Electrospray ionization mass spectrometry: a major tool to investigate reaction mechanisms in both solution and the gas phase

Electrospray ionization mass spectrometry (ESI-MS), in conjunction with its tandem version ESI-MS/MS, is now established as a major tool to study reaction mechanisms in solution. This suitability results mainly from the ability of ESI to "fish" ions directly from solution to the gas phase environment of mass spectrometers. In this review, we summarize recent studies from our laboratory on the use of on-line monitoring by ESI-MS ion fishing of several types of reactions that permitted us to follow how these reactions progress as a function of both time and conditions using the ultra-high sensitivity and speed of ESI-MS to detect and even characterize transient reaction intermediates. We also show that the intrinsic reactivity of each key gaseous species fished by ESI can be further investigated via ESI-tandem mass spectrometry experiments, searching for the most active species via gas-phase ion/molecule reactions. In the gas-phase, solvent and counter-ion effects are absent. These studies often permit a detailed overview of major steps via the interception, characterization and reactivity investigation of key reaction players.     

Cine-substitution in the Stille coupling: evidence for the carbenoid reactivity of sp3-gem-organodimetallic iodopalladio-trialkylstannylalkane intermediates

Two complementary routes to sp3-gem-organodimetallic iodopalladio-trialkylstannanylalkanes are presented. Such intermediates have been proposed as Pd-carbenoid precursors in the Busacca-Farina cine-substitution mechanism in the Stille coupling. The decomposition of iodomethyltrialkylstannanes by Pd(0) catalysts was monitored by 1H, 2D, and 119Sn NMR. The formation of ethylene, trace formaldehyde, and iodotrialkylstannanes was detected. When the reaction was carried out in the presence of norbornene, the corresponding cyclopropane was produced in good yield. These observations are consistent with the intermediacy of a Pd-carbenoid species. sp3-gem-Organodimetallic iodopalladio-trialkylstannanylalkane complexes were also prepared under stoichiometric conditions via transmetalation from tin to Pd(II). Me3SnCH2Sn(CH2CH2CH2)3 reacted with [(D-t-BPF)PdI]+I-, yielding the (D-t-BPF)Pd(II)ICH2SnMe3 complex that dimerized to form ethylene and cyclopropanated norbornene. The carbenoid reactivity of iodopalladio-trialkylstannanylalkanes complexes validates the Busacca-Farina mechanism of the cine-substitution in the Stille coupling.     

Study of the Transient Reactive Pd(IV) Intermediate in the Pd(OAc)2‐Catalyzed Oxidative Coupling Reaction System by Electrospray Ionization Tandem Mass Spectrometry

Pd‐catalyzed oxidative coupling reaction was of great importance in the aromatic C? H activation and the formation of new C? O and C? C bonds. Sanford has pioneered practical, directed C? H activation reactions employing Pd(OAc)₂ as catalyst since 2004. However, until now, the speculated reactive Pd(IV) transient intermediates in these reactions have not been isolated or directly detected from reaction solution. Electrospray ionization tandem mass spectrometry (ESI‐MS/MS) was used to intercept and characterize the reactive Pd(IV) transient intermediates in the solutions of Pd(OAc)₂‐catalyzed oxidative coupling reactions. In this study, the Pd(IV) transient intermediates were detected from the solution of Pd(OAc)₂‐catalyzed oxidative coupling reactions by ESI‐MS and the MS/MS of the intercepted Pd(IV) transient intermediate in reaction system was the same with the synthesized authentic Pd(IV) complex. Our ESI‐MS(/MS) studies confirmed the presence of Pd(IV) reaction transient intermediates. Most interestingly, the MS/MS of Pd(IV) transient intermediates showed the reductive elimination reactivity to Pd(II) complexes with new C? O bond formation into product in gas phase, which was consistent with the proposed reactivity of the Pd(IV) transient intermediates in solution.     

