Plasmonic nanostructures, like noble metal, have gained large attention due to their plasmonic properties so they can reach areas like electronics, photo-catalysis, biomedicine, and sensing. Plasmonic nanomaterials are known for their local surface plasmon resonance and enhanced electromagnetic field and wavelength dependence. The higher the electromagnetic field at the surface of the nanoparticles can interact with nearby molecules, the bigger the influence is on the intensity of the molecule signals. This phenomenon is called surface-enhanced Raman scattering (SERS) and plasmonic enhanced fluorescence (PEF), which enable the plasmonic nanomaterials as a signal amplifier. By using these plasmonic nanostructures as a signal amplifier, SERS and PEF have become ultrasensitive methods in biomedicine and biosensing. Plasmonic biosensing is fast and label-free detection of biologically relevant analytes in real time. The objective of my doctoral dissertation focusses on developing new plasmonic nanostructures for detecting biomarkers related to cancers and some other diseases based on hybrid platforms. In this work, a newly spiky nanostructure was developed, internal standard Raman molecules were embedded into the nanostructure for quantitative SERS detection of polycyclic aromatic hydrocarbons molecules. Then the morphology and dispersity of this nanostructure were optimized to get an approximately fusiform shape, which showed a stable, reproducible and high SERS signals. This nanostructure was furtherly functionalized by double strand DNA and aptamer, showing a good performance in drug delivery and detecting circulating tumor cells. Inspired by the mechanism of SERS, a SERS and PEF dual model sensor based on plasmonic nanostructures and newly synthesized probe molecules was developed. This dual model sensor combined the advantages of SERS and PEF and exhibited a lower limit of detection of γ-glutamyl transferase in living cells. This dissertation contains the fabrication of newly plasmonic nanostructures and utilizing them in bioanalysis.

Bioanalysis is a subdivision of analytical chemistry and deals with biological analytes such as metabolites, proteins, nucleic acids, small molecules, virus particles and entire cells. The rationale of my thesis was to achieve two goals: (i) develop a set of ready to use methods (ii) which are capable providing exact concentrations of analytes as well as kinetic and thermodynamic parameters of their interactions. To investigate interactions between biomolecules special conditions are required which do not interfere with the course if biomolecule interactions. Establishing these conditions and optimization of separation and detection parameters can be tedious and can take longer than actual analysis of samples. I developed a variety of Capillary Electrophoresis – Mass Spectrometry (CE-MS) methods suitable for bionalalysis. CE-MS establishes a new paradigm that separation methods together with MS detection can be used as comprehensive kinetic tools. Most previous attempts to use chromatography and electrophoresis for studying nucleic acid interactions were restricted to assuming slow or no equilibrium between reactants. Kinetic CE (KCE) shows that non-zero kinetics and structural dynamics must be taken into account when separation happens. KCE-MS could be a valuable supplement to IM-MS due to the separation of ions in solution according to their size-to-charge ratio. These methods allowed to reveal new facts about biomolecules and added novel data to the bank of the mankind knowledge. For the best of my knowledge, kinetic parameters for TG2 and thrombin G-quadruplex folding were reported for the first time. I developed a homogeneous method to determine kon, koff and Kd of fast and weak noncovalent interactions between multiple unlabeled ligands (small molecule drugs) and an oligosaccharide (α- or β-cyclodextrin) simultaneously in one capillary microreactor. It has been shown for the first time that KCE can be used to separate and detect the slowly interconverting open and closed conformations of human TG2. It allowed the first direct measurement of the Kd value for calcium binding. Sixteen new substrates were discovered for three aminotransferases (AAT, BCAT, and DAAT). In addition, Viral qCE showed a feasibility to analyse both the count of intact viral particles and sample nucleic acid contamination.

