1.IntroductionMost of the trace elements in biological systems are bound to biomolecules. Metal-binding compounds play essential roles acting as biological catalysts that regulate reactions and physiological functions in cells and organs. For instance, the metalloproteins which catalyse biological reactions, called metalloenzymes, participate in important biological processes.1Metal-binding biomolecules are also present in degeneration processes: traces of Fe, Cu and Zn are involved in the growth of neurotoxic amyloid fibrils which allow the progression of Alzheimer’s disease.2Furthermore, drugs containing metal atoms are frequently employed in medicine.3Bi complexes have been successfully used therapeutically in antiulcer treatments;4Au compounds are used on the treatment of rheumatoid arthritis,5also exhibitingin vivoantitumor activity when gold is in oxidation state +1 or +3;6and, finally, compounds with Ga,7Ru,8Rh,9Sn10and As11present antitumor properties. Nowadays, the most powerful metallodrugs used are the Pt-containing compounds cisplatin, carboplatin or oxaliplatin, which are applied worldwide in the clinical practice.The antitumor properties of cisplatin (cis-diaminedichloroplatinum(ii)) were discovered in the 60’s by Rosenberget al.12and its clinical use was approved by the FDA in 1978. When DNA reacts with cisplatin, cross-linked adducts are produced, resulting in the distortion of its double-helix structure, with a global bending in the duplex of 35–40° and a local unwinding of 25° in the double helix.13As a consequence of the DNA damage several cellular processes are disrupted, including transcription and replication, being cell death, either by apoptosis or necrosis, finally induced.14As it has been recently summarised, there are four consecutive stages involved in the inhibition of the transcription by Pt-containing drugs: (1) cellular accumulation by both passive and active uptake; (2) activation of the Pt(ii) complex; (3) binding to nucleic acids to form a variety of Pt-DNA adducts, and (4) the cellular response to DNA damage.14Clinical treatments are limited by the toxic side effects such as nephrotoxicity, emetogenesis and neurotoxicity. Nephrotoxicity may be partially inhibited using administration protocols including a previous hydration of the patients with saline solutions15as well as varying the extension of the infusion times, volume and number of the dosages.16Efforts have been made towards the improvement of cisplatin therapeutic properties, looking for minimising its side effects, and as a result, many new Pt-based drugs have been developed, some of them already approved for clinical use or under consideration for approval by regulatory authorities. Carboplatin (cis-diamine(1,1-cyclobutanedicarboxylato)platinum(ii)), which was authorised by the FDA in 1989, is a second generation drug widely used to treat ovarian and lung cancers. Compared to cisplatin, carboplatin presents lower activity and toxicity, can be used more easily in combined therapies and is active against the same type of tumours. Oxaliplatin ((trans-RR-cyclohexane-1,2-diamine)oxalatoplatinum(ii)), a third generation Pt-drug approved by the FDA in 2004, improves the toxic behaviour of cisplatin, does not present cross-resistance with cisplatin and is very effective against colon cancer.17The main side effects of carboplatin and oxaliplatin are myelosupression and neuropathy, respectively.Fig. 1shows some other important Pt-based drugs. Tetraplatin, iproplatin and satraplatin (JM216) are Pt(iv) water soluble compounds, and therefore, suitable for oral administration. Specifically, satraplatin has been extensively studied18and now is under consideration for approval by the FDA for hormone-refractory prostate cancer.19Other promising Pt-drugs are nedaplatin, lobaplatin and picoplatin, the latter in Phase III of clinical trials for small-cell lung cancer.20New generation Pt-based drugs that do not follow the traditional structure–activity rules of platinum cytoxicity established by Cleare and Hoeschele in 197321aretrans-compounds (JM335) or even polynuclear complexes (BBR3464).Structures of the most important Pt-based drugs.The evolution of the number of publications involving cisplatin, oxaliplatin and carboplatin analysis in the last decades shows a great increase. This tendency is due not only to the current interest of the scientific community in Pt-based drugs, but also to the improvement of the analytical tools and methodologies used in such studies. A new discipline, Metallomics,22is being developed to study the evolution of metal or metalloid species and their interaction in time and space with other type of molecules, including biomolecules, focusing both in qualitative and quantitative aspects. Consequently, the goals of metallomics are the determination of the metallome, which is defined as the entirety of metal and metalloid species within a cell or tissue type, and the elucidation of the physiological roles and functions in which any metallo-species may be implicated in a biological system.23Analytical Chemistry methodologies, techniques and instrumentation are essential for