Using sno-lncRNAs as potential markers for Prader-Willi syndrome diagnosis.

The genetic disorder Prader-Willi syndrome (PWS) is mainly caused by the loss of multiple paternally expressed genes in chromosome 15q11-q13 (the PWS region). Early diagnosis of PWS is essential for timely treatment, leading to effectively easing some clinical symptoms. Molecular approaches for PWS diagnosis at the DNA level are available, but the diagnosis of PWS at the RNA level has been limited. Here, we show that a cluster of paternally transcribed snoRNA-ended long noncoding RNAs (sno-lncRNAs, sno-lncRNA1-5) derived from the SNORD116 locus in the PWS region can serve as diagnostic markers. In particular, quantification analysis has revealed that 6,000 copies of sno-lncRNA3 are present in 1 µL whole blood samples from non-PWS individuals. sno-lncRNA3 is absent in all examined whole blood samples of 8 PWS individuals compared to 42 non-PWS individuals and dried blood samples of 35 PWS individuals compared to 24 non-PWS individuals. Further developing a new CRISPR-MhdCas13c system for RNA detection with a sensitivity of 10 molecules per µL has ensured sno-lncRNA3 detection in non-PWS, but not PWS individuals. Together, we suggest that the absence of sno-lncRNA3 represents a potential marker for PWS diagnosis that can be detected by both RT-qPCR and CRISPR-MhdCas13c systems with only microlitre amount of blood samples. Such an RNA-based sensitive and convenient approach may facilitate the early detection of PWS.

Monitoring of sirolimus in the whole blood samples from pediatric patients with lymphatic anomalies.

In recent years, off-label use of sirolimus (SIR) has been gaining attention in the clinical practice. However, since it is critical to achieve and maintain therapeutic blood levels of SIR during treatment, the regular monitoring of this drug in individual patients must be implemented, especially in off-label indications of this drug. In this article, a fast, simple, and reliable analytical method for determining SIR levels in whole blood samples is proposed. Sample preparation based on dispersive liquid-liquid microextraction (DLLME) followed by liquid chromatography-mass spectrometry (LC-MS/MS) was fully optimized toward the analysis of SIR and proposed as a fast, simple, and reliable analytical method for determining the pharmacokinetic profile of SIR in whole-blood samples. In addition, the practical applicability of the proposed DLLME-LC-MS/MS method was evaluated by analyzing the pharmacokinetic profile of SIR in whole blood samples obtained from two pediatric patients suffering from lymphatic anomalies, receiving this drug as off-label clinical indication. The proposed methodology can be successfully applied in routine clinical practice for the fast and precise assessment of SIR levels in biological samples, thus allowing SIR dosages to be adjusted in real time during pharmacotherapy. Moreover, the measured SIR levels in the patients indicate the need for monitoring between doses to ensure the optimal pharmacotherapy of patients.

Rapid quantification of phenobarbital and barbital in human whole blood by liquid-liquid extraction combined with DART-orbitrap-HRMS.

PURPOSE: This study aims to develop and validate a rapid, simple, and efficient bioanalytical method for the simultaneous quantification of phenobarbital and barbital in human whole blood using liquid-liquid extraction combined with direct analysis in real time (DART) and high-resolution mass spectrometry (HRMS). METHOD: Phenobarbital-d5 and aprobarbital were selected as internal standards (ISs) of phenobarbital and barbital, respectively. A mixed solvent of o-xylene and ethyl acetate at a ratio of 1:6 was used to extract analytes of interest and ISs from 100 µL of human whole blood samples. Phenobarbital and barbital were detected by DART-HRMS. The proposed method has been validated in accordance with United States Food and Drug Administration Guidelines for Bioanalytical Method Validation in terms of selectivity, linearity, accuracy, precision, matrix effect, recovery, stability, and dilution integrity. RESULTS: The lower limits of quantification (LLOQs) of phenobarbital and barbital were both 10 ng/mL. The linearities were in the range of 10-1000 ng/mL (R2 ≥ 0.99). The mean recovery values of phenobarbital and barbital were 99.7% and 88.1%, respectively. The interday and intraday precision values were less than 10.4%, and the interday and intraday accuracy values ranged from 87.6 to 106.7%. Furthermore, the validated method was applied to four cases of phenobarbital poisoning at the Shanghai Institute of Forensic Science. CONCLUSION: The developed and fully validated method enabled the simultaneous quantification of phenobarbital and barbital in human whole blood and was successfully applied to authentic cases.

