Recent advances of drugs monitoring in oral fluid and comparison with blood.

The use of alternative matrices in toxicological analyses has been on the rise in clinical and forensic settings. Oral fluid (OF), as non-invasive fluid, has attracted attention in the field of drug screening, both for therapeutic and forensic purposes, as well as for medical diagnosis, clinical management, on-site (real time) doping and for monitoring environmental exposure to toxic substances. A good correlation between OF and blood is now established for drug concentrations. Therefore, OF might be a potential substitute of blood, especially for long-term surveillance (e.g., therapeutic drugs) or to screen a large number of patients, as well as for the development of salivary point-of-care technologies. In this review, we aimed to summarize and critically evaluate the current literature that focused on the comparison of drugs detection in OF and blood specimens.

GC-MS analysis of soil faecal biomarkers uncovers mammalian species and the economic management of the archeological site "Le Colombare di Negrar".

The identification of the mammalian species based on faecal sediments in modern and ancient environments is the aim of the research of archaeologists, forensic scientists and ecologists. Here, we set up and validated an optimized gas chromatography-mass spectrometry (GC-MS) method, characterized by a time-saving sample preparation protocol, for the simultaneous analysis of faecal biomarkers (6 sterols/stanols and 5 bile acids) in 14 soil samples from the archaeological site of "Le Colombare di Negrar" in northern Italy. Although the archaeological sediment samples examined are numerically exiguous, a comparative reading of our faecal biomarkers findings with new studies on faunal materials collected in the same stratigraphic detail during recent excavation campaigns will allow to better clarify the economic interest of the animal species farmed in the Colombare site (such as bovines, goats, sheep and pigs) and to shed light on the management of breeding. Together with archaeozoological and archaeobotanical analyses, the investigation of faecal biomarkers can increase our knowledge of how ancient local communities exploited natural resources and may allow us to deduce what their impact on the landscape was.

Involvement of the endocannabinoid system in current and recurrent periodontitis: A human study.

OBJECTIVE: The aim of the present study was to assess if the endocannabinoid system is involved differently in patients with recurrent and non-recurrent periodontal disease and if in sites that have a predisposition for reactivation, levels of anandamide (AEA) change after periodontal therapy. BACKGROUND: Periodontal disease (PD) may be due to a dysregulation of the endocannabinoid system. METHODS: Periodontal patients were recruited, treated for PD and monitored. Gingival samples from these patients with recurrent (n = 10) and non-recurrent (n = 10) periodontal disease were harvested before and after treatment and compared to those of periodontally healthy (n = 10) subjects. Levels of CB1 and CB2, AEA and CBs receptor activation were assessed in healthy and inflamed samples using immunohistochemistry, chromatography and autoradiography. In healed sites, AEA levels were also assessed. RESULTS: The number of CBs in inflamed sites of recurrent patients was significantly higher than in those with non-recurrent disease and also higher than those in healthy subjects. Inflamed sites of recurrent patients had significantly lower CBs receptor activation than those of healthy subjects. Levels of AEA in inflamed sites of non-recurrent patients were significantly higher than those found both in inflamed recurrent sites and in healthy sites. Otherwise, the amount of AEA in healthy subjects and recurrent inflamed sites was similar. After periodontal therapy, levels of AEA were significantly lower in both periodontal groups. In recurrent sites, they resulted significantly lower than in non-recurrent and even in healthy subjects. CONCLUSIONS: The endocannabinoid system seems involved differently in subjects with recurrent and non-recurrent periodontal disease.

Hexahydrocannabinol on the Light Cannabis Market: The Latest "New" Entry.