The ethical and practical issues of obtaining a blood sample pose a significant challenge to performing pharmacokinetic studies in children, infants and neonates. Dried blood spot analysis, based on the collection of a micro blood sample has potential to overcome these difficulties. There are at present a limited number of reports on the utility of dried blood spot analysis in clinical pharmacokinetic studies. The studies described in this thesis were undertaken to investigate the accuracy and precision of dried blood spot sampling coupled with mass spectrometry detection for drug quantification, and clinically validate the robustness and feasibility of this technique for pharmacokinetic studies in preterm neonates. Dried blood spot methods were developed for application to pharmacokinetic studies of test drugs dexamethasone and caffeine. Investigations were focused on the blood collection system, analyte recovery and optimisation of the detection system. In-vitro validation results indicated developed methods were precise, accurate and selective in accordance with the Food and Drug Administration regulatory guidelines on the assessment of bioanalytical methods. Results were not significantly affected by small variations in the blood volume spotted or the presence of petroleum jelly, which is often used on the sampling site during capillary blood collection in neonates. Variability in haematocrit was determined to be the single most important factor affecting assay accuracy. Stability assessments by comparison with freshly prepared samples verified the suitability of sample drying, storage and post sample extraction conditions. An investigation of method transferability between different analytical instruments was undertaken with caffeine to provide an assessment of the robustness of dried blood spot analysis. Results generated from a single and triple quadrupole mass spectrometer were comparable with an expected lower limit of quantification with the latter technique most likely due to a greater ionisation and detection efficiency. Intravenous dexamethasone pharmacokinetics was determined in 5 preterm neonates receiving treatment for chronic lung disease. Individual pharmacokinetic analyses were performed using a one compartment model to estimate primary pharmacokinetic parameters, clearance (mean, 0.18 l/h/kg) and volume of distribution (mean, 1.33 l/kg). The whole blood derived mean estimates were similar to previous plasma clearance and volume estimates of 0.14 l/h/kg and 1.91 l/kg, respectively reported in neonates (n=7). This highlights the potential for dried blood spot analysis as an alternative to conventional plasma based methods for dexamethasone dose optimisation studies in neonates. The population pharmacokinetics of oral / intravenous caffeine was determined in 67 preterm neonates. A one compartment model was used to describe the blood concentration-time data. Model evaluation using a bootstrapping technique confirmed the robustness and stability of the developed model. Pharmacokinetic parameters derived from dried blood spot drug measurements were estimated with precision (relative standard error < 10%) and were comparable to estimates of plasma clearance (mean, 7.3 vs. 7.0 ml/h/kg) and volume of distribution (mean, 593 vs. 851 ml/kg) from a previous population study in neonates (n=110). Weight and postnatal age were the most influential covariates in the clearance model which is in agreement with previous population studies. These results demonstrate that dried blood spot analysis is a practical technique, with significant potential as a robust method for use in clinical pharmacokinetic studies in vulnerable populations such as preterms. Haematocrit related effects on paper will need to be accounted for if this potential is to be realised. Further investigations to determine the reproducibility of capillary blood sampling in neonates and the impact of using blood drug measurements on pharmacokinetic parameter estimation will be necessary before widespread use of the technique is possible.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-05-28T17:55:37Z No. of bitstreams: 2 Dissertação - Aline Rosa Fernandes - 2018.pdf: 2708071 bytes, checksum: 09cf08ef257f815da8d21193826fc32f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-29T11:24:27Z (GMT) No. of bitstreams: 2 Dissertação - Aline Rosa Fernandes - 2018.pdf: 2708071 bytes, checksum: 09cf08ef257f815da8d21193826fc32f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-29T11:24:27Z (GMT). No. of bitstreams: 2 Dissertação - Aline Rosa Fernandes - 2018.pdf: 2708071 bytes, checksum: 09cf08ef257f815da8d21193826fc32f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-02 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Paper spray ionization (PSI) is an environmental ionization technique in mass spectrometry (MS), where the sample is deposited on triangular paper moistened with solvent and the ionization process occurs by the application of high voltage on the paper. An important factor that can affect its performance is ion suppression. Aiming to reduce this effect and the exclusion of macromolecules in complex samples, the RAM-PSI-MS method for the analysis of dopamine and epinephrine in plasma was developed and validated. A methodology for analyzing creatinine in the urine was also developed using graphene oxide (GO) modified paper. The methods developed were efficient and promising in improving the role for PSI-MS analysis. The graphene oxide coating was able to improve the time and stability of the spray, in addition to reducing the effect of ion suppression. Already coating the paper with ISRP-RAM phase made it possible to analyze a complex sample, such as plasma, without any sample preparation, obtaining very satisfactory results. Showing that it is possible, practically, to eliminate the inherent problems of PSI-MS, only with the paper coating. / Paper spray ionization (PSI) é uma técnica de ionização ambiente em espectrometria de massas (MS), onde a amostra é depositada sobre papel triangular umedecido com solvente e o processo de ionização ocorre pela aplicação de alta voltagem no papel. Um importante fator que pode afetar seu desempenho é a supressão iônica. Visando a diminuição desse efeito e a exclusão de macromoléculas em amostras complexas foi desenvolvido e validado o método RAM-PSI- MS para analise de dopamina e epinefrina em plasma. Foi também desenvolvida uma metodologia para análise de creatinina na urina utilizando um papel modificado com óxido de grafeno (GO). Os métodos desenvolvidos foram eficientes e promissores na melhora do papel para analises de PSI-MS. O revestimento com óxido de grafeno foi capaz de melhorar o tempo e a estabilidade do spray, além de diminuir o efeito de supressão iônica. Já revestindo o papel com fase ISRP-RAM tornou possível analisar uma amostra complexa, como o plasma, sem nenhum preparo de amostra, obtendo resultados bastante satisfatórios. Mostrando que possível, praticamente, eliminar os problemas inerentes do PSI-MS, apenas com o revestimento do papel.