Circulating LncRNAs landscape as potential biomarkers in breast cancer.

BACKGROUND: In Iran, the delay in diagnosis and treatment of breast cancer results in low survival rates. AIM: It is essential to characterize new therapeutic targets and prognostic breast cancer biomarkers. The rising evidence suggested that long non-coding RNAs (lncRNAs) expression levels are deregulated in human cancers and can use as biomarkers for the rapid diagnosis of breast cancer. METHODS: In the present study, a quantitative real-time polymerase chain reaction (qRT-PCR) technique was used to measure 20 oncogenic and tumor suppressor lncRNAs expression levels in whole blood samples of female breast cancer patients and healthy women. Receiver operating characteristic curve (ROC) was used to assess the diagnostic value of each selected lncRNA as a biomarker. RESULTS: The results revealed that some circulating lncRNAs (MEG3, NBAT1, NKILA, GAS5, EPB41L4A-AS2, Z38, and BC040587) were significantly down-regulated in breast cancer patients compared to healthy women. In contrast, other circulating lncRNAs (H19, SPRY4-IT1, XIST, UCA1, AC026904.1, CCAT1, CCAT2, ITGB2-AS, and AK058003) were significantly up-regulated in breast cancer patients compared to controls. It was shown that the expression levels of NKILA, and NBAT1 lncRNAs were related to tumor size, and BC040587 expression level related to age, node metastasis, tumor size, and grade (p < .05). The association between H19 and SPRY4-IT1 lncRNAs with HER-2 was confirmed statistically (p < .05). ROC curves illustrated that the blood levels of SPRY4-IT1, XIST, and H19 lncRNAs have excellent potential in discriminating breast cancer from the healthy controls, showing an AUC of 1.0 (95% CI 1.0-1.0, p = .00), 0.898 (95% CI 0.815-0.981, p = .00), and 0.848 (95% CI 0.701-0.995, p = .01), respectively. CONCLUSION: In conclusion, the expression levels of circulating H19 and SPRY4-IT1 lncRNAs in breast cancer patients could consider as the prognostic biomarkers and therapeutic targets in breast cancer, because of their excellent power in discriminating breast cancer from healthy individuals and the significant correlation of H19, and SPRY4-IT1 lncRNAs with clinicopathological traits. We also suggest the possible application of BC040587 lncRNA as a diagnostic and prognostic indicator to assess tumor progression in case of verification in larger patients' cohorts.

Selective detection of manganese(II) ions based on the fluorescence turn-on response via histidine functionalized carbon quantum dots.

Herein, water-soluble emissive carbon quantum dots (His-CQDs) were synthesized from pyrolysis of sodium citrate in the presence of histidine under hydrothermal conditions. The as-synthesized His-CQDs were characterized using Fourier transform infrared (FT-IR), fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM) techniques. The obtained His-CQDs display a strong emission peak at 534 nm when excited at 476 nm with a high quantum yield (61.8 %). The as-synthesized His-CQDs were applied as a new platform for highly selective determination of Mn(II) based on the fluorescence "turn-on" response with a limit of detection of 1.85 µg L-1 (at 3σ) and a linear range of 3.50-35.5 µg L-1 in aqueous solution. The sensing mechanism of the His-CQDs probe for the detection of Mn(II) was studied via density functional theory (DFT), FT-IR, and EDTA complexation methodology. In addition, His-CQDs were successfully applied to determine the accurate amounts of Mn(II) in whole blood control material. More importantly, the integrating such an efficient sensor with point-of-care technology can enable portable, easy-to-use, and rapid sensing systems for better biological and clinical applications.

A Retrospective Metabolomics Analysis of Gamma-Hydroxybutyrate in Humans: New Potential Markers and Changes in Metabolism Related to GHB Consumption.