Introduction: Hexahydrocannabinols (HHCs), referred to as (9R)-HHC and (9S)-HHC diastereoisomers, are poorly studied cannabinoids naturally found in small concentrations in the pollen and the seeds of the hemp plants. Aim: In this study, for the first time, we describe the finding of (9R)-HHC and (9S)-HHC in two commercialized hemp derived products. Methods: The achievement of reference standards by semisynthetic or isolation approach allows us to develop and validate a gas chromatography mass spectrometry method for the identification and quantification of HHCs in hemp-derived resin. Results: The two analyzed samples showed percentage of 42.5 and 41.5 for (9R)-HHC and of 23.6 and 23.6 for (9S)-HHC. Conclusions: Despite the lack of in-depth studies about HHCs activity, potency, toxicity, and safety, these cannabinoids are emerging on the light-cannabis (hemp) market probably because legislations still do not clearly regulate them. Since analytical assay for hemp-derived products usually include only Δ9-THC, THC-A, CBD, and CBD-A, a thorough investigation could be carried out to reveal the possible addition of "new" compounds that might be a matter of safety.

Lipidomics of Cell Secretome Combined with the Study of Selected Bioactive Lipids in an In Vitro Model of Osteoarthritis.

Analytical advancements in lipidomics have enabled large-scale investigations of lipid biology. Herein, we focused on four bioactive lipid families, namely polyunsaturated fatty acids, eicosanoids, endocannabinoids, and N-acylethanolamines, and their involvement in the mesenchymal stem cells (MSC)-related inflammatory scenario. Since MSC secretome may represent a valid therapeutic alternative, here, the complete secretome and its vesicular component from adipose- and bone marrow-derived MSC and dermal fibroblasts were characterized by targeted mass spectrometry lipidomics. The 2-arachidonoylglycerol (2AG) and the palmitoylethanolamide (PEA), previously quantified in the MSC's secretome, were further investigated by assessing hypothetical effects in an in vitro model of osteoarthritis (OA) based on human primary articular chondrocytes (CH) stimulated with tumor necrosis factor alpha (TNFα). TNFα enhances the release of the inflammatory lipid prostaglandin E2 (PGE2), and an additional increment was observed when CH were treated with both TNFα and 2AG. In contrast, PEA downmodulates the PGE2 release to the levels of unstimulated CH suggesting a protective effect. TNFα also increases the expression of cyclooxygenase 2 (COX2), in particular when combined with 2AG, while PEA partly blunts TNFα-induced COX2 expression. In addition, TNFα-stimulated CH produce significantly higher levels of the inflammatory mediator nitric oxide (NO) both in the presence and in the absence of 2AG, and PEA was able to partially reduce NO release. Our results show a first partial lipidomic profile of MSC and DF secretome and suggest a possible implication of bioactive lipids in the OA scenario and in the future use of these cell-free products as innovative therapeutics.

Determination of cannabinoids in hair: Indicators for illegal vs CBD-rich cannabis use.

Cannabis products rich in cannabidiol (CBD) and low in Δ9-tetrahydrocannabinol (THC) (e.g., light cannabis in Italy) are becoming widely popular and available on the market as replacements for THC preparations and tobacco for their recreational and/or therapeutic benefits. In this paper, which aims to establish alternative discrimination parameters between hair samples from CBD-rich and THC-prevalent cannabis users, cannabinoid concentrations, such as THC, CBD, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) were quantified in 127 hair samples by a GC-MS/MS technique. Initially, this analysis was able to discriminate two cohorts: cohort 1 (individuals with THC values ≥ 0.05 ng/mg and THC-COOH ≥ 0.2 pg/mg or THC-positive users, n = 60) and cohort 2 (individuals with THC values ranging between 0.01 and 0.05 ng/mg and THC-COOH or 11-OH-THC ≥ LOQs, n = 67). The evaluation of CBD/THC ratio in cohort 2 identified two further sub-cohorts 2a (CBD/THC<<1 or ~ 1, THC-prevalent cannabis users) and 2b (CBD/THC>>1, suspected CBD-rich and THC-low cannabis users). The latter showed unusual profiles for THC metabolites, in particular for 11-OH-THC. Statistical evaluation of the data of cohort 1, cohort 2a and cohort 2b yielded significant differences in CBD/THC and THC/11-OH-THC. Based on the analysis of 337 seized cannabis samples and 630 CBD-rich/light cannabis samples by GC-FID and GC-MS, respectively, we also evaluated statistical differences in the CBD/THC ratio between biological (hair) and plant-derived samples. Considering the legal implications of a positive result, the obtained findings could be relevant for the interpretation of cannabinoid concentrations in hair. Further studies are necessary to elucidate the reason behind the unusual metabolic ratios.

