Volabolomic Fingerprinting for Post-Mortem Interval Estimation: A Novel Physiological Approach.

Death is a multifaceted process wherein each individual cell and tissue has a metabolic homeostasis and a time of functional cessation defined by the dying process as well as by intrinsic and extrinsic factors. Decomposition is physiologically associated with the release of different types of volatile organic compounds (VOCs), and these form volaboloma mortis. The main purpose of this study was to record the volabolomic fingerprint produced by volatile molecules during the physiological decomposition process of human tissue and muscle cells. The volatile chemical signature has important implications for an open issue in forensics and pathology, namely the estimation of the postmortem interval (PMI), which decreases in accuracy with the passage of time. Volatile metabolites emitted from human tissues and muscle cells at 0, 24, 48, and 72 h were recorded in real time with an electronic nose sensor device. The key findings were the continuous sampling of VOCs emitted from tissues and cells. These showed a common behavior as time progressed; particularly, after 48 h the distributions became dispersed, and after 72 h they became more variable. Volabolomic fingerprinting associated with time progression relevant to the study of PMIs was reconstructed. Additionally, there may be broader applications, such as in dog training procedures for detecting human remains, and perhaps even for studying scavenger and insect attractants.

Impact of TNF and IL-33 Cytokines on Mast Cells in Neuroinflammation.

Mast cells (MCs) are derived from hematopoietic progenitors, mature in vascularized tissues, and participate in innate and acquired immunity. Neuroinflammation is a highly debated topic in the biomedical literature; however, the impact of tumor necrosis factor (TNF) and IL-33 on MCs in the brain has not been widely addressed. MCs can be activated by IgE binding to FcεRI, as well as by different antigens. After activation, MCs mediate various immunological and inflammatory responses through TNF and IL-33. TNF has two receptors: TNFR1, a p55 molecule, and TNFR2, a p75 molecule. This cytokine is the only one of its kind to be stored in the granules of MCs and can also be generated by de novo synthesis via mRNA. In the central nervous system (CNS), TNF is produced almost exclusively by microglial cells, neurons, astrocytes, and, minimally, by endothelial cells. After its release into brain tissue, TNF rapidly induces the adhesion molecules endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. TNF causes the chemoattraction of neutrophils by inducing several molecules, including CXC chemokines (IL-8). Both MCs and microglial cells act as a primary barrier against foreign molecules in the CNS, producing pro-inflammatory cytokines such as IL-33. IL-33 belongs to the IL-1 family, is activated through the ST2L/IL1-RAcP receptor complex, and mediates both the innate and adaptive immune response. IL-33 is a nuclear transcription factor expressed in the brain, where it induces pro-inflammatory cytokines (TNF and IL-1) and chemokines (CCL2, CCL3, CCL5, and CXCL10). Therefore, MCs and microglia in the CNS are a source of pro-inflammatory cytokines, including TNF and IL-33, that mediate many brain diseases. The inhibition of TNF and IL-33 may represent a new therapeutic approach that could complement existing neuroinflammatory therapies.

Applications of electrophoresis for small enantiomeric drugs in real-world samples: Recent trends and future perspectives.

Separation and identification of chiral molecules is a topic widely discussed in the literature and of fundamental importance, especially in the pharmaceutical and food fields, both from industrial and laboratory points of view. Several techniques are used to carry out these analyses, but high-performance liquid chromatography is often the "gold standard." The high costs of chiral columns, necessary for this technique, led researchers to look for an alternative, and capillary electrophoresis (CE) is a technique capable of overcoming some of the disadvantages of liquid chromatography, often providing comparable results in terms of sensitivity and robustness. We addressed this topic, already widely discussed in the literature, providing an overview of the last 6 years of the most frequent and recent applications of CE. To make the manuscript more effective, we decided to divide it into paragraphs that represent the main field of application, from enantioseparation in complex matrices (pharmacokinetic studies or toxicological dosage of drugs, analysis of environmental pollutants, and analyses of foods) to quality control analyses on pharmaceutical formulas. About these, which are the fields of most meaningful use, we mentioned some of the most innovative and performing methods, with a look to the future on the application of new materials used, such as chiral selectors, that can make these types of analyses accessible to all, reducing cost, time, and excessive use of toxic solvents.

Activation of Mast Cells by Neuropeptides: The Role of Pro-Inflammatory and Anti-Inflammatory Cytokines.