GHB is an endogenous short-chain organic acid presumably also widely applied as a rape and knock out drug in cases of drug-facilitated crimes or sexual assaults (DFSA). Due to the endogenous nature of GHB and its fast metabolism in vivo, the detection window of exogenous GHB is however narrow, making it challenging to prove use of GHB in DFSA cases. Alternative markers of GHB intake have recently appeared though none has hitherto been validated for forensic use. UHPLC-HRMS based screening of blood samples for drugs of abuse is routinely performed in several forensic laboratories which leaves an enormous amount of unexploited data. Recently we devised a novel metabolomics approach to use archived data from such routine screenings for elucidating both direct metabolites from exogenous compounds, but potentially also regulation of endogenous metabolism and metabolites. In this paper we used UHPLC-HRMS data acquired over a 6-year period from whole blood analysis of 51 drivers driving under the influence of GHB as well as a matched control group. The data were analyzed using a metabolomics approach applying a range of advanced analytical methods such as OPLS-DA, LASSO, random forest, and Pearson correlation to examine the data in depth and demonstrate the feasibility and potential power of the approach. This was done by initially detecting a range of potential biomarkers of GHB consumption, some that previously have been found in controlled GHB studies, as well as several new potential markers not hitherto known. Furthermore, we investigate the impact of GHB intake on human metabolism. In aggregate, we demonstrate the feasibility to extract meaningful information from archived data here exemplified using GHB cases. Hereby we hope to pave the way for more general use of the principle to elucidate human metabolites of e.g. new legal or illegal drugs as well as for applications in more global and large scale metabolomics studies in the future.

Homogeneous two-dimensional visual and fluorescence analysis of circulating tumor cells in clinical samples via steric hindrance regulated enzymes recognition cleavage and elongation.

Oncology detection technology is significant for the early detection of tumors. The current study reports a new method that uses folate receptor (FR) as circulating tumor cells (CTCs) marker and only folate modified T30 as a probe. This method also uses dual-enzyme assisted amplification strategy for homogeneous fluorescence as well as two-dimensional visual (color and distance) detection of SMMC-7721 liver cancer cells from clinical blood samples. This work was based on the steric hindrance caused by binding between FR and folate to regulate cleavage of folate-T30 by exonuclease I (Exo I) and to inhibit subsequent polymerization and extension reaction of the cleavage product by terminal deoxynucleotidyl transferase (TdT). It explores the use of CdTe QDs to selectively identify Cu2+ and polyT-template Cu NPs as a bridge combined with inkjet printing technology to make test strips that can be read through distance changes. Under fluorometer mode, limit of detection as low as 1 cells/mL was achieved. The color and distance reading modes can identify cells with concentrations as low as 5 and 1 cells/mL, respectively. This CTCs detection approach of fluorescence mode was further validated by using 50 clinical samples of liver cancer patients (19 negative and 31 positive). The results were in good agreement with FR-polymerase chain reaction (FR-PCR) kits, radiologic and pathological techniques. In addition, the quantitative results of distance reading test strips of CTCs in 22 clinical samples (8 negative and 14 positive) were also in 100% agreement with the findings of clinical kits, computed tomography (CT) and pathological tests.

Homogeneous Visual and Fluorescence Detection of Circulating Tumor Cells in Clinical Samples via Selective Recognition Reaction and Enzyme-Free Amplification.

Here we report a simple all-nucleic-acid enzyme-free catalyzed hairpin assembly assisted amplification strategy with quantum dots (QDs) as the nanoscale signal reporter for homogeneous visual and fluorescent detection of A549 lung cancer cells from clinical blood samples. This work was based on the phenomenon that CdTe QDs can selectively recognize Ag+ and C-Ag+-C and by using mucin 1 as the circulating tumor cells (CTCs) marker and aptamer as the recognition probe. Under optimized conditions, the limits of detections as low as 0.15 fg/mL of mucin 1 and 3 cells/mL of A549 cells were achieved with fluorescence signals. A 1 fg/mL concentration of mucin 1 and 100 cells/mL of A549 can be distinguished by the naked eye. This method was used to quantitatively analyze CTCs in 51 clinical whole blood samples of patients with lung cancer. The levels of CTCs detected in clinical samples by this method were consistent with those obtained using the folate receptor-polymerase chain reaction clinical test kit and correlated with radiologic and pathological findings.

Tumor-infiltrating lymphocytes in tissue material combined with systemic lymphocyte inflammation in patients with colorectal cancer.

