Real-world applications of the new grading system in lung adenocarcinoma: A study of 907 patients focusing on revealing the relationship between pathologic grade and genetic status.

BACKGROUND: The status of the lung adenocarcinoma (LUAD) grading system and the association between LUAD differentiation, driver genes, and clinicopathological features remain to be elucidated. METHODS: We included patients with invasive non-mucinous LUAD, evaluated their differentiation, and collected available clinicopathological information, gene mutations, and analyzed clinical outcomes. RESULTS: Among the 907 patients with invasive non-mucinous LUAD, 321 (35.4 %) were poorly differentiated, 422 (46.5 %) were moderately differentiated, and 164 (18.1 %) were well differentiated. EGFR mutation was more common in the LUADs accompanied without CGP (complex glandular pattern) than LUADs with CGP (p < 0.001). Correlation analysis between mutations and clinical characteristics showed that EGFR gene mutation (p < 0.001), KRAS gene mutation (p < 0.05), and ALK gene rearrangement (p < 0.001) were significantly related to the degree of tumor differentiation, and the KRAS and ALK gene mutation frequencies were higher in the low-differentiation group than in the high and medium differentiation groups. The EGFR mutation frequency was higher in the well/moderately differentiated adenocarcinoma group. CONCLUSIONS: Our study adds to the evidence regarding the role of the grading system in prognosis. EGFR, KRAS, and ALK are related to the degree of tumor differentiation.

The observation records from whale and dolphin watching inshore of Hualien, eastern Taiwan.

Background: The eastern waters of Taiwan have been lacking baseline and research data for several years. This study was initiated by Kuroshio Ocean Education Foundation (KOEF) in collaboration with the Turumoan whale-watching company since 1998, collecting long-term ecological data for cetaceans in the inshore of Hualien, eastern Taiwan. This dataset includes 10,675 records of cetacean sightings from June 1998 to December 2021. Collection of cetacean sighting records was paused for one year in 2001 due to budgetary reasons. All of the sighting records were collected by whale-watching boat guides that were trained by KOEF. Following a standardised protocol, guides used a handheld GPS device and cetacean sighting record sheets to document information about the cetacean species identification, location, time, number of individuals, the presence of mother-calf pairs and mixed-species groups and other states of each sighting during a whale-watching tour. The collection of citizen-science data during this period has significantly advanced Taiwan's cetacean baseline data in the study area. Additionally, we make data available to the public in the form of citizen-science, making a substantial contribution to the advancement of ocean scientific research. We have published the dataset on Global Biodiversity Information Facility, allowing users around the world to download the dataset. New information: This is currently the largest dataset of cetacean sighting records in Taiwan (last updated on 2023-09-05). We have also recorded several species on the International Union for Conservation of Nature (IUCN) Red List categorised as "Data Deficient" during our survey inshore of Hualien, eastern Taiwan, including Ginkgo-toothed beaked whales (Mesoplodonginkgodens Nishiwaki & Kamiya, 1958), Omura's whales (Balaenopteraomurai Wada, Oishi & Yamada, 2003) and killer whales (Orcinusorca (Linnaeus, 1758)). There are also sperm whales (Physetermacrocephalus Linnaeus, 1758), categorised as "Vulnerable" in the IUCN Red List and false killer whales (Pseudorcacrassidens (Owen, 1846)), categorised as "Near Threatened". This study is also the first and only long-term study that has documented cetaceans in the study area.

Bronchial salivary gland-type intraductal carcinoma with KIAA1217::RET gene fusion composed of intercalated and oncocytic components.

Salivary gland-type intraductal carcinoma (IC) is a rare malignant salivary gland neoplasm. Primary salivary gland-type IC has never been described in the lung. Herein, we present a primary pulmonary IC in a 63-year-old woman. The tumor originated in the bronchus wall of the right middle lobe. The tumor consisted of two histological types, intercalated component and oncocytic component. The intercalated component showed tubular/cystic pattern composed of column to cube-shaped cells and scattered mucous cells. The oncocytic component showed solid nests composed of large cells with abundant eosinophilic granular cytoplasm. Immunohistochemically, both histological components were positive for cytokeratin 7 (CK7), S-100 protein, SOX10, and mammaglobin. The rimming myoepithelial cells were highlighted by p63 and smooth muscle actin (SMA). The tumor cells were negative for androgen receptor (AR), HER-2, Dog-1, TTF-1, napsin A, GCDFP-15, and GATA3. In the present case, we detected KIAA1217::RET fusion via DNA-based next-generation sequencing (NGS) and RT-PCR, which established the diagnosis of IC at a molecular level. The present case expands the categories of bronchopulmonary salivary gland-type tumors.

Yolk Sac Tumor Originating From Cervical Adenocarcinoma: A Case Predominated by Enteroblastic Differentiation.

The fetal gut-like phenotype can be found in yolk sac tumors and adenocarcinomas with enteroblastic differentiation (AEBDs). We report a cervical yolk sac tumor in a 44-yr-old woman. The tumor has similar morphology, immunophenotype, and molecular features to the AEