also determined that acetic acid as the mobile-phase additive provided the highest signal intensities, while ammonium acetate should not be used as an additive for the determination of amoxicillin at residue levels. Using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS), fragmentation of the protonated molecules and the protonated methanol-adduct ions, in both positive and negative ion mode, has been performed. The fragmentation was stable and strong product ion spectra were obtained. The linearity of the MS detector response, and that of the chromatographic method, was tested. Due to the linear behaviour it was concluded that the protonated methanol-adduct ion can be used for analytical purposes, i.e. for identification and quantification of amoxicillin at trace levels.     

Analysis of a commercial preparation of erythromycin estolates by tandem mass spectrometry and high performance liquid chromatography/electrospray ionization tandem mass spectrometry using an ion trap mass spectrometer

Because of the lack of a UV chromophore and their much smaller abundances in comparison with the major component, the minor components in erythromycin estolate preparations are difficult to analyze by high performance liquid chromatography ultraviolet (HPLC-UV). Tentative assignment of the major and minor components can be achieved with the combination of full scan and ZoomScan using an ion trap mass spectrometer. Tandem mass spectrometry (MS/MS) provided an effective method to quickly identify most components without chromatographic separation, and all the related compounds, except the isobaric pair ECE and PdMeEA, could be identified in this way. The best result was obtained by using liquid chromatography/tandem mass spectrometry (LC/MS/MS) operated in selected reaction monitoring mode. The major compound, the estolate of erythromycin A (EAE), and seven other minor components, could be separated and identified, with semiquantitative estimates of relative concentrations.     

Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry

The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non-covalent protein complexes has been demonstrated repeatedly. However, questions about correlation of the solution and gas-phase structures of these complexes still produce vigorous scientific discussion. Here, we demonstrate the evaluation of the gas-phase binding of non-covalent protein complexes formed between bovine pancreatic trypsin inhibitor (BPTI) and its target enzymes over a wide range of dissociation constants. Non-covalent protein complexes were detected by ESI-MS. The abundance of the complex ions in the mass spectra is less than expected from the values of the dissociation constants of the complexes in solution. Collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) and a collision model for ion activation were used to evaluate the binding of non-covalent complexes in the gas phase. The internal energy required to induce dissociation was calculated for three collision gases (Ne, Ar, Kr) over a wide range of collision gas pressures and energies using an electrospray ionization source. The order of binding energies of the gas-phase ions for non-covalent protein complexes formed by the ESI source and assessed using CAD-MS/MS appears to differ from that of the solution complexes. The implication is that solution structure of these complexes was not preserved in the gas phase.     

A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation

Reversed-phase microcapillary chromatography (RP-microLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) is one of two prevailing techniques in proteomic analysis, the other being matrix-assisted laser desorption/ionization (MALDI). Despite the arguably better dynamic range obtainable with ESI, MALDI is increasingly popular due to ease of use, ruggedness and the ability to decouple separation from ionization. By contrast, in order to take advantage of the sensitivity and dynamic range afforded by the concentration-dependent nature of ESI, it is directly coupled to separations that take place in small i.d. RP-microLC columns. This gain in sensitivity often comes at a loss of ruggedness due to clogging of the small i.d. RP-microLC columns, one result of which is limited sample throughput. Here we describe a combined micropre-column-microLC-ESI device that is sensitive, rugged and modular in design allowing facile construction and troubleshooting. Due to low signal-to-noise as little as 1 attomole of a peptide can be selected by data-dependent methods for collision-induced dissociation. Importantly, the resulting tandem mass spectrum is of high enough quality to identify the peptide sequence by a database search against a complex database using SEQUEST. Finally, the device is demonstrated to be rugged as judged by >60 consecutive reversed-phase microLC separations on complex peptide mixtures before chromatographic resolution is degraded.     

Investigation of the interaction between arsenic species and thiols via electrospray ionization tandem mass spectrometry