Towards Secretome Standardization: Identifying Key Ingredients of MSC-Derived Therapeutic Cocktail.

The therapeutic potential of the conditioned medium (CM) derived from MSCs (mesenchymal stem/stromal cells) in disparate medical fields, from immunology to orthopedics, has been widely suggested by in vitro and in vivo evidences. Prior to MSC-CM use in clinical applications, appropriate quality controls are needed in order to assess its reproducibility. Here, we evaluated different CM characteristics, including general features and precise protein and lipid concentrations, in 3 representative samples from adipose-derived MSCs (ASCs). In details, we first investigated the size and distribution of the contained extracellular vesicles (EVs), lipid bilayer-delimited particles whose pivotal role in intercellular communication has been extensively demonstrated. Then, we acquired Raman signatures, providing an overlook of ASC-CM composition in terms of proteins, lipids, and nucleic acids. At last, we analyzed a panel of 200 molecules including chemokines, cytokines, receptors, and inflammatory and growth factors and searched for 32 lipids involved in cell signalling and inflammation. All ASC-CM contained a homogeneous and relevant number of EVs (1.0 × 109 ± 1.1 × 108 particles per million donor ASCs) with a mean size of 190 ± 5.2 nm, suggesting the appropriateness of the method for EV retaining and concentration. Furthermore, also Raman spectra confirmed a high homogeneity among samples, allowing the visualization of specific peaks for nucleic acids, proteins, and lipids. An in depth investigation that focused on 200 proteins involved in relevant biological pathways revealed the presence in all specimens of 104 factors. Of these, 26 analytes presented a high degree of uniformity, suggesting that the samples were particularly homogenous for a quarter of the quantified molecules. At last, lipidomic analysis allowed the quantification of 7 lipids and indicated prostaglandin-E2 and N-stearoylethanolamide as the most homogenous factors. In this study, we assessed that ASC-CM samples obtained with a standardized protocol present stable features spanning from Raman fingerprint to specific marker concentrations. In conclusion, we identified key ingredients that may be involved in ASC-CM therapeutic action and whose consistent levels may represent a promising quality control in the pipeline of its preparation for clinical applications.

Bioactive Lipids in MSCs Biology: State of the Art and Role in Inflammation.

Lipidomics is a lipid-targeted metabolomics approach that aims to the comprehensive analysis of lipids in biological systems in order to highlight the specific functions of lipid species in health and disease. Lipids play pivotal roles as they are major structural components of the cellular membranes and energy storage molecules but also, as most recently shown, they act as functional and regulatory components of intra- and intercellular signaling. Herein, emphasis is given to the recently highlighted roles of specific bioactive lipids species, as polyunsaturated fatty acids (PUFA)-derived mediators (generally known as eicosanoids), endocannabinoids (eCBs), and lysophospholipids (LPLs), and their involvement in the mesenchymal stem cells (MSCs)-related inflammatory scenario. Indeed, MSCs are a heterogenous population of multipotent cells that have attracted much attention for their potential in regulating inflammation, immunomodulatory capabilities, and reparative roles. The lipidomics of the inflammatory disease osteoarthritis (OA) and the influence of MSCs-derived lipids have also been addressed.

Endogenous 1-H-Pyrrole-2,3,5-Tricarboxylic Acid (PTCA) in Hair and Its Forensic Applications: A Pilot Study on a Wide Multi-Ethnic Population.