On-line monitoring of Brookhart polymerization by electrospray ionization mass spectrometry

For the first time, an electrospray ionization (ESI) mass spectrometer coupled on-line with a microreactor permitted the fast screening of polymerization growth of alkenes in the liquid phase mediated by palladium Brookhart catalyst and allowed the direct mass spectrometric identification of alkyl chains still bonded to Pd. The method also allowed us to intercept the catalytically active cationic intermediates of the Brookhart polymerization of alkenes, and to prove on-line their catalytic activity through ion/molecule reactions. The technique is fast and sensitive, requires no sample preparation and manipulation, and is therefore suitable for the fast screening of catalyst/polymer growth by MS identification, with potential applications in areas such as new catalyst development, polymer chemistry, and product quality monitoring in manufacturing.     

Electrospray ionization mass spectrometry monitoring of indigo carmine degradation by advanced oxidative processes

The degradation of the dye indigo carmine in aqueous solution induced by two oxidative processes (H(2)O(2)/iodide and O(3)) was investigated. The reactions were monitored by electrospray ionization mass spectrometry in the negative ion mode, ESI(-)-MS, and the intermediates and oxidation products characterized by ESI(-)-MS/MS. Both oxidative systems showed to be highly efficient in removing the color of the dye aqueous solutions. In the ESI(-)-MS of the indigo carmine solution treated with H(2)O(2) and H(2)O(2)/iodide, the presence of the ions of m/z 210 (indigo carmine in its anionic form, 1), 216, 226, 235, and 244 was noticeable. The anion of m/z 235 was proposed to be the unprecedented hydroperoxide intermediate 2 formed in solution via an electrophilic attack by hydroxyl and hydroperoxyl radicals of the exocyclic C=C bond of 1. This intermediate was suggested to be rapidly converted into the anionic forms of 2,3-dioxo-1H-indole-5-sulfonic acid (3, m/z 226), 2-amino-alpha-oxo-5-sulfo-benzeneacetic acid (4, m/z 244), and 2-amino-5-sulfo-benzoic acid (5, m/z 216). In the ESI(-)-MS of the indigo carmine solution treated with O(3), two main anions were detected: m/z 216 (5) and 244 (4). Both products were proposed to be produced via an unstable ozonide intermediate. Other anions in this ESI(-) mass spectrum were attributed to be [4 - H + Na](-) of m/z 266, [4 - H](2-) of m/z 121.5, and [5 - H](2-) of m/z 107.5. ESI-MS/MS data were consistent with the proposed structures for the anionic products 2-5.     

新型包覆Pd纳米粒子液相色谱-串联质谱法用于鉴定沙特阿拉伯原油中的含氧化合物(英文)

Saudi Arabian crude oil is a super complex mixture and,up to now,there has been little research into its heteroatom-containing compounds.First,oxygenated compounds(OCs)were isolated from Saudi Arabian oil using a Pd nanoparticle exchange complex,which formed between the nano-Pds and the oxygenated ligands.Normally,polycyclic aromatic sulphur heterocycles(S-PAHs)are separated from petroleum oil via the same method.The obtained results reveal that all the OC formulations with S-PAHs can be separated from the pre-isolated aromatic fraction of crude oil via this approach.S-PAHs are mixtures of benzothiophene and dibenzothiophene congeners.The isolated OCs are composed mainly of hydroxyl compounds.The liquid chromatography(LC)/electrospray ionization(ESI)in positive ion mode ESI(+)/tandem mass spectrometry(MS/MS)technique was used to assign the molecular weight distribution and identify the isolated OCs.The LC/ESI(+)-MS/MS technique differentiates S-PAHs and OCs using protonated ions.Thus,LC/ESI(+)-MS/MS can be used to assign molecular weight distributions for both the groups as a single mixture.MS/MS in precursor ion mode was used for the immediate identification of the target S or O analytes.     