Arsenic species have been known to participate in a number of chemical and biological reactions, including oxidation-reduction reactions, acid-base reactions, covalent interactions, and methylation-demethylation reactions because of the element's multiple and interconvertible oxidation states. Little is known about the structure or bonding behavior between arsenic species and thiolcontaining biomolecules. Therefore, a better understanding of the bonding behavior and detailed information on the molecular structure for arsenic-thiol complexes is needed. As a result, we have investigated the interaction between arsenic species (arsenate (As^V), arsenite (As^III), monomethylarsonic acid (MMA^V), and dimethylarsinic acid (DMA^V)) with biomolecules containing thiol groups (glutathione and cysteine) by electrospray ionization mass spectrometry (ESI-MS). These compounds were dissolved in methanol/water solution and introduced into the MS instrument in order to elucidate the direct bonding behavior of thiol group of biomolecules with arsenic species. In addition, further detailed structural information on this complex was obtained by collision-induced dissociation (CID) measurements.In each mass spectrum for mixture solutions between arsenic species and thiol compounds, various peaks such as protonated arsenic-thiol complexes, protonated noncomplexed thiol compounds, sodium bound cluster ions, and proton bound cluster ions were observed. In these mass spectra, the arsenic complexes were formed by interaction with thiol groups on the cysteine residues. These arsenic-thiol complexes produced a variety of fragment ions by cleavage of chemical bonds, and by interaction of other binding site on thiol compounds in tandem mass spectrometry experiments.     

Determination of carnitine and acylcarnitines in plasma by high-performance liquid chromatography/electrospray ionization ion trap tandem mass spectrometry

A high-performance liquid chromatography/mass spectrometry method was developed for the determination of carnitine, its biosynthetic precursor butyrobetaine, and eight acylcarnitines in plasma. The procedure includes a solid-phase extraction for carnitine and short- and medium-chain acylcarnitines, and a liquid-liquid extraction for protein-bound long-chain acylcarnitines, followed by separation on a reversed-phase column in the presence of a volatile ion-pairing reagent. Detection was achieved using an ion-trap mass spectrometer run in the tandem mass spectrometry (MS/MS) mode. The choice of the matrix for calibrators, used for quantification of these endogenous compounds, was also investigated. Validation was performed for standard quality controls diluted with 4% bovine serum albumin solution and for spiked plasma quality control samples at concentrations between 0.5 and 80 micromol/L, depending on the compound. Intra- and inter-day precisions for the determination of carnitine were below 3.4% and accuracies were between 95.2 and 109.0%. Application of the method to the diagnosis of pathological acylcarnitine profiles of metabolic disorders in a patient suffering from methylmalonic aciduria is presented. The method allows quantification of carnitine, butyrobetaine, acetylcarnitine and propionylcarnitine, and semiquantitative analysis of medium- and long-chain acylcarnitines. In contrast with other methods, no derivatization step is needed.     

Confirmation of gentamicin and neomycin in milk by weak cation-exchange extraction and electrospray ionization/ion trap tandem mass spectrometry

A new procedure for the confirmation of two aminoglycoside antibiotics in milk was developed and validated. This work is among the early applications of ion trap mass spectrometry for regulatory methodology, and it incorporates a novel weak cation-exchange extraction. The procedure was validated for the confirmation of both gentamicin and neomycin at 30 ng ml(-1) and above. Milk is first treated with acid and centrifuged. The supernate, excluding the fat layer, is buffered with sodium citrate to neutral pH. The extract is applied to a weak cation-exchange solid-phase extraction column. Aminoglycosides are eluted with acidified methanol. Following separation by ion-pair liquid chromatography, analytes are ionized with an electrospray interface. Protonated molecular ions are selectively stored in an ion trap mass spectrometer, then collisionally dissociated to yield unique product ion spectra. Confirmation is based on matching spectral responses between samples and comparison standards consisting of a bona fide standard spiked into control extracts. Method performance was demonstrated with replicate samples of control milk, fortified milk, and milk containing incurred residues of each compound.     

Multi-parameter investigation of tandem mass spectrometry in a linear ion trap using response surface modelling

The feasibility of experimental design in combination with subsequent response surface modelling was illustrated for the prediction and interpretation of tandem mass spectrometric (MS/MS) fragmentation data using a linear quadrupole ion trap under various experimental conditions. The instrumental parameters included were (i) the pressure of the collision gas, (ii) the collision energy, (iii) the fill time of the linear ion trap and (iv) the scan rate. The spectral intensity and width of five fragment ions of the doubly charged neuro-active peptide bombesin were used for evaluation, and all experiments were performed so as to resemble the results obtained from a liquid chromatographic peak. The reported results show how fairly simple mathematical tools can be utilized successfully to describe fundamental mechanisms associated with multiple collisional activation and collision-induced dissociation processes without an extensively controlled experimental environment. Most beneficial, using the suggested approach, is the ability to study interaction (synergistic) effects between various parameters. As was realized from the results, many interaction effects are indeed significant. For example, the effect on the signal intensity of different collision gas pressure settings is strongly dependent on the settings of the other parameters. The described approach can easily be adopted for optimization purposes of any MS/MS experiment.     