The efficacy of cancer immunotherapy has been actively explored in the treatment of various malignant neoplasms of the gastrointestinal tract. In light of recent reports, the present study aimed to investigate the combination of the absolute lymphocyte count (ALC), percentage of tumor-infiltrating lymphocyte (TILs) and tumor progression status in patients with colorectal cancer (CRC) who underwent surgery. The medical records of 160 patients diagnosed with CRC were retrospectively reviewed. TILs were determined as a percentage of mononuclear inflammatory cells in the total intratumoral or stromal area as determined in five high power fields (magnification, x200-400), at the invasive front and in the centre of the tumour. Blood samples were obtained within 3 days prior to and 7 days following the surgical treatment. The assessment of the TIL percentage was performed in the tissue at the invasive front and in the centre of the primary tumour mass in combination with the determination of ALC in whole blood samples. The samples were obtained prior to and after surgery from patients with CRC, and the tumour progression status was also assessed (TILs/ALC/tumour progression status). A significant association was observed between the percentage of TILs in the main mass of tumour and the tumour size (P=0.031), the pT stage (P=0.049) and the incidence of necrosis (P=0.037) following surgery. The histological type was associated with the evaluated combined parameters prior to surgery (P=0.046). Lymph node pouch invasion was associated with TILs at the invasive front of tumour and with ALC prior to and after surgery (P=0.006 and P=0.037). Furthermore, the data indicated that the percentage of TILs located on the invasive front and centre of the tumour, and the ALC prior to and following surgery correlated with the treatment status (P=0.032, P=0.018, P≤0.001 and P≤0.001). A significant association was noted between eight features and evaluated combined parameters following surgery. These included the tumour size (P=0.021), TNM stage (P<0.001), tumour deposits (P=0.001), incidence of necrosis (P=0.042) and lymph node metastasis (P<0.001). Furthermore, the degree of invasion of venous (P<0.001), lymphatic (P<0.001) and perineural (P<0.001) sites was also significantly associated with TILs, ALC obtained after surgical treatment and tumor progression status. The data demonstrated that local and systemic chronic inflammation was associated with tumour progression in patients with CRC.

Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples.

Phosphorylation of H2AX histone (γH2AX) represents an early event in the DNA damage response against double-strand breaks (DSB); hence, its measurement provides a surrogate biomarker of DSB. Recently, we reported initial steps in the standardization of γH2AX assay in peripheral blood leukocytes (PBL), addressing the possibility of using cryopreserved samples, and the need of phytohaemagglutinin (PHA) stimulation prior analysis (Toxicol Sci 2015, 144:406-13). Validating the use of whole blood samples as cell specimen for this assay would be particularly useful for human population studies. Hence, in the current study we determined for the first time the feasibility of whole blood samples, both fresh and frozen, to be used in the γH2AX assay, evaluated by flow cytometry, and the convenience of PHA stimulation. Freshly collected and cryopreserved whole blood samples were treated with bleomycin (BLM), actinomycin-D (Act-D) and mitomycin C (MMC); half of the samples were previously incubated with PHA. Results were compared with those from PBL. Negative responses in MMC treatments were probably due to the quiescence of unstimulated cells, or to the short treatment time in PHA stimulated cells. Fresh whole blood samples exhibited a more intense response to BLM and Act-D treatments in stimulated cells, probably due to DSB indirectly produced from other less relevant types of DNA damage. Results obtained in frozen whole blood samples indicate that PHA stimulation is not advisable. In conclusion, this study demonstrates that whole blood samples can be used to assess DSB-related genotoxicity by the flow cytometry γH2AX assay.

A rapid high-throughput sequencing-based approach for the identification of unknown bacterial pathogens in whole blood.

High-throughput DNA sequencing (HTS) of pathogens in whole blood samples is hampered by the high host/pathogen nucleic acids ratio. We describe a novel and rapid bacterial enrichment procedure whose implementation is exemplified in simulated bacteremic human blood samples. The procedure involves depletion of the host DNA, rapid HTS and bioinformatic analyses. Following this procedure, Y. pestis, F. tularensis and B. anthracis spiked-in samples displayed an improved host/pathogen DNA ratio of 2.5-5.9 orders of magnitude, in samples with bacteria spiked-in at 103-105 CFU/ml. The procedure described in this study enables rapid and detailed metagenomic profiling of pathogens within 8-9 h, circumventing the challenges imposed by the high background present in the bacteremic blood and by the unknown nature of the sample.

Fast screening method for molecular recognition of islet amyloid polypeptide from whole blood samples collected from diabetic patients with disposable stochastic sensors obtained by nanolayer, and nanolayer by nanolayer deposition using cold plasma.