新型包覆Pb纳米粒子液相色谱-串联质谱法用于鉴定沙特阿拉伯原油中的含氧化合物（英文）

Saudi Arabian crude oil is a super complex mixture and,up to now,there has been little research into its heteroatom-containing compounds.First,oxygenated compounds(OCs)were isolated from Saudi Arabian oil using a Pd nanoparticle exchange complex,which formed between the nano-Pds and the oxygenated ligands.Normally,polycyclic aromatic sulphur heterocycles(S-PAHs)are separated from petroleum oil via the same method.The obtained results reveal that all the OC formulations with S-PAHs can be separated from the pre-isolated aromatic fraction of crude oil via this approach.S-PAHs are mixtures of benzothiophene and dibenzothiophene congeners.The isolated OCs are composed mainly of hydroxyl compounds.The liquid chromatography(LC)/electrospray ionization(ESI)in positive ion mode ESI(+)/tandem mass spectrometry(MS/MS)technique was used to assign the molecular weight distribution and identify the isolated OCs.The LC/ESI(+)-MS/MS technique differentiates S-PAHs and OCs using protonated ions.Thus,LC/ESI(+)-MS/MS can be used to assign molecular weight distributions for both the groups as a single mixture.MS/MS in precursor ion mode was used for the immediate identification of the target S or O analytes.     

The mechanism of Sandmeyer's cyclization reaction by electrospray ionization mass spectrometry

Using electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) spectrometric experiments, the Sandmeyer reaction was monitored on-line, and key intermediates were intercepted and characterized for the first time. The mechanistic information provided by on-line ESI-MS(/MS) is in accordance with Sandmeyer's proposal, and was made possible by coupling a microreactor on-line to the ESI ion source, which allowed reactions to be screened from 0.7-2.0 s, identifying and characterizing all intermediates that were formed and consumed during the reaction.     

Studies on CH3CN-assisted decomposition of 1st Grubbs catalyst by electrospray ionization tandem mass spectrometry

The CH(3)CN-assisted decomposition reaction of the 1(st) Grubbs catalyst (1) was studied using electrospray ionization tandem mass spectrometry (ESI-MS/MS). We detected a series of Ru-intermediates and decomposition products by off-line and on-line ESI-MS(/MS) monitoring of the decomposition process. In particular, an on-line microreactor method was applied with ESI-MS/MS to profile the change and relationship of various Ru-intermediates by controlling the reaction within the first 30 s of its time scale. The main fast decomposition mechanism of Ru-catalyst 1 in the presence of CH(3)CN was similar to that proposed by Grubbs, and this was confirmed by detecting the (PhCH(2)PCy(3))(+) ion at m/z 371 as the major decomposition products with ESI-MS. We also studied the time evolution of the transient reactive Ru-intermediate ions step by step with ESI-MS/MS and detected the C-H bond activation products of toluene--dehydrogenated PCy(3), such as P(Cy)(2)(C(6)H(9)), P(Cy)(2)Ph--by analyzing the decomposition reaction solution by gas chromatography (GC)/MS. The mechanism of another minor decomposition pathway involving the phosphine activation of catalyst 1 was proposed on the basis of the ESI-MS(/MS) interception and characterization of the transient reactive Ru-species in the decomposition reaction solution. Finally the coordination effect of the CH(3)CN in assisting the decomposition and stabilizing the transient Ru-complexes is discussed.     

ESI‐MS Studies on Alcoholysis Mechanism of Electron‐deficient Cyclopropane Derivatives

Electrospray ionization mass spectrometry (ESI‐MS) was utilized to perform monitoring of the intermediates in the reaction of 1,2,3‐trisubstituted electron‐deficient cyclopropane derivatives, cis‐1‐thien‐2′‐oyl‐2‐(p‐subustituted phenyl‐6,6‐dimethyl)‐5,7‐dioxaspiro[2.5]‐4,8‐octadiones, with methanol. Key intermediates, either cationic or protonated forms of neutral species, were intercepted and characterized by ESI‐MS and its tandem version (ESI‐MS/MS). Therefore, the mechanism of the ring‐opening process for electron‐deficient cyclopropane derivatives was fully confirmed by the intermediates monitored.     