Revised and Expanded Handbook Provides Comprehensive Introduction and Complete Instruction for Sample Preparation in Vital Category of Bioanalysis Following in the footsteps of the previously published Handbook of LC-MS Bioanalysis, this book is a thorough and timely guide to all important sample preparation techniques used for quantitative Liquid Chromatography–Mass Spectrometry (LC-MS) bioanalysis of small and large molecules. LC-MS bioanalysis is a key element of pharmaceutical research and development, post-approval therapeutic drug monitoring, and many other studies used in human healthcare. While advances are continually being made in key aspects of LC-MS bioanalysis such as sensitivity and throughput, the value of research/study mentioned above is still heavily dependent on the availability of high-quality data, for which sample preparation plays the critical role. Thus, this text provides researchers in industry, academia, and regulatory agencies with detailed sample preparation techniques and step-by-step protocols on proper extraction of various analyte(s) of interest from biological samples for LC-MS quantification, in accordance with current health authority regulations and industry best practices. The three sections of the book with a total of 26 chapters cover topics that include: Current basic sample preparation techniques (e.g., protein precipitation, liquid-liquid extraction, solid-phase extraction, salting-out assisted liquid-liquid extraction, ultracentrifugation and ultrafiltration, microsampling, sample extraction via electromembranes) Sample preparation techniques for uncommon biological matrices (e.g., tissues, hair, skin, nails, bones, mononuclear cells, cerebrospinal fluid, aqueous humor) Crucial aspects of LC-MS bioanalytical method development (e.g., pre-analytical considerations, derivation strategies, stability, non-specific binding) in addition to sample preparation techniques for challenging molecules (e.g., lipids, peptides, proteins, oligonucleotides, antibody-drug conjugates) Sample Preparation in LC-MS Bioanalysis will prove a practical and highly valuable addition to the reference shelves of scientists and related professionals in a variety of fields, including pharmaceutical and biomedical research, mass spectrometry, and analytical chemistry, as well as practitioners in clinical pharmacology, toxicology, and therapeutic drug monitoring.

Polymeric microchips have received increasing attention in chemical analysis because polymers have attractive properties, such as low cost, ease of fabrication, biocompatibility and high flexibility. However, commercial polymers usually exhibit analyte adsorption on their surfaces, which can interfere with microfluidic transport in, for example, chemical separations such as chromatography or electrophoresis. Usually, surface modification is required to eliminate this problem. To perform stable and durable surface modification, a new polymer, poly(methyl methacrylate-co-glycidyl methacrylate) (PGMAMMA) was prepared for microchip fabrication, which provides epoxy groups on the surface. Whole surface atom transfer radical polymerization (ATRP) and in-channel ATRP approaches were employed to create uniform and dense poly(ethylene glycol) (PEG)-functionalized polymer brush channel surfaces for capillary electrophoresis (CE) separation of biomolecules, such as peptides and proteins. In addition, a novel microchip material was developed for bioanalysis, which does not require surface modification, made from a PEG-functionalized copolymer. The fabrication is easy and fast, and the bonding is strong. Microchips fabricated from this material have been applied for CE separation of small molecules, peptides, proteins and enantiomers. Electric field gradient focusing (EFGF) is an attractive technique, which depends on an electric field gradient and a counter-flow to focus, concentrate and separate charged analytes, such as peptides and proteins. I used the PEG-functionalized copolymer to fabricate EFGF substrates. The separation channel was formed in an ionically conductive and protein resistant PEG-functionalized hydrogel, which was cast in a changing cross-sectional cavity in the plastic substrate. The hydrogel shape was designed to create linear or non-linear gradients. These EFGF devices were successfully used for protein focusing, and their performance was optimized. Use of buffers containing small electrolyte ions promoted rapid ion transport in the hydrogel for achieving the designed gradients. A PEG-functionalized monolith was incorporated in the EFGF separation channel to reduce dispersion and improve focusing performance. Improvement in peak capacity was proposed using a bilinear EFGF device. Protein concentration exceeding 10,000-fold was demonstrated using such devices.