Metal ion complexes in the structural analysis of phospholipids by electrospray ionization tandem mass spectrometry

The effects of metal cationization on collisionally activated dissociation (CAD) of phospholipids were investigated by electrospray ionization with quadrupole ion trap tandem mass spectrometry. The metal ions include Li(+), Na(+), K(+), Sr(2+), Ba(2+), and the first transition series. CAD of the transition metal ion-bound lipid complexes gave significant yields of product ions that identify the positions of the two fatty acyl substituents on the glycerophospholipid backbone. The cobalt(II) ion, which has a single naturally occurring isotope, was expected to be a better cationization reagent as it produces simpler mass spectra than other transition metal ions. CAD of the cobalt(II) ion complexes of glycerophosphoethanolamines, glycerophosphoglycerols and glycerophosphoserines yielded product ions that revealed information regarding both the lipid classes and the regiospecific positions of the two fatty acyl substituents.     

Confirmatory analysis of beta-lactam antibiotics in kidney tissue by liquid chromatography/electrospray ionization selective reaction monitoring ion trap tandem mass spectrometry

Eleven beta-lactam antibiotics were analyzed in fortified and incurred beef kidney tissue using high-performance liquid chromatography/electrospray ionization/selective reaction monitoring-ion trap tandem mass spectrometry (LC/ESI-SRM-MS(n)). The analytes included: deacetylcephapirin, amoxicillin, cephapirin, desfuroylceftiofur cysteine disulfide (DCCD, a biomarker of ceftiofur), ampicillin, cefazolin, Pen G, oxacillin, cloxacillin, naficillin and dicloxicillin. Analytes were extracted with acetonitrile and water. Clean-up was performed by solid-phase extraction. Limits of confirmation in fortified tissue are as follows (tolerances or target levels in parentheses): deacetylcephapirin: 10-50 ng/g (100 ng/g); amoxacillin: 50-100 ng/g (10 ng/g); cephapirin: 10 ng/g (100 ng/g); DCCD: 500 ng/g (8000 ng/g); ampicillin: 10 ng/g (10 ng/g); cefazolin: 10 ng/g (10-50 ng/g); Pen G: 10 ng/g (50 ng/g); oxacillin: 10 ng/g (10-50 ng/g); cloxacillin: 10 ng/g (10 ng/g); naficillin: 10 ng/g (10-50 ng/g); dicloxacillin: 100-500 ng/g (10-50 ng/g). The present method was also tested on incurred kidney tissue that had previously been analyzed using a microbial assay. Good correspondence was found between the results from this new method and the bioassay. However, the present method is much more specific and, in several cases, more sensitive than the bioassay. In addition, the time of analysis is significantly shorter than the bioassay. We also found that SRM MS(n) was superior in the analysis of unknown incurred tissue than full spectrum MS(n). We also obtained an MS/MS spectrum of DCCD that is significantly at variance with previously published fragmentation spectra.     

Evaluation of the metal binding properties of a histidine-rich fusogenic peptide by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) was used to investigate metal ion interactions of the 18 amino acid peptide fragment B18 (LGLLLRHLRHHSNLLANI), derived from the membrane-associated protein bindin. The peptide sequence B18 represents the minimal membrane-binding motif of bindin and resembles a putative fusion peptide. The histidine-rich peptide has been shown to self-associate into distinct supramolecular structures, depending on the presence of Zn(2+) and Cu(2+). We examined the binding of B18 to the metal ions Cu(2+), Zn(2+), Mg(2+), Ca(2+), Mn(2+) and La(3+). For Cu(2+), we compared the metal binding affinities of the wild-type B18 peptide with those of its mutants in which one, two or three histidine residues have been replaced by serines. Upon titration of B18 with Cu(2+) ions, we found sequential binding of two Cu(2+) ions with dissociation constants of approximately 34 and approximately 725 micro M. Mutants of B18, in which one histidine residue is replaced by serine, still exhibit sequential binding of two copper ions with affinities for the first Cu(2+) ion comparable to that of wild-type B18 peptide, but with a greatly reduced affinity for the second Cu(2+) ion in mutants H112S and H113S. For mutants in which two histidines are replaced by serines, the affinity for the first Cu(2+) ion is reduced approximately 3-10 times in comparison with B18. The mutant in which all three histidine residues are replaced by serines exhibits an approximately 14-fold lower binding for the first Cu(2+) ion compared with B18. For the other metal ions under investigation (Zn(2+), Mg(2+), Ca(2+), Mn(2+) and La(3+)), a modest affinity to B18 was detected binding to the peptide in a 1 : 1 stoichiometry. Our results show a high affinity of the wild-type fusogenic peptide B18 for Cu(2+) ions whereas the Zn(2+) affinity was found to be comparable to that of other di- and trivalent metal ions.     