Electrospray ionization mass spectrometry fingerprinting of beer

After just simple degassing, dilution, pH adjustment and direct flow injection, characteristic fingerprint spectra of beer samples have been obtained by fast (few seconds) electrospray ionization mass spectrometry (ESI-MS) analysis in both the negative and positive ion modes. A total of 29 samples belonging to the two main beer types (lagers and ales) and several beer subtypes from USA, Europe and Brazil could be clearly divided into three groups both by simple visual inspection of their ESI(+)-MS and ESI(-)-MS fingerprints as well as by chemometric treatment of the MS data. Diagnostic ions with contrasting relative abundances in both the positive and negative ion modes allow classification of beers into three major types: P = pale (light) colored (pilsener, pale ale), D = dark colored (bock, stout, porter, mild ale) and M = malt beer. For M beers, samples of a dark and artificially sweetened caramel beer produced in Brazil and known as Malzbiers were used. ESI-MS/MS on these diagnostic beer cations and anions, most of which are characterized as arising from ionization of simple sugars, oligosaccharides, and iso-alpha-acids, yield characteristic tandem mass spectra adding a second and optional MS dimension for improved selectivity for beer characterization by fingerprinting. Direct ESI-MS or ESI-MS/MS analysis can therefore provide fast and reliable fingerprinting characterization of beers, distinguishing between types with different chemical compositions. Other unusual polar components, impurities or additives, as well as fermentation defects or degradation products, could eventually be detected, making the technique promising for beer quality control.     

Dicationic ion-pairing of phosphoric acid diesters post-liquid chromatography and subsequent determination by electrospray positive ionization-tandem mass spectrometry

Several organophosphate triesters are widely used as flame retardants and can be metabolized to dibutyl (DBP), diphenyl (DPhP), di(2-ethylhexyl) (DEHP) and di(1,3-dichloro-2-propyl) (or bis(1,3-dichloro-2-propyl); DDCPP) phosphoric acid, respectively. A highly sensitive liquid chromatography-electrospray ionization(+)-triple quadrupole mass spectrometry (LC-ESI(+)-QQQ-MS/MS) based analysis method was presently developed. In this method the target compounds were separated with a C(18)-based reversed phase LC column, and decamethonium hydroxide (dicatonic reagent) was introduced post-LC to form ion-pairs, which were subsequently detected by ESI(+). For the phosphate acid diester ion-pairs, the mass spectra showed the most abundant ion to be [(CH(3))(2)N(CH(2))(10)N(CH(3))(3)](+), with lesser abundances of [[M-H](-)[(CH(3))(3)N(CH(2))(9)CH(2)](2+)](+) and [CH(2)CH(CH(2))(8)N(CH(3))(3)](+). For DDCPP, the fragment ions of [[Cl](-)[(CH(3))(3)N(CH(2))(10)N(CH(3))(3)](2+)](+) and [[Cl](-)[(CH(3))(3)N(CH(2))(9)CH(2)](2+)](+) could also be observed. The limits of quantitation (LOQs) by LC-ESI(+)-MS/MS (based on multiple reaction monitoring) were 0.14, 0.03, 0.14 and 0.02 ng/mL for DPhP, DBP, DDCPP and DEHP, respectively. The response was highly linearly correlated (r>0.995) with concentration over the range of the LOD to 1000 ng/mL. The matrix effect on ESI+ was negligible for the samples in experiment of in vitro metabolism using rat liver microsomes.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Development of a method for simultaneous determination of eflucimibe and its three major metabolites in rat plasma by liquid chromatography/electrospray tandem mass spectrometry: a preliminary study

A high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the simultaneous determination of eflucimibe, a powerful acyl-coenzyme A cholesterol O-acyltransferase (ACAT) inhibitor, and its main metabolites, in plasma. The ESI and MS/MS parameters were investigated and optimised for each of the four compounds in the positive ion mode. Plasma samples were deproteinised by precipitation with acetonitrile and directly analysed by HPLC/ESI-MS/MS in less than 4 min. Quantitation was performed in the multiple reaction monitoring (MRM) mode for highest sensitivity, selecting the protonated molecules [M+H](+) as precursor ions. The method was demonstrated to be specific and sensitive, and a linear response was observed within a 1-25 ng/mL concentration range. Correlation coefficients (r(2)) greater than 0.9960 were obtained by least-squares regression, and limits of detection down to 0.2 ng/mL were calculated. Therefore, this HPLC/ESI-MS/MS method appears to be an efficient tool, able to provide valuable information for a pharmacological purpose.     