Mast cells (MCs) are tissue cells that are derived from bone marrow stem cells that contribute to allergic reactions, inflammatory diseases, innate and adaptive immunity, autoimmunity, and mental disorders. MCs located near the meninges communicate with microglia through the production of mediators such as histamine and tryptase, but also through the secretion of IL-1, IL-6 and TNF, which can create pathological effects in the brain. Preformed chemical mediators of inflammation and tumor necrosis factor (TNF) are rapidly released from the granules of MCs, the only immune cells capable of storing the cytokine TNF, although it can also be produced later through mRNA. The role of MCs in nervous system diseases has been extensively studied and reported in the scientific literature; it is of great clinical interest. However, many of the published articles concern studies on animals (mainly rats or mice) and not on humans. MCs are known to interact with neuropeptides that mediate endothelial cell activation, resulting in central nervous system (CNS) inflammatory disorders. In the brain, MCs interact with neurons causing neuronal excitation with the production of neuropeptides and the release of inflammatory mediators such as cytokines and chemokines. This article explores the current understanding of MC activation by neuropeptide substance P (SP), corticotropin-releasing hormone (CRH), and neurotensin, and the role of pro-inflammatory cytokines, suggesting a therapeutic effect of the anti-inflammatory cytokines IL-37 and IL-38.

LC-MS/MS Application in Pharmacotoxicological Field: Current State and New Applications.

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.

When the Helmet Is Not Enough: Forensic Multidisciplinary Reconstruction of a Deadly Motorcycle Accident.

We report the case of a 54-year-old man who died in a motorcycle accident due to loss of control of the vehicle on a viaduct. No other vehicles were apparently involved, except for a car hit by the motorcycle after it fell. A post-mortem CT scan (computed tomography scan) was performed showing complex head trauma with a subarachnoid hemorrhage and multiple skull and facial bone fractures. A forensic cinematic reconstruction performed by an engineer was needed to exclude other incident causes other than the loss of control. The multidisciplinary approach that included autopsy findings, a cinematic reconstruction, a helmet test and an examination played a key role in clarifying the dynamics of the accident, allowing us to explain how the death occurred despite the motorcyclist's helmet use. The cause of death was identified as a penetrating head trauma with cerebral material exposure, produced by the impact of the head against a fixed bolt in the guardrail base. Despite the use of the helmet, the impact force was enough to render the protection ineffective and allowed the bolt to penetrate through the helmet and the skull.

Antigenicity Preservation Is Related to Tissue Characteristics and the Post-Mortem Interval: Immunohistochemical Study and Literature Review.

The main aim of this study was to investigate the post-mortem proteolytic degradation process of selected tissue antigens and correlate it to the post-mortem interval. During the autopsy of 12 cadavers (time interval ranging 1 day-2 years after death) samples of skin, liver, kidney, and spleen were collected. All samples were formalin-fixed and paraffin-embedded. Four µm paraffin sections were used for hematoxylin-eosin staining and immunohistochemical analysis (Ki67, Vimentin, Pan cytokeratin, and CD20). Data reported here show that immunohistochemical reactivity preservation was related to the characteristics of the tissues. In particular, the most resistant tissue was the skin, where the autolysis phenomena were not appreciable before 5 days. On the contrary, the liver and the spleen underwent early autolysis, while the kidney displayed an early autolysis of the tubules and a late one of the glomeruli. As concerns specific antigens, immunoreactivity was lost earliest for nuclear antigens as compared to cytoplasmic ones. In conclusion, our results demonstrate that immunohistochemical detection of specific antigens may be useful in estimating the post-mortem interval, especially when we need to know whether the post-mortem interval is a few days or more than 7-10 days.

New Perspectives for Postmortem Human Satellite Cells of Different Embryological Origin.

Human postmortem skeletal muscles are a unique source of satellite cells for skeletal muscle regenerative studies. Presomite and somite satellite cells obtained by postmortem muscles have been established as populations of human skeletal muscle precursor cells able to proliferate and differentiate in vitro. It is extremely interesting to have access to a large amount of postmortem human skeletal muscle precursor cells, especially from craniofacial as well as limb skeletal muscles in order to evaluate their potential application not only for the fundamental understanding of muscle physiology and diseases but also for drug testing in a challenging 3D-shaping muscles like skeletal muscle microphysiological systems.

Long persistence of severe acute respiratory syndrome coronavirus 2 swab positivity in a drowned corpse: a case report.