Analysis of polyaromatic quinones in a complex environmental matrix using gas chromatography ion trap tandem mass spectrometry

GC/MS and GC/MS/MS in a quadrupole ion trap were used to analyze for anthraquinone, alkyl anthraquinones, benz[a]anthracene-7, 12-dione and 9-fluoranone in a sediment obtained from an aluminum smelter settling pond contaminated with polycyclic aromatic hydrocarbons. By standard GC/MS analysis many of these target compounds were either undetectable or their confirmation uncertain because of matrix interferences. Detection and identification were greatly improved by using GC/MS/MS. GC/MS/MS analyses were performed by selecting the molecular ion (M) of a target compound and fragmenting it via collision induced dissocation (CID) to yield product ions corresponding to loss of CO for unsubstituted compounds or CO plus CH(3) for alkylated compounds. The CID conditions were optimized using anthraquinone and 2-methylanthraquinone standards by varying the CID excitation energy and RF storage levels to yield optimum amounts of fragment ions. CID experiments were performed using both resonant and non-resonant wave forms. Although both excitation techniques gave comparable results for the removal of matrix interferences, non-resonant excitation provided more characteristic spectra for the alkylated anthraquinones. Monitoring of secondary fragmentation products, such as M-2CO, provided greater discrimination from matrix interferences than the use of primary fragmentation products, such as M-CO.     

Effects of heavy gases on the tandem mass spectra of peptide ions in the quadrupole ion trap

Heavy gases (xenon, argon, krypton, methane) have been used to improve the performance of the quadrupole ion trap when performing collision-induced dissociation on peptides. MS/MS spectra reveal that increased amounts of internal energy can be deposited into peptide ions and more structural information can be obtained. Specifically, the pulsed introduction of the heavy gases (as reported previously by Doroshenko, V. M.; Cotter, R. J.Anal. Chem. 1996, 68, 463) provides greater energy deposition without the deleterious effects that static pressures of heavy gas have on spectra. Internal energy deposition as indicated by a qualitative evaluation of MS/MS spectra shows pulsed introduction of heavy gases enables ions to obtain more internal energy than possible by using static pressures of the same heavy gases. A linear correlation is observed between the percentage of heavy gas added and the ratio of product ions used to reflect internal energy deposition. Results here also show that upon pulsed introduction of heavy gases, empirical optimization of a single frequency resonant excitation signal is no longer needed to obtain good MS/MS spectrometry efficiency. The presence of many low mass-to-charge ratio ions and the absence of side chain cleavages in the MS/MS spectra of peptides suggests that the propensity for consecutive fragmentations is increased with the pulsed introduction of heavy gases. In addition, by varying the delay time between introduction of the gas and application of the resonant excitation signal, the amount of fragmentation observed in MS/MS spectra can be changed.     

Potentials of ion trap collisional spectrometry for liquid chromatography/electrospray ionization tandem mass spectrometry determination of buprenorphine and nor-buprenorphine in urine, blood and hair samples

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the analysis of buprenorphine (BUP) and nor-buprenorphine (NBUP) in biological fluids. Analytes are isolated from urine and blood, after addition of d4-buprenorphine (d4-BUP) as internal standard, by solid-phase extraction. Preparation of hair involves external decontamination, mechanical pulverization, overnight incubation in acidic medium, and neutralization prior to extraction. Enzymatic hydrolysis with beta-glucuronidase may be performed to distinguish between free and total BUP. Chromatographic separation is accomplished by gradient elution on a cyanopropyl 2.1 x 150 mm column. Positive ion ESI and MS analyses are carried out in an ion trap mass spectrometer. The use of this mass analyzer allows effective collisional experiments to be performed on ESI-generated MH+ species. Abundant product ions are produced, which can be monitored together with precursor ions without losing sensitivity. Thus, assay selectivity is definitely increased with respect to LC/ESI-MS/MS methods in which only precursor ions are monitored. The method has good linearity (calibration curves were linear in the range 0.1-10 ng/mL in urine and blood, in the range 10-160 pg/mg in hair) and limits of detection of 0.05 ng/mL for both BUP and NBUP in blood and urine samples, of 4 pg/mg for both analytes in hair. Both intra- and inter-assay precision and accuracy were satisfactory at three concentrations studied: relative standard deviations were <13.7% in urine, <17.3% in blood, <17.8% in hair; percent deviation of the mean from the true value was always <10.5% in urine and blood, <16.1% in hair. The method can be used to determine both analytes in the urine and hair of drug addicts on replacement therapy, and in post-mortem blood specimens when there is suspicion of drug-related death.