Photolytic degradation of the insecticide thiamethoxam in aqueous medium monitored by direct infusion electrospray ionization mass spectrometry

Photodegradation of the insecticide thiamethoxam (1), 3-[(2-chloro-5-thiazolyl)methyl]tetrahydro-5-methyl-N-nitro-4H-1,3,5-oxadiazin-4-imine, in an aqueous medium was monitored by electrospray ionization mass spectrometry in the positive ion mode, ESI(+)-MS. An aqueous solution of (1) was incessantly exposed to a UV radiation source and aliquots were taken after reaction times of 1, 2, 3, and 4 h. Analysis by GC/NCI-MS revealed that (1) was continuously degraded under these experimental conditions. However, the total organic carbon (TOC) content remained practically constant during the exposition period, thereby indicating that 1 was not mineralized but continuously converted into other compounds. ESI(+)-MS monitoring revealed that whereas the intensity of the ions of m/z 292/294 ([1 + H](+)) constantly decreased, there was the emergence of other ions of m/z 247/249, 197, 168, and 116 whose intensities simultaneously increased. Their structures were proposed on the basis of: (1) the data of their ESI(+)-MS/MS; (2) their high resolution m/z values; and (3) a plausible reactivity of the thiamethoxam molecule exposed to UV radiation in aqueous solution. Finally, these data allowed us to suggest a reaction route for the photodegradation of 1 in an aqueous medium.     

ESI-MS detection of proposed reaction intermediates in the air-promoted and ligand-modulated oxidative Heck reaction

Electrospray ionization mass spectrometry (ESI-MS) and subsequent MS/MS analyses were used to directly detect palladium-containing cationic reaction intermediates in a ligand controlled palladium(II)-catalyzed oxidative Heck arylation. All potential intermediates were observed as dmphen-ligated palladium(II) species, suggesting that the dmphen bidentate ligand is attached to the metal center during the entire catalytic cycle. The study supports previous mechanistic propositions and provides new information regarding the composition of aryl-containing Pd(II) complexes in an ongoing oxidative Heck reaction. In addition, sodium acetate was found to be a useful base alternative to previously used tertiary amines.     

Analysis of triacetone triperoxide (TATP) and TATP synthetic intermediates by electrospray ionization mass spectrometry

The explosive triacetone triperoxide (TATP) has been analyzed by electrospray ionization mass spectrometry (ESI-MS) on a linear quadrupole instrument, giving a 62.5 ng limit of detection in full scan positive ion mode. In the ESI interface with no applied fragmentor voltage the m/z 245 [TATP + Na](+) ion was observed along with m/z 215 [TATP + Na - C(2)H(6)](+) and 81 [(CH(3))(2)CO + Na](+). When TATP was ionized by ESI with an applied fragmentor voltage of 75 V, ions at m/z 141 [C(4)H(6)O(4) + Na](+) and 172 [C(5)H(9)O(5) + Na](+) were also observed. When the precipitates formed in the synthesis of TATP were analyzed before the reaction was complete, a new series of ions was observed in which the ions were separated by 74 m/z units, with ions occurring at m/z 205, 279, 353, 427, 501, 575, 649 and 723. The series of evenly spaced ions is accounted for as oligomeric acetone carbonyl oxides terminated as hydroperoxides, [HOOC(CH(3))(2){OOC(CH(3))(2)}(n)OOH + Na](+) (n = 1, 2 ... 8). The ESI-MS spectra for this homologous series of oligoperoxides have previously been observed from the ozonolysis of tetramethylethylene at low temperatures. Precipitates from the incomplete reaction mixture, under an applied fragmentor voltage of 100 V in ESI, produced an additional ion observed at m/z 99 [C(2)H(4)O(3) + Na](+), and a set of ions separated by 74 m/z units occurring at m/z 173, 247, 321, 395, 469 and 543, proposed to correspond to [CH(3)CO{OOC(CH(3))(2)}(n)OOH + Na](+) (n = 1,2 ... 5). Support for the assigned structures was obtained through the analysis of both protiated and perdeuterated TATP samples.     