BACKGROUND: Since the beginning of the worldwide spread of severe acute respiratory syndrome coronavirus 2 to date, important knowledge has been obtained about the virus behavior in living subjects and on inanimate surfaces; however, there is still a lack of data on virus persistency on dead bodies and the risk of contagion from cadavers. CASE PRESENTATION: The present case shows the persistency of the severe acute respiratory syndrome coronavirus 2 viral genome in nasopharyngeal swabs performed on a drowned Caucasian man, aged 41 years old, who was completely asymptomatic when he was alive, up to 41 days after death. Specific real-time reverse transcriptase-polymerase chain reaction (TaqMan 2019-nCoV Assay Kit v2; Thermo Fisher Scientific, Italy and Realquality RQ-SARS-CoV-2, AB Analytical) was used to evaluate the swabs. CONCLUSIONS: This data reflect the importance of postmortem swabs in all autopsy cases, and not only in potential severe acute respiratory syndrome coronavirus 2-related death, and also highlight the necessity to evaluate virus positivity a long time after the moment of death, even if a low initial viral load was assessed.

Fast Quantitative LC-MS/MS Determination of Illicit Substances in Solid and Liquid Unknown Seized Samples.

Narcotic and psychotropic substances are natural, synthetic, or semisynthetic compounds that are present in both solid and liquid illicit products. The alterations effects on the central nervous system related to their use can be psycholeptic, psychoanaleptic, or psychodiseptic and are able to generate tolerance, addiction, or dependence phenomena, creating social and public order problems. In this scenario, the analytical evaluations that aim to determine these analytes in seized nonbiological samples, and which assume the character of judicial evidence, must meet high analytical requirements of reliability, transparency, and procedures uniformity at a national level. For the first time in the literature, the herein validated method is able to provide the simultaneous quantitative determination of 37 of the most common narcotic substances as well as the most commonly used excipients/adulterants found in seized illicit material. Additionally, the validated method can process both solid and liquid samples maintaining the precision and trueness levels (intraday and interday) in accordance with the U.S. Food and Drug Administration and European Medicines Agency international guidelines (<14.31 and <13.41%, respectively). Furthermore, it provides a simple and fast procedure for sample preparation using the dilute and shoot approach, exploiting the sensitivity and selectivity of the LC-MS/MS instrument configuration used and the signal acquisition in multiple reaction monitoring (MRM) mode (both positive and negative polarization modes).

Therapeutic Drug Monitoring of Antiseizure Medications Using Volumetric Absorptive Microsampling: Where Are We?

Therapeutic drug monitoring (TDM) of antiseizure medications (ASMs) represents a valuable tool to establish an appropriate patient therapy, to collect important information about drugs' interactions and to evaluate patient's metabolic capabilities. In recent years, a new volumetric absorptive microsampling technique using VAMS® technology and Mitra® devices, consisting of a sampling technique for the collection of fixed-volume capillary blood, was developed. These new devices provide a new home-sampling technique for whole blood that has been spread out to simplify sample collection from finger-pricks. This review is aimed to compare published articles concerning the application of VAMS® in epilepsy and to identify the strengths and improvement points for the TDM of antiseizure medications. VAMS® allowed a minimally invasive blood sampling even in the absence of trained personnel. Good stability data have indicated that storage and delivery can be facilitated only for specific ASMs. Trueness and precision parameters have been evaluated, and the hematocrit (HCT) effect was minimized.

Forensic Biochemical Markers to Evaluate the Agonal Period: A Literature Review.

Currently, forensic research is multidisciplinary with new methods and parameters useful to define the cause and time of death as well as survival/agony times. The identification of biochemical markers able to estimate agonal period has been studied by many forensic researchers. It is known that the estimation of agonal time in different types of death is not always easy, hence our interest in literature's data. The studies analyzed in this review confirm the important role of thanatobiochemistry for the estimation of survival times. Regardless of the death cause, the survival/agony time between the primary event and death influences markers concentrations in biological samples (e.g., blood, urine, cerebrospinal fluid). Different biomarkers can be used for qualitative evaluations in deaths with short and long agony (e.g., C-reactive protein, ferritin, GFAP, etc.). Instead, the quantitative interpretation showed limits due to the lack of reference cut-offs. Thanatobiochemistry is a useful tool to confirm what emerged from autopsies findings (macroscopic and histological analysis), but further studies are desirable to confirm the evidence emerging from our review of the literature.

Fabric-Phase Sorptive Membrane Array As a Noninvasive In Vivo Sampling Device For Human Exposure To Different Compounds.