Le projet concerne des molécules hétérocycliques, de faible poids moléculaire, présentant des activités cytotoxiques comparables à celles des meilleurs anticancéreux actuellement sur le marché. Ces molécules sont originales, protégées par un brevet international et un brevet de sélection déposé en décembre 2014. La synthèse des premières molécules leaders est maîtrisée et l’exemplification de la diversité moléculaire est en cours. Les études menées pour définir leur profil d’activité permettent d’identifier des caractéristiques tout à fait originales. Le projet, en phase de développement préclinique académique, a permis l’identification de composés leaders présentant des potentiels de développement en tant qu’anticancéreux. Le mécanisme exact des molécules développées est encore en cours d’étude et permettra de définir s’il s’agit d’un mode d’action unique ou multiple. Plusieurs têtes de séries ont pu être identifiées avec visiblement des modes d’actions différents. En effet, les composés EAPB0203 et EAPB0503 montrent un effet dose à partir de 1 µM sur la polymérisation de la tubuline mais la molécule EAPB02303, la plus active sur la lignée A375 (CI50 = 10 nM, de dix fois à cent fois plus active que les deux précédentes), ne montre aucune fixation à la tubuline à la dose de 1µM suggérant un mécanisme d’action différent et original. Le sujet de recherche présenté concerne le développement des études précliniques de ces molécules à visée anticancéreuse. Le premier axe de travail a été de mettre au point une formulation galénique de l'EAPB0503 sous forme de nanocapsules lipidiques. Afin d’optimiser la biodisponibilité des composés, sans perdre leur activité intrinsèque, nous avons ensuite réalisé une modulation chimique sur la structure la plus active des Imiqualines : l’EAPB02303. Afin d’améliorer la balance globale hydrophilie/lipophilie (HLB) des composés dérivés de l’EAPB02303, nous avons greffé un résidu acide aminé en position 4. Enfin, la mise au point d’une méthode de dosage de l’EAPB02303 et l’EAPB02302 en milieu plasmatique en vue d’une étude pharmacocinétique a été le dernier axe du travail de thèse. / The project relates to heterocyclic molecules, low molecular weight, having cytotoxic activities similar to those presented by the best anticancer agents currently on the market. These molecules are originals, protected by an international patent and a selection patent filed in December 2014. The synthesis of the first molecules leader is under control and the exemplification of molecular diversity is underway. Studies to define their activity profile allow to identify quite original features. The project, in its academic preclinical development phase, enabled the identification of leader’s compounds with potential for development as anticancer. The exact mechanism of the molecules developed is still under study and will determine whether there is a single or multiple mode of action. Several series leads were identified with apparently different modes of action. Indeed, compounds EAPB0203 and EAPB0503 show a dose effect from 1 µM on tubulin polymerization but compound EAPB02303, the more active on A375 cell line (IC50 = 10 nM, ten times to hundred times more active than the preceding two), shows no binding to tubulin at a dose of 1 µM suggesting a different and original mechanism of action. The research topic presented concerns the preclinical development of these anticancer molecules. The first line of work was to develop a galenic formulation of EAPB0503 in the form of lipid nanocapsules. To optimize the bioavailability of the compounds, without losing their intrinsic activity, we achieved chemical modulation on the most active structure Imiqualines: EAPB02303. To improve the overall balance hydrophilic / lipophilic (HLB) of compounds derived from EAPB02303, we considered grafting amino acids on position 4. Finally, the development of a method for assaying EAPB02303 and EAPB02302 in plasma for a pharmacokinetic study has been the final focus of the thesis work.