Over the years, several studies have shown that many factors are likely to affect the results of forensic hair analyses and complicate their interpretation. Among these factors, one of the major drawbacks in hair analysis is the affectability of deposited xenobiotics by cosmetic treatments, which could be eventually used to adulterate the sample. It is well known that some cosmetic treatments containing hydrogen peroxide, such as permanent dyeing or bleaching, lead to the formation of 1-H-pyrrole-2,3,5-tricarboxylic acid (PTCA), a melanin degradation product. Considering that PTCA is also an endogenous compound, spontaneously formed by natural oxidation of melanin, its only detection in hair is not enough to confirm a cosmetic oxidative treatment. For this reason, the aim of the present work was to develop and validate a reliable liquid-liquid extraction method in ultra-high-performance liquid chromatographic-tandem mass spectrometry for the determination of endogenous PTCA in hair from a wide multi-ethnic population (African, Arab, Asian-Pacific, Caucasian, Hispanic and Indian). According to previous studies, untreated hair samples showed a PTCA content of 8.54 ± 5.72 ng/mg (mean ± standard deviation [SD]), ranging between 0.44 and 23.7 ng/mg; after in vitro cosmetic bleaching, PTCA increased to 16.8 ± 6.95 ng/mg (range: 4.16-32.3 ng/mg). Comparing baseline PTCA levels of each subgroup with the others, we could not observe any statistically significant difference, except for Caucasians (P < 0.05), wherein the concentrations were lower. Further studies and a wider sampling are necessary to elucidate the role of PTCA as diagnostic marker of cosmetic hair treatment in forensic field.

PTCA (1-H-Pyrrole-2,3,5-Tricarboxylic Acid) as a Marker for Oxidative Hair Treatment: Distribution, Gender Aspects, Correlation with EtG and Self-Reports.

Hair analysis is an important and reliable resource for the assessment of alcohol or drug abstinence in both clinical and forensic toxicology. Recently, it has been demonstrated that hair oxidative cosmetic treatments lead to the reduction in incorporated xenobiotics in hair, such as ethyl glucuronide (EtG), a marker of alcohol abuse, and the formation of 1-H-pyrrole-2,3,5-tricarboxylic acid (PTCA), a degradation product of melanin. The aim of the present study was to investigate PTCA trends in a large number of samples in order to evaluate the reliability of this biomarker in recognizing previous cosmetic treatment in forensic analyses. Therefore, a single-step extraction followed by an high-performance liquid chromatography--tandem mass spectrometry (HPLC--MS-MS) method was established and validated for the simultaneous determination of EtG and PTCA. This method was applied to 1,219 scalp hair samples from two groups, namely self-reported untreated and in vivo treated hair, exhibiting a concentration range of 6.7 to 440.0 pg/mg for EtG (mean 26.8 pg/mg, median 14.6 pg/mg) and 0.009 to 49.8 ng/mg for PTCA (mean 0.66 ng/mg, median 0.02 ng/mg). The PTCA content was significantly different among the two experimental groups, with the in vivo treated group showing significantly higher levels of PTCA than the untreated group. Finally, an in vitro bleaching was performed and the results confirmed that a strong hair oxidative treatment may negatively affect EtG test results (false negative), whereas the mean PTCA content increased showing statistically significant differences between untreated and in vitro oxidative treated samples. The present study suggests that the determination of PTCA in routine hair analysis procedure could be useful in order to discover previous cosmetic treatment including oxidation.

Quantitative Lipidomic Analysis of Osteosarcoma Cell-Derived Products by UHPLC-MS/MS.