This study introduces an innovative device for the noninvasive sampling and chromatographic analysis of different compounds present in exhaled breath aerosol (EBA). The new sampling device, especially in light of the recent COVID-19 pandemic that forced many countries to impose mandatory facemasks, allows an easy monitoring of the subject's exposure to different compounds they may come in contact with, actively or passively. The project combines the advantages of a fabric-phase sorptive membrane (FPSM) as an in vivo sampling device with a validated LC-MS/MS screening procedure able to monitor more than 739 chemicals with an overall analysis time of 18 min. The project involves the noninvasive in vivo sampling of the EBA using an FPSM array inserted inside an FFP2 mask. The study involved 15 healthy volunteers, and no restrictions were imposed during or prior to the sampling process regarding the consumption of drinks, food, or drugs. The FPSM array-LC-MS/MS approach allowed us to effectively exploit the advantages of the two complementary procedures (the convenient sampling by an FPSM array and the rapid analysis by LC-MS/MS), obtaining a powerful and green tool to carry out rapid screening analyses for human exposure to different compounds. The flexible fabric substrate, the sponge-like porous architecture of the high-efficiency sol-gel sorbent coating, the availability of a large cache of sorbent coatings, including polar, nonpolar, mixed mode, and zwitterionic phases, the easy installation into the facemask, and the possibility of sampling without interrupting regular activities provide FPSMs unparalleled advantages over other sampling techniques, and their applications are expected to expand to many other clinical or toxicological studies.

A fatal case of post endobronchial ultrasound transbronchial needle aspiration endobronchial haemorrhage: a case report and review of the literature.

Notwithstanding life-threatening haemorrhagic complications, endobronchial ultrasound-guided transbronchial needle aspiration represents a cornerstone in the evaluation of NSCLC patients. Due to its low invasiveness and satisfactory tolerability even in high-risk patients, it is usually scheduled in one-day surgery hospital stay. Moreover, EBUS-TBNA offers a viable alternative to other conventional endoscopic procedures such as mediastinoscopy with the addiction to gain access also to hilar nodes. We report an unexpected and rare event of post-bronchoscopy fatal endobronchial haemorrhage in a 67-year-old female patient with a right S1 solitary pulmonary nodule and concomitant multiple bilateral lymphadenopaties. According to clinical staging the patient was unfit for upfront surgery and endoscopic procedure for tissue diagnosis was scheduled. The immediate postoperative period was uneventful as no intraoperative injuries were reported. Twenty-four hours later, the patient claimed respiratory distress and, after admission to the Emergency Department, a massive and uncontrolled bleeding coming from the lower respiratory tract was diagnosed leading to a death. Although one-day surgery EBUS-TBNA is the standard of care in patients undergoing endoscopic exploration, current guidelines seem to be murky about proper case stratification in order to plan an exhaustive observation time especially in high-risk patients.

Ethanol Determination in Post-Mortem Samples: Correlation between Blood and Vitreous Humor Concentration.

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.

A new LC-MS/MS confirmation method for the determination of 17 drugs of abuse in oral fluid and its application to real samples.

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation method for the direct analysis of 17 drugs starting from 200µL of diluted oral fluid (OF), in a single chromatographic run, was developed and validated. Cocaine, benzoylecgonine (BEG), cocaethylene, Δ-9-tetrahydrocannabinol (Δ-9-THC), buprenorphine, 6-acetylmorphine (6AM), morphine, codeine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), 3,4-Methylenedioxy-N-ethylamphetamine (MDE), ketamine, N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) were determined in a chromatographic run of 12min only with no sample pre-treatment, after the addition of 15 different internal standards (ISs). The method met all requirements in terms of linearity, accuracy (precision and trueness), recovery, and stability requested by FDA guidelines. Carry-over and interferences were negligible, as well as the matrix effects. LLOQs are below the limits defined by European guidelines and Italian national laws. The original oral fluid collections are stable at least six months at -20°C and one week at +4°C.

Novel MIPs-Parabens based SPE Stationary Phases Characterization and Application.

In this work, the synthesis, characterization, and application of novel parabens imprinted polymers as highly selective solid-phase extraction (SPE) sorbents have been reported. The imprinted polymers were created using sol-gel molecular imprinting process. All the seven parabens were considered herein in order to check the phase selectivity. By means of a validated HPLC-photodiode array detector (PDA) method all seven parabens were resolved in a single chromatographic run of 25 min. These SPE sorbents, in-house packed in SPE empty cartridges, were first characterized in terms of extraction capability, breakthrough volume, retention volume, hold-up volume, number of theoretical plates, and retention factor. Finally, the device was applied to a real urine sample to check the method feasibility on a very complex matrix. The new paraben imprinted SPE sorbents, not yet present in the literature, potentially encourage the development of novel molecularly imprinted polymers (MIPs) to enhance the extraction efficiency, and consequently the overall analytical performances, when the trace quantification is required.

Progression in migraine: Role of mast cells and pro-inflammatory and anti-inflammatory cytokines.