abstract: Biological fluids contain information-rich mixtures of biochemicals and particles such as cells, proteins, and viruses. Selective and sensitive analysis of these fluids can enable clinicians to accurately diagnose a wide range of pathologies. Fluid samples such as these present an intriguing challenge to researchers; they are packed with potentially vital information, but notoriously difficult to analyze. Rapid and inexpensive analysis of blood and other bodily fluids is a topic gaining substantial attention in both science and medicine. Current limitations to many analyses include long culture times, expensive reagents, and the need for specialized laboratory facilities and personnel. Improving these tests and overcoming their limitations would allow faster and more widespread testing for disease and pathogens, potentially providing a significant advantage for healthcare in many settings. Both gradient separation techniques and dielectrophoresis can solve some of the difficulties presented by complex biological samples, thanks to selective capture, isolation, and concentration of analytes. By merging dielectrophoresis with a gradient separation-based approach, gradient insulator dielectrophoresis (g-iDEP) promises benefits in the form of rapid and specific separation of extremely similar bioparticles. High-resolution capture can be achieved by exploiting variations in the characteristic physical properties of cells and other bioparticles. Novel implementation and application of the technique has demonstrated the isolation and concentration of blood cells from a complex biological sample, differentiation of bacterial strains within a single species, and separation of antibiotic-resistant and antibiotic-susceptible bacteria. Furthermore, this approach allows simultaneous concentration of analyte, facilitating detection and downstream analysis. A theoretical description of the resolving capabilities of g-iDEP was also developed. This theory explores the relationship between experimental parameters and resolution. Results indicate the possibility of differentiating particles with dielectrophoretic mobilities that differ by as little as one part in 100,000,000, or electrophoretic mobilities differing by as little as one part in 100,000. These results indicate the potential g-iDEP holds in terms of both separatory power and the possibility for diagnostic applications. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A rapid, specific and highly sensitive liquid chromatography-tandem mass spectrometry method was developed to determine nimodipine in human plasma using dibucaine as the internal standard (IS) is described. The analyte (m/z 418,6 > 342,6) and IS (m/z 344,2 > 271,0) were extracted from plasma samples by liquid-liquid extraction using hexane-ethyl acetate (1:1v/v). Chromatography was performed on a Varian Polaris C18 analytical column (3 micrometer, 50 x 2,0 mm) and pre-column SecurityguardTM C18 (4,0 x 3,0 mm). The phase mobile consisted of Acetonitrile-Ammonium acetate 0.02 ml/L (80:20v/v). The method had a chromatographic run time of 4.5 min and linear calibration curve over the range 0.1- 40 ng/mL (r2 > 0.9938). The limit of quantification (LQ) was 0.1 ng/mL. The intra-day precision for the limit quantification was 0.00% (batch 01), 5.71% (batch 02) and 5.27% (batch 03); for the quality controls low (QCL), middle (QCM) and high (QCH) the results were respectively 8.57, 0.81 and 1.37%. The inter-day precision for LQ and QCL, QCM and QCH were respectively: 7% and 5.46, 4.12 and 3.37%. The intra-day accuracy for LQ was 110, 96 and 104%; for QCL, QCM and QCH the results were100.67, 109.09 and 109.72% respectively. The results of the inter-day accuracy for LQ, QCL, QCM and QCH were respectively and 110.0, 96.0, 104.0% for the limit of quantification and 8.57, 0.81, 1.37% and 100.67, 109.09, 109.72% respectively: 103% e 102.89, 106.60, 109.69%. This validated method was successfully applied for the pharmacokinetic profiles of nimodipine tablets administered to 24 healthy volunteerâs participant of bioavailability comparative study. Geometric mean of Test formulation/Refernce formulation individual percent