Changes in lipid metabolism are involved in several pathological conditions, such as cancer. Among lipids, eicosanoids are potent inflammatory mediators, synthesized from polyunsaturated fatty acids (PUFAs), which coexist with other lipid-derived ones, including endocannabinoids (ECs) and N-acylethanolamides (NAEs). In this work, a bioanalytical assay for 12 PUFAs/eicosanoids and 20 ECs/NAEs in cell culture medium and human biofluids was validated over a linear range of 0.1-2.5 ng/mL. A fast pretreatment method consisting of protein precipitation with acetonitrile followed by a double step liquid-liquid extraction was developed. The final extracts were injected onto a Kinetex ultra-high-performance liquid chromatography (UHPLC) XB-C18 column with a gradient elution of 0.1% formic acid in water and methanol/acetonitrile (5:1; v/v) mobile phase. Chromatographic separation was followed by detection with a triple-quadrupole mass spectrometer operating both in positive and negative ion-mode. A full validation was carried out in a small amount of cell culture medium and then applied to osteosarcoma cell-derived products. To the best of our knowledge, this is the first lipid profiling of bone tumor cell lines (SaOS-2 and MG-63) and their secretome. Our method was also partially validated in other biological matrices, such as serum and urine, ensuring its broad applicability as a powerful tool for lipidomic translational research.

Urine Endocannabinoids as Novel Non-Invasive Biomarkers for Bladder Cancer at Early Stage.

Due to the involvement of the endocannabinoid system (ECS) in cancer onset and progression and the less studied connection between ECS and bladder cancer, here an evaluation of the ECS modifications associated with bladder cancer is reported. Urine samples were collected from healthy volunteers and patients with bladder cancer at different grades. Endocannabinoids (ECs) and N-acylethanolamides (NAEs) were quantified by HPLC-MS/MS and results normalized for creatinine content. An increase in the urine concentrations of four ECs and NAEs analyzed was observed with a statistically significant increase in the arachidonoylethanolamide (AEA) and stearoylethanoamide (SEA) associated with bladder cancer. Receiver operating characteristic curves built with AEA and SEA data allowed the selection of 160 pg/mL for SEA (area under the curve (AUC) = 0.91, Selectivity (SE) 94%, Specificity (SP) 45%) and 8 pg/mL for AEA (AUC = 0.85, SE 94%, SP 61%) as the best cut-off values. Moreover, data from bladder cancer samples at different grades were derived from The Cancer Genome Atlas, and the expressions of thirteen different components of the "endocannabinoidome" were analyzed. Statistical analysis highlights significant variations in the expression of three enzymes involved in EC and NAE turnover in bladder cancer.

Production of melatonin and other tryptophan derivatives by Oenococcus oeni under winery and laboratory scale.

Malolactic fermentation (MLF) in Valtellina Superiore DOCG red wine was monitored in 4 cellars and the final products were analysed to determine the content of melatonin (MEL) and other tryptophan (TRP) derivatives, including tryptophan ethyl ester (TEE) and MEL isomers (MISs), and to isolate predominant O. oeni strains. MEL and TEE significantly increased in wines after MLF from two cellars out of four. Six strains were isolated during the MLF of red wines and under laboratory scale, in rich and synthetic wine cultural media, together with other four O. oeni strains able to trigger the MLF. Results showed that the presence of stressful growth factors, like ethanol and acid pH, has a pivotal role in triggering the release of TEE by oenococci. Indeed, all the strains became capable to produce also MEL and MISs, together with TEE. under harsh growth conditions, as in a synthetic wine medium. The production of these compounds was strain-dependent and a maximum amount of 0.0078 ±â€¯0.0023 ngT/mL (UMB472) and 619.85 ±â€¯196.16 ngT/mL (UMB436) of MEL and TEE was obtained, respectively. In particular, different MISs were detected under oenological and laboratory scale suggesting that other factors (i.e. technological and/or physico-chemical) could affect the synthesis of TRP derivatives.

11-OH-THC in hair as marker of active cannabis consumption: Estimating a reliable cut-off by evaluation of 672 THC-positive hair samples.