Nanolayer and nanolayer by nanolayer deposition of nanofilms of Ag and C using cold plasma in sequences (Ag, Ag-C, Ag-Ag-C), on porous paper, were used to design three disposable stochastic sensors for the assay of amyloid polypeptide from whole blood. The nanofilms were modified with α-cyclodextrin. The test developed using the nanofilm-based disposable stochastic sensors is used for early detection of diabetes. The wider linear concentration range (1.00 × 10-6-1.00 ng mL-1) and the lower limit of quantification (1.00 × 10-6ng mL-1) were obtained using the disposable stochastic sensors based on Ag-C and Ag-Ag-C, while the highest sensitivity (3.19 × 104 s-1/µg mL-1) was recorded using the disposable stochastic sensor based on Ag-Ag-C. The screening methods were fully validated using whole blood samples from confirmed patients, when the recovery of the islet amyloid polypeptide was higher than 98.00%. Graphical abstract.

Effects of culturing technique on human peripheral blood lymphocytes response to proton and X-ray radiation.

Purpose: The main aim of this study was to comparatively investigate the effects of culturing methods on the response of human peripheral blood lymphocytes to irradiation exposure.Materials and methods: Whole blood and isolated lymphocytes were ex vivo exposed to two radiation sources (60 MeV proton or 250 kV X-ray radiation) with different doses (0.3, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 Gy), and genotoxic markers were subsequently assayed. The observed effects were compared as dose-response relationships using two end points (CBMN and PCC tests) and different biomarkers (NDI, PCC index, MNi frequency and excess PCC fragments).Results and conclusions: The results showed different effects of the culturing techniques on the response of human peripheral blood lymphocytes to radiation. The MNi frequency and excess PCC fragments were significantly higher when lymphocytes were cultured after being isolated. After irradiation, no differences were seen in the NDI between the lymphocytes of the two culturing techniques; however, there were differences in the PPC index. When planning or performing cytogenetic studies, the possibility of such effects and their potential to impact the variability of the results of human biomonitoring studies should be considered important and taken into account.

Enantioanalysis of tryptophan in whole blood samples using stochastic sensors-A screening test for gastric cancer.

Tryptophan is a key amino acid related to metabolomics in gastric cancer. To date, methods were developed only for the assay of l-tryptophan, the role of d-tryptophan being not yet established. Therefore, four stochastic sensors based on different graphene materials modified with ß-cyclodextrins, 2,2-diphenyl-1-picrylhydrazyl, and protoporphyrin IX were designed and used for enantioanalysis of tryptophan in whole blood samples. High sensitivities, and reliabilities were recorded when the stochastic sensors were used for the enantioanalysis of tryptophan in whole blood samples. The paper opened a new chapter in early detection of gastric cancer, based on establishing the role of d-tryptophan in metabolomics, and in early diagnosis of gastric cancer.

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS.

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL-1 and 3.2 to 160 ng mL-1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and - 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract.

Associations of self-reported smoking, cotinine levels and epigenetic smoking indicators with oxidative stress among older adults: a population-based study.

Tobacco smoking and oxidative stress (OS) are both related to a wide spectrum of adverse age-related health outcomes, but their association is not yet well-established. We examined the associations of self-reported smoking indicators, serum cotinine levels and smoking-related DNA methylation biomarkers with two urinary proxy markers of OS, 8-isoprostane (8-iso) and 8-hydroxy-2'-deoxyguanosine (8-oxodG), in two independent subsets of older adults recruited in Germany (discovery set: n = 978, validation set: n = 531). We obtained DNA methylation profiles in whole blood samples by Illumina Human Methylation450K Beadchip and measured the urinary levels of both OS markers using commercial ELISA kits. After controlling for potential confounders, current smoking, cumulative smoking exposure (pack-years) and serum cotinine levels (ng/ml) were strongly associated with 8-iso levels (p values <0.0001, 0.004 and 0.001, respectively). Of 151 previously identified smoking-related CpG sites, 71 loci were associated with 8-iso levels after correction for multiple testing (FDR < 0.05) in the validation phase and were designated as loci related to 8-iso levels defined OS. In addition, serum cotinine levels, cumulative smoking exposure and a smoking index (SI) based on the 71 identified loci manifested monotonic associations with 8-iso levels. However, we did not observe any associations between these smoking indicators and 8-oxodG levels. In conclusion, this study suggests that smoking-related epigenetic alterations are closely correlated with smoking-induced OS. The identified CpG sites could potentially be prognostic epigenetic markers of OS and OS-related health outcomes. Our findings and the underlying mechanisms should be followed up in further, preferably longitudinal studies.