Pharmacokinetic measurements of IDN 5390 using electrospray ionization tandem mass spectrometry: structure characterization and quantification in dog plasma

In this report, electrospray ionization tandem mass spectrometry (ESI-MS/MS) for a pharmacokinetic study of IDN 5390, a novel C-seco taxane derivative, which is under preclinical evaluation, has been investigated. Our results showed that IDN 5390 and other taxanes including paclitaxel and IDN 5109 could ionize well in not only positive-, but also in negative-ion mode. Under collision-induced dissociation (CID) conditions, these compounds could fragment into similar M- (molecular), T- (taxane ring) and S- (side chain) series ions. In positive-ion ESI, the formation of both T- and S-series ions involved the breaking of the C-13 ester bond. In negative-ion ESI, however, while the formation mechanism of S-series ions remained the same, the breaking of the C-1' carboxylic ester bond resulted in T-series ions. At optimum collision energy (CE) values, M-, T- and S-series ions of IDN 5390 in both positive- and negative-ion ESI-MS/MS spectra had good intensity. This phenomenon makes both positive- and negative-ion ESI-MS/MS good methods for IDN 5390 metabolite structural characterization, i.e. to reveal the location of modification groups in IDN 5390 metabolites versus IDN 5390 either on the side chain or the taxane ring. A liquid chromatography (LC)/ESI-MS/MS method using the multiple-reaction monitoring (MRM) technique was thereafter developed to quantify IDN 5390 in dog plasma using paclitaxel as internal standard. The method was validated using a concentration range between 5 and 1000 ng/mL and had a limit of detection of 1 ng/mL. The inter-day %CV (%coefficient of variation) of the calibration standards ranged between 4.36 and 9.64%, the intra-day %CV of the calibration standards between 0.61 and 13.44%, and the mean % accuracy of the quality control samples at the low, middle and high end of the concentration curves were 12.5, 6.8 and 9.6%, respectively.     

Electrospray ionization mass spectrometry fingerprinting of whisky: immediate proof of origin and authenticity

Authentic samples of whisky produced in Scotland and USA and counterfeit whisky samples commercialized in Brazil have been directly submitted to electrospray ionization mass spectrometry (ESI-MS) analysis in both the negative and positive ion modes to assess the potential of this technique for simple and rapid quality control and proof of authenticity of whisky samples. ESI in the negative ion mode yields the most characteristic whisky fingerprinting mass spectra in just a few seconds by direct infusion of the samples, detecting the most polar or acidic components of each sample in their deprotonated anionic forms. No pre-treatment of the sample, such as extraction or derivatization or even dilution, is required. The analysis of the ESI(-)-MS data both by simple visual inspection but more particularly by chemometric data treatment enables separation of the whisky samples into three unequivocally distinct groups: Scotch, American and counterfeit whisky, whereas single malt and blended Scotch whiskies are also distinguished to some extent. As indicated by ESI-MS/MS analysis, the diagnostic anions are simple sugars, disaccharides and phenolic compounds. Direct infusion ESI-MS therefore provides immediate chemical fingerprinting of whisky samples for type, origin and quality control, as demonstrated herein for American, Scottish and counterfeit samples, whereas ESI-MS/MS analysis of diagnostic ions adds a second dimension of fingerprinting characterization when improved selectivity is desired.     

Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.