Hair analysis for the assessment of cannabis active use from passive consumption may be failed when performed by the sole detection of compounds present in plant material as well as in cannabis smoke like Δ-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN). For this reason, the determination of 11-nor-9-carboxy-Δ-9-tetrahydrocannabinol (THC-COOH) has been proposed by the Society of Hair Testing (SoHT) in order to prove active cannabis consumption. The identification of THC-COOH in hair will continue to be complicated by its acidic nature and the critical low concentration due to the preferential incorporation of basic compounds into hair shaft. Alternatively, 11-OH-THC may be considered as a complementary marker for THC administration. Our recent study reported an accurate validated procedure for THC, CBD, CBN and 11-OH-THC in hair, based on a GC/MS-MS method in electron ionization mode. However, unlike THC-COOH, a cut-off level for 11-OH-THC in hair has not been fixed yet. For this reason, the aim of this study is to propose a concentration value for 11-OH-THC in hair analysis in order to discriminate between chronic use and external contamination. Receiver operating characteristics (ROC) analysis was applied for cut-off evaluation after 11-OH-THC quantification in a pool of 672 THC-positive hair samples. Results have shown a concentration range between 0.01-5.34 ng/mg for THC (mean 0.34 ng/mg, median 0.12), 0.00-19.2 pg/mg for THC-COOH (mean 0.72 pg/mg, median 0.19 pg/mg) and 0.01-13.33 ng/mg for 11-OH-THC (mean 1.09 ng/mg, median 0.51 ng/mg) for scalp hair and between 0.03-6.32 ng/mg for THC (mean 0.82 ng/mg, median 0.30), 0.00-42.1 pg/mg for THC-COOH (mean 2.70 pg/mg, median 1.08 pg/mg) and 0.00-7.88 ng/mg for 11-OH-THC (mean 1.70 ng/mg, median 0.89 ng/mg) for body hair. Considering these experimental data collected in our laboratory, we propose a cut-off level of 0.5 for scalp and body hair, as indicative of cannabis active consumption. The ROC curve AUCs for 11-OH-THC were 0.873 and 0.884 in 590 scalp hair and 82 body hair samples, respectively. The comparison of the results for THC-COOH (control method) and 11-OH-THC (test method) was also made by means of the Cohen's kappa statistics providing a good agreement according to both Landis & Koch and Fleiss scales. Additionally, we suggest that the detection of both THC-COOH and 11-OH-THC should be mandatory in order to prove active intake and exclude false positive results from external contamination.

Correction: Ottria, R., et al. Quantitative Characterization of Olaparib in Nanodelivery System and Target Cell Compartments by LC-MS/MS. Molecules 2019, 24, 989.

The authors wish to make the following corrections to this paper [...].

Analysis of hydroxy-cocaine metabolites as evidence of cocaine consumption: Identification by parent ion search and quantitation by UHPLC-MS/MS in hair.

Over the last decade, hair analysis has become a routine procedure in most forensic laboratories and, complementary to blood and urine, hair is a unique biological matrix which gives the opportunity to establish a temporal consumption profile. Despite hair is widely used to identify drug use, environmental contamination continues to represent a challenging factor of this procedure, especially for cocaine (COC). In the last few years several strategies have been proposed in order to distinguish between actual use and external contamination, however the commonly detected COC metabolites probably are insufficient for demonstrating cocaine use through hair testing. Thus, the aim of this study is to develop an ultra high performance liquid cromatography - tandem mass spectrometry (UHPLC-MS/MS) method able to detect and quantify hydroxy-COC metabolites, as specific markers of COC abuse, in hair samples from COC consumers, thus enabling unambiguous evidence of COC consumption. At the beginning, since no commercial reference materials were available, COC-positive hair samples were tested using parent ion scan-based analysis to extract hydroxy COC metabolites target ions. Once identified, the reference materials were synthesized by our analytical laboratory allowing the development of the first UHPLC-MS/MS validated method to quantify p- and m-isomers of hydroxy COC, as well as hydroxy benzoylecgonine (BE) and hydroxy norcocaine (NCOC). The method was successfully applied to a large number of COC-positive hair samples and introduced into a routine procedure for testing drug ingestion in order to evaluate for the first-time hydroxy metabolites of COC ranges in hair and their correlation with COC and BE.

Assessment of Tryptophan, Tryptophan Ethylester, and Melatonin Derivatives in Red Wine by SPE-HPLC-FL and SPE-HPLC-MS Methods.