Parallel artificial liquid membrane extraction of new psychoactive substances in plasma and whole blood.

Parallel artificial liquid membrane extraction (PALME) was combined with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the potential for screening of new psychoactive substances (NPS) was investigated for the first time. PALME was performed in 96-well format comprising a donor plate, a supported liquid membrane (SLM), and an acceptor plate. Uncharged NPS were extracted from plasma or whole blood, across an organic SLM, and into an aqueous acceptor solution, facilitated by a pH gradient. MDAI (5,6-methylenedioxy-2-aminoindane), methylone, PFA (para-fluoroamphetamine), mCPP (meta-chlorophenylpiperazine), pentedrone, methoxetamine, MDPV (methylenedioxypyrovalerone), ethylphenidate, 2C-E (2,5-dimethoxy-4-ethylphenethylamine), bromo-dragonfly, and AH-7921 (3,4-dichloro-N-{[1-(dimethylamino)cyclohexyl]methyl}benzamide) were selected as representative NPS. Optimization of operational parameters was necessary as the NPS were novel to PALME, and because PALME was performed from whole blood for the very first time. In the PALME method developed for plasma, NPS were extracted from a 250µL alkalized donor solution consisting of 125µL plasma sample, 115µL 40mM NaOH, and 10µL internal standard. In the PALME method from whole blood, the 250µL alkalized donor solution consisted of 100µL whole blood, 50µL deionized water, 75µL 80mM NaOH, and 25µL internal standard. In both methods, extraction was accomplished across an SLM of 5µL dodecyl acetate with 1% trioctylamine (w/w), and further into an acidic acceptor solution of 50µL 20mM formic acid. The extraction was promoted by agitation at 900rpm and was carried out for 120min. Method validation was performed and the following parameters were considered: linearity, limits of quantification (LOQ), intra- and inter-day precision, accuracy, extraction recoveries, carry-over, and matrix effects. The validation results were in accordance with FDA guidelines.

Rapid determination of nine barbiturates in human whole blood by liquid chromatography-tandem mass spectrometry.

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the qualitative and quantitative analysis of nine barbiturates (barbital, phenobarbital, pentobarbital, amobarbital, secobarbital, thiopental, butalbital, butabarbital, and hexobarbital) in human whole blood. Barbiturates were extracted from 100 µL of human whole blood samples using a simple liquid-liquid extraction (LLE) procedure, and detected by LC-MS/MS. An UPLC C18 (2.1 mm × 100 mm, 1.7 µm) column was used at 40 °C for the separation and acetonitrile/water system was used as the mobile phase with gradient elution. This method showed excellent accuracy (86-111%) and precision (relative standard deviation <15%). The limits of detection (LODs) were 0.2 ng/mL for barbital and secobarbital and 0.5 ng/mL for the other barbiturates. The linearity ranged from 2 ng/mL to 2000 ng/mL, with r2 > 0.99 over the range. This method achieved the separation and detection of pentobarbital and amobarbital at the same time in a convenient way. Moreover, it was both simple and sensitive for the determination of nine most commonly used barbiturate drugs, which was meaningful in the field of clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of endogenous GHB concentrations in blood and hair in death cases with emphasis on the post mortem interval.

Gamma-hydroxybutyric acid (GHB) is an endogenous compound which has a story of clinical use and illicit abuse since the 1960's. The possibility to use a multi-sample approach for GHB evaluation, including whole blood and hair, to better characterize a forensic toxicology case and evaluate a possible causal association with the death is an exciting up-to-date issue. In addition, its post-mortem behaviour, namely regarding degradation and metabolism, has been increasingly investigated as a putative biomarker for post-mortem interval (PMI) estimation. Thus, in order to contribute to clarification of this specific aspect, whole blood and hair post-mortem GHB levels were evaluated in 32 real cases with previous information on death and autopsy data. The results obtained suggest that the PMI (until 5 days between death and sampling) influences GHB whole blood concentration, but not GHB levels in hair samples. No differences were encountered for the other parameters evaluated, including age, gender, cause of death and presence or absence of substances. This study brings new insights regarding the usefulness of GHB levels in forensic toxicology, which might be further strengthened with larger, but comparable, studies from other laboratories and institutions in the context of legal medicine.

A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia.