Nowadays, it is vital to have new, complete, and rapid methods to screen and follow pharmacotoxicological and forensic cases. In this context, an important role is undoubtedly played by liquid chromatography-tandem mass spectrometry (LC-MS/MS) thanks to its advanced features. This instrument configuration can offer comprehensive and complete analysis and is a very potent analytical tool in the hands of analysts for the correct identification and quantification of analytes. The present review paper discusses the applications of LC-MS/MS in pharmacotoxicological cases because it is impossible to ignore the importance of this powerful instrument for the rapid development of pharmacological and forensic advanced research in recent years. On one hand, pharmacology is fundamental for drug monitoring and helping people to find the so-called “personal therapy” or “personalized therapy”. On the other hand, toxicological and forensic LC-MS/MS represents the most critical instrument configuration applied to the screening and research of drugs and illicit drugs, giving critical support to law enforcement. Often the two areas are stackable, and for this reason, many methods include analytes attributable to both fields of application. In this manuscript, drugs and illicit drugs were divided in separate sections, with particular attention paid in the first section to therapeutic drug monitoring (TDM) and clinical approaches with a focus on central nervous system (CNS). The second section is focused on the methods developed in recent years for the determination of illicit drugs, often in combination with CNS drugs. All references considered herein cover the last 3 years, except for some specific and peculiar applications for which some more dated but still recent articles have been considered.

In recent years, major attention has been focused on microextraction procedures that allow high recovery of target analytes, regardless of the complexity of the sample matrices. The most used techniques included liquid-liquid extraction (LLE), solid-phase extraction (SPE), solid-phase microextraction (SPME), dispersive liquid-liquid microextraction (DLLME), microextraction by packed sorbent (MEPS), and fabric-phase sorptive extraction (FPSE). These techniques manifest a rapid development of sample preparation techniques in different fields, such as biological, environmental, food sciences, natural products, forensic medicine, and toxicology. In the biological and forensic fields, where a wide variety of drugs with different chemical properties are analyzed, the sample preparation is required to make the sample suitable for the instrumental analysis, which often includes gas chromatography (GC) and liquid chromatography (LC) coupled with mass detectors or tandem mass detectors (MS/MS). In this review, we have focused our attention on the biological and forensic application of these innovative procedures, highlighting the major advantages and results that have been accomplished in laboratory and clinical practice.

Ethanol (ethylic alcohol) represents the most commonly used drug worldwide and is often involved in clinical and forensic toxicology. Based on several reports, excessive alcohol consumption is the main contributing factor in traffic accidents, drownings, suicides, and other crimes. For these reasons, it becomes essential to analyze the alcohol concentration during autopsy. Although blood is usually used for alcohol analysis in post-mortem cases, it could suffer alterations, putrefaction, and microbial contaminations. As an alternative to whole blood, vitreous humor has been successfully used in medico-legal studies. In this work, post-mortem specimens were analyzed for ethanol determination. The analysis of blood and vitreous humor were carried-out using gas chromatography-flame ionized detector (GC-FID) with a total run time of 6 min. The method was validated in terms of limit of detection, limit of quantification, dynamic range, sensibility, recovery, precision and trueness. A linear regression analysis indicated a coefficient of determination (R²) of 0.9981. The study confirmed no statistically differences between alcohol concentration in blood and vitreous humor, leading vitreous humor as an excellent matrix that could be used as an alternative to whole blood in toxicological analysis in cases where blood is not available.

Nowadays, the challenges in analytical chemistry, and mostly in quantitative analysis, include the development and validation of new materials, strategies and procedures to meet the growing need for rapid, sensitive, selective and green methods. In this context, considering the constantly updated International Guidelines, constant innovation is mandatory both in the pre-treatment procedures and in the instrumental configurations to obtain reliable, true, and reproducible information. In this context, additionally to the classic plasma (or serum) matrices, biopsies, whole blood, and urine have seen an increase in the works that also consider non-conventional matrices. Obviously, all these studies have shown that there is a correlation between the blood levels and those found in the new matrix, in order to be able to correlate and compare the results in a robust way and reduce any bias problems. This review provides an update of the most recent developments currently in use in the sample pre-treatment and instrument configurations in the biological/clinical fields. Furthermore, the review concludes with a series of considerations regarding the role and future developments of Analytical Chemistry in light of the forthcoming challenges and new goals to be achieved.

The primary cause of poor and ambiguous results obtained from the bioanalytical process is the sample pre-treatment, especially in clinical analysis because it involves dealing with complex sample matrices, such as whole blood, urine, saliva, serum, and plasma. So, the aim of this review is to focus attention on the classical and new techniques of pre-treatment for biological samples used in the bioanalytical process. We discussed the methods generally used for these types of complex samples. Undoubtedly, it is a daunting task to deal with biological samples because the analyst may encounter a substantial loss of the analytes of interest, or the overall analysis may be too time-consuming. Nowadays, we are inclined to use green solvents for the environment, but without sacrificing analytical performance and selectivity. All the characteristics mentioned above should be added to the difficulty of the withdrawal of samples like blood because it can be an invasive practice. For these reasons, now we can also find in the literature the use of saliva as alternative biological samples and new techniques that do not require substantial sample pre-treatment, such as fabric phase sorptive extraction (FPSE). The text has been divided into the following two distinct parts: firstly, we described clinical applications under different subsections, such as anticancer drugs, antibiotics, vitamins, antivirals, non-steroidal anti-inflammatory drugs, statin, imidazoles, and triazoles. The second part is dedicated to sample preparation techniques for diagnostic purposes and is divided into the following different sample preparation techniques: solid-phase microextraction (SPME), microextraction by packed sorbent (MEPS), dispersive liquid–liquid microextraction (DDLME), and fabric phase sorptive extraction (FPSE).

One of the most discussed topics concerns the sample preparation before the analysis and, therefore, all the operations necessary to eliminate the interferents, clean up the specimens, and extract the analytes of interest, reducing the matrix effect. This review highlights the fundamental steps in the treatment of postmortem samples used in forensic analysis. Through critical literature research, it was possible to choose among the countless works that could provide a general overview of the state-of-the-art in this field. Different biological matrices have been considered; blood and urine (the traditional biological fluids) are used to investigate the presence of substances that may have caused death, whilst other body fluids, such as bile and oral fluids, are still under discussion for their usability (and suitability). In the second part of the review, all the solid matrices obtained after autopsy were further divided into conventional and unconventional matrices to facilitate proper understanding. The choice of literature was also made according to the most widely used pretreatment techniques and the most representative innovative techniques.