ratio was 104,56% for AUC0-48h and 55,73% for Cmax. The 90% for the confidence intervals (CI) were 94,80-115,32% e 44,73-69,42%, respectively. The values of half-life and Cmax for test formulation and reference formulation were 27,83;32,78h and 9,48;18,76ng/mL, respectively. The values of Tmax were 2,34;0,98h for the formulations test and reference respectively. Since the 90% CI for Cmax and AUC0-48h, were within the 80-125% interval proposed by the âFood and Drug Administrationâ and ANVISA, it was concluded that the two formulations of nimodipine 30mg tablets were not bioequivalent, according to the rate of absorption after single dose administration. / Um mÃtodo rÃpido, sensÃvel e especÃfico de Cromatografia LÃquida de Alta EficiÃncia acoplada à Espectrometria de Massa (LC-MS-MS) foi desenvolvido para determinar nimodipino (analito) em plasma humano usando dibucaÃna como padrÃo interno (PI). O analito (m/z 418,6 > 342,6) e o PI (m/z 344,2 > 271,0) foram extraÃdos de amostras de plasma atravÃs de extraÃÃo lÃquido-lÃquido utilizando hexano-acetato de etila (1:1v/v). As corridas cromatogrÃficas foram executadas utilizando-se uma coluna analÃtica Varian Polaris C18 (3 micrÃmetros, 50 x 2,0 mm) e uma prÃ-coluna SecurityguardTM C18 (4,0 x 3,0 mm). A fase mÃvel consistiu de acetonitrila-soluÃÃo de acetato de amÃnio 0,02 mol/L (80:20v/v). O mÃtodo teve um tempo total de corrida de 4,5 min e uma curva de calibraÃÃo linear que variou de 0,1-40 ng/mL. O limite de quantificaÃÃo de 0,1 ng/mL. A precisÃo intra-ensaio para o limite de quantificaÃÃo (LQ) foi 0,00% (lote 01), 5,71% (lote 02) e 5,27% (lote 03); para os controles de qualidade baixo (CQB), mÃdio (CQM) e alto (CQA) os resultados foram respectivamente: 8,57, 0,81 e 1,37%. A precisÃo interensaio para o LQ e os CQB, CQM e CQA foram respectivamente de: 7% e 5,46, 4,12 e 3,37%. A exatidÃo intra-ensaio para o LQ foi 110, 96 e 104%; para CQB, CQM e CQA os resultados foram 100,67, 109,09 e 109,72% respectivamente. Os resultados da exatidÃo interensaio para o LQ, CQB, CQM e CQA foram respectivamente de: 103% e 102,89, 106,6, 109,69%. Este mÃtodo foi aplicado para a avaliaÃÃo do perfil farmacocinÃtico do nimodipino administrado em 24 voluntÃrios sadios participantes de um estudo de biodisponibilidade comparativa. A mÃdia geomÃtrica da FormulaÃÃo teste/FormulaÃÃo referÃncia para as porcentagens individuais foi 104,56% para ASC0-48h e 55,73% para Cmax. Os intervalos obtidos a partir do intervalo de confianÃa (IC) de 90% foram 94,80-115,32% e 44,73-69,42% respectivamente. Os valores de meia-vida e Cmax para as formulaÃÃes teste e referÃncia foram de 27,83;32,78h e 9,48;18,76ng/mL, respectivamente. Os valores de Tmax foram de 2,34;0,98h para as formulaÃÃes teste e referÃncia, respectivamente. Considerando o IC de 90% para Cmax e ASC0-48h dentro da variaÃÃo de 80-125% proposto pelo Food and Drug Administration e ANVISA, as duas formulaÃÃes de nimodipino 30mg nÃo sÃo bioequivalentes quanto à taxa de absorÃÃo (Cmax) apÃs uma Ãnica administraÃÃo.

Liquid chromatography, coupled with tandem mass spectrometry, presents a powerful tool for the quantification of the sex steroid hormones 17-β estradiol, progesterone and testosterone from biological matrices. The importance of accurate quantification with these hormones, even at endogenous levels, has evolved with our understanding of the role these regulators play in human development, fertility and disease risk and manifestation. Routine monitoring of these analytes can be accomplished by immunoassay techniques, which face limitations on specificity and sensitivity, or using gas chromatography–mass spectrometry. LC–MS/MS is growing in capability and acceptance for clinically relevant quantification of sex steroid hormones in biological matrices and is able to overcome many of the limitations of immunoassays. Analyte specificity has improved through the use of novel derivatizing agents, and sensitivity has been refined through the use of high-resolution chromatography and mass spectrometric technology. This review highlights these innovations, among others, in LC–MS/MS steroid hormone analysis captured in the literature over the last decade.