the study of metallomes.24The chemical forms and amounts of trace elements present in biological systems are responsible for their bioavailability, toxicity or functionality. For that reason, elemental biospeciation and structural determination studies are essential for the comprehension of the effects produced by metal-containing compounds in living organisms. The high complexity of Pt-based drugs metallomics studies arise from factors like the poor stability of the drugs and drug–biomolecule adducts, their low concentrations in real samples, the different complex sample matrices to be analysed and the high amount of potential biomolecule targets.25,26Pt(ii), as a soft Lewis acid, presents a high affinity for soft bases with donor atoms. It shows a preferential binding toS-donor groups, but also other groups such asN-donors represent significant coordination points, being the affinity to the latter higher than forO-donors. Thus, aside from DNA, peptides and proteins withS- andN-containing aminoacidic residues are potentially reactive molecules towards Pt-drugs.26It has been suggested that on reaction with proteins, cisplatin may bind as a bidentate ligand, for instance, with one of the leaving groups replaced by aS-donor and the other by a neighbouringN- orO- donor. Since the SH → SS bonding is important for the protein conformation, the binding of Pt-drugs to cystines may produce an alteration on the structure and the biological functions of proteins. This fact may be related to the side effects observed during the antitumor treatment.When Pt-based drugs enter the blood stream, a series of interactions with blood components and later on with other molecules on cell membranes and inside the cells take place. The low chloride concentration in cytosols triggers the hydrolysis of cisplatin, producing a series of adducts as a result of the interaction of the evolved drug with cytoplasmic biomolecules. It is believed that some of these adducts are the key to understand the antitumor and toxic effects of Pt-drugs. The extent of the DNA-adducts formation may be limited by the interaction of the drug with cytosolic biomolecules like MT (metallothioneins) or GSH (glutathione), resulting in detoxification or resistance effects. Moreover, the binding of the drugs to blood proteins or the efficacy of the cell uptake could also alter the supply of the drug to the DNA. Other proposed resistance mechanism is related with the capacity of the cell to recognise and repair the damage produced by the drug–DNA adducts avoiding cell death.The low concentration and the high diversity of drug–biomolecule adducts require the use of mass spectrometry analytical techniques, both elemental and molecular, coupled to different separation techniques, for the quantification and structural determination of Pt-adducts.27Depending on the objective, blood, urine, tissues or cell fractions must be analysed, using at least one high resolution separation technique.28Both analysis conditions and sample treatment, such as pre-concentration or clean-up steps, must ensure the preservation of the Pt-species identities. Nevertheless, the difficulty to analyse Pt-adducts at the trace levels present in real samples, preserving their identity along the several steps needed to separate and detect the adducts, has precluded, in general,in vivoexperiments in bio-speciation. Alternatively, many of the reported articles consist inin vitroexperiments that simplify the sample matrix and allow working with more concentrated species. The concordance betweenin vitroandin vivoexperiments is usually a problem because slight differences in the reaction media and analyte concentration results in great differences in the identity and stability of the Pt-species. Results obtained fromin vitroexperiments must be carefully reviewed evaluating their reproducibility in biological living systems, being in any case a previous step to tackle the challenge of analysing real samples.Not only speciation studies are important to evaluate the Pt-based drugs mechanism of action. Accumulation and distribution studies of the drug among different organs, tissues and cell compartments impose the use of cell fractionation techniques and cut-off filters to determine the affinity of the drug and to allow the identification of their main biological targets.29The use of samples from patients with different sort of tumours, ages, administration dosages,etc., forin vivoexperiments, have allowed to draw a distribution map of Pt-based drugs in organisms and are reported in a great number of works.29,30The information collected in this review will help the reader to clarify and sum up the reported data related with the speciation of Pt-containing drugs and the Pt adducts they form with biomolecules and also concerning the determination of total Pt content after the administration of the aforementioned drugs. The most commonly employed analytical tools are presented firstly, including sample preparation, separation and detection techniques. According to the sample matrix, the last section contains a complete summary of the most interesting reported analytical articles.