Migraine is a common painful neurovascular disorder usually associated with several symptoms, such as photophobia, phonophobia, nausea, vomiting and inflammation, and involves immune cells. Mast cells (MCs) are immune cells derived from hematopoietic pluripotent stem cells which migrate and mature close to epithelial, blood vessels, and nerves. In almost all vascularized tissues there are MCs that produce, contain and release biologically active products including cytokines, arachidonic acid compounds, and proteases. In addition, MCs participate in innate and adaptive immune responses. Innate responses in the central nervous system (CNS) occur during neuroinflammatory phenomena, including migraine. Antigens found in the environment have a crucial role in inflammatory response, causing a broad range of diseases including migraine. They can be recognized by several innate immune cells, such as macrophages, microglia, dendritic cells and MCs, which can be activated trough Toll-like receptor (TLR) signaling. MCs reside close to primary nociceptive neurons, associate with nerves, and are capable of triggering local inflammation. MCs are involved in the pathophysiology of various tissues and organs, especially where there is an increase of angiogenesis. Activated MCs release preformed mediators include histamine, heparin, proteases (tryptase, chimase), hydrolases, cathepsin, carboxypeptidases, and peroxidase, and they also generate pro-inflammatory cytokines/chemokines. In addition, activated macrophages, microglia and MCs in the CNS release pro-inflammatory cytokines which provoke an increase of arachidonic acid product levels and lead to migraine and other neurological manifestations including fatigue, nausea, headaches and brain fog. Innate immunity and pro-inflammatory interleukin (IL)-1 cytokine family members can be inhibited by IL-37, a relatively new member of the IL-1 family. In this article, we report that some pro-inflammatory cytokines inducing migraine may be inhibited by IL-37, a natural suppressor of inflammation, and innate and acquired immunity.

Anomalous Fusion of Right Pulmonary Artery to Aortic Arch: Case Report of a Rare and Fatal Congenital Malformation in a Newborn and a Literature Review.

BACKGROUND We present a report of a rare cardiac malformation case as well as a review of the literature. In addition, the diagnostic features are discussed. CASE REPORT The case of a female newborn who died on her third day of life was studied at the Institute of Legal Medicine, University of Chieti-Pescara (Italy). The investigations around her death revealed a cardiac congenital malformation, seen as a rare variant of a common arterial trunk, in which the aorta was fused with the right branch of the pulmonary artery. The ascending aorta showed hypoplasia, while the coronary arteries were free of any pathological findings. The atrial septum showed a closed foramen ovale and the ventricular septum did not show any defect. Only an isolated right ventricular hypertrophy and dilation with no other cardiac abnormalities was found. The cause of death was acute respiratory failure on the third day of extrauterine life when the ductus Botalli closed. The karyotype analysis performed in this case was normal, and the fluorescent in situ hybridization analysis did not show the 22q11.2 microdeletion suggestive of the DiGeorge syndrome. CONCLUSIONS These findings underline the value of 3-dimensional/4-dimensional ultrasound imaging when added to a fetal cardiology screening program, and the need for improvements in postnatal screening routines by using pulse oximetry in order to discover isolated vascular defects before circulatory collapse occurs, as well as to reduce the medico-legal disputes in cases of missed diagnosis. We found the relevant literature search lacked a description of this congenital malformation, which supports our deeper perinatal investigation.

FPSE-HPLC-DAD method for the quantification of anticancer drugs in human whole blood, plasma, and urine.

The present work describes a fast, sensitive and selective procedure for the analyses of aromatase inhibitors including anastrozole, letrozole and exemestane used in the treatment of metastatic breast cancer by high performance liquid chromatography (HPLC) in human whole blood, plasma, and urine samples succeeding an extraction by innovative fabric phase sorptive extraction (FPSE). These drugs were successfully determined using a Luna C18 column at 25 °C using acetonitrile and phosphate buffer. The analytical method was validated, using weighted-matrix matched standard calibration curves. The intra- and inter-day accuracy values (precision and trueness) fulfill the criteria of International Guidelines on Bioanalytical Methods Validation. The analytical performances were further tested on real human biological samples. To the best of our knowledge, this is the first FPSE procedure applied to human whole blood, plasma, and urine samples for the concurrent analysis of aromatase inhibitors possessing a wide range of polarity index values and could be easily adopted as a rapid and green analytical protocol for clinical and pharmaceutical applications. The proposed HPLC method is very innovative since in the literature there are only methods dealing with a single antitumoral drug, and no process has been described so far for these three antitumoral drugs together directly from the whole blood.