Melatonin (MEL) is an indoleamine produced mainly by the pineal gland in vertebrates. It plays a significant role in the regulation of circadian rhythms, mitigation of sleeping disorders, and jet lag. This compound is synthetized from tryptophan (TRP) and it has been found in seeds, fruits, and fermented beverages, including wine. Wine is also a source of other tryptophan derivatives, the tryptophan ethylester (TEE) and MEL isomers (MISs), for which the biological properties need to be elucidated. An analytical method for the simultaneous quantification of TRP, TEE, and MEL was developed by a Solid Phase Extraction (SPE) of a preconcentration of wine followed by high performance liquid chromatography (HPLC) analysis either with fluorescence or mass spectrometer detectors. The analytical method showed a relative standard deviation (RSD) lower than 8%, except for TRP (RSD 10.5% in wine). The recovery was higher than 76%. The versatility of SPE preconcentrations allowed for the adequate preconcentration of wine sample as well as detection of low concentrations, an important aspect especially for MEL (detection limit 0.0023 µg/L). The proposed method proved to be suitable for assessing the investigated compounds in some red wine samples, where 74.4⁻256.2 µg/L and 0.038⁻0.063 µg/L of TEE and MEL were detected, respectively. Five MISs were also found in wine samples in concentrations up to 1.97 µg/L.

Lactonization Method To Assign the Anomeric Configuration of the 3,4-Unsaturated Congeners of N-Acetylneuraminic Acid.

Assigning the correct configuration at C2 in sialosides is a standing problem because of the absence of an anomeric hydrogen. All different empirical rules that have been proposed over the years lack general applicability. In particular, the correct configuration of several 3,4-unsaturated derivatives of N-acetylneuraminic acid (Neu5Ac), which have been recently shown to be novel sialidase/neuraminidase inhibitors, could only be tentatively assigned by similarity with the reported 3,4-unsaturated 2O-methyl sialosides. In this work, we overcome this problem as we devised a rapid synthetic method to unequivocally resolve the anomeric configuration of the 3,4-unsaturated Neu5Ac derivatives through the synthesis of the corresponding unreported unsaturated 1,7-lactones. Moreover, we discovered a diagnostic 13C nuclear magnetic resonance signal that allows the formulation of a new empirical rule for the direct assignment of the C2 stereochemistry of these molecules, even when only one of the two C2 epimers is available.

Quantitative Characterization of Olaparib in Nanodelivery System and Target Cell Compartments by LC-MS/MS.

Olaparib, an orally active inhibitor of poly(ADP-ribose)polymerase(PARP), is the drug of choice in the treatment of gBRCA1/2+ metastatic breast cancers. Unfortunately, Olaparib is poorly soluble with low bioavailability and tumor accumulation; nano-delivery could be a good choice to overcome these disadvantages. Here, a rapid and robust HPLC-ESI⁻MS/MS method for the quantification of Olaparib in ferritin nano-carriers led to the development of cells compartments, different tissues, plasma and urines and were validated to assess the effects of nano-delivery on cell compartment distribution of the drug. This method allows the quantification of Olaparib within the linear range of 0.1⁻10ng/mL in cells culture medium and cell cytoplasm, of 0.5⁻10ng/mL in nuclei, of 0.5⁻100ng/mL in plasma and urine and of 10⁻500ng/mL in tissue samples (kidney and liver). The limit of quantification was found to be 1.54 ng/mL for liver, 2.87 ng/mL for kidney, and lower than 0.48 ng/mL for all matrices. The method has been applied to quantify Ola encapsulated in ferritin-nano-carriers during the nano-drug development. The application of the method to human BRCA-mutated cell model to quantify the Olaparib distribution after incubation of free or ferritin-encapsulated Olaparib is also reported. This sensitive method allows the quantification of low concentrations of Olaparib released from nano-carriers in different cell compartments, leading to the determination of the drug release and kinetic profile of an essential parameter to validate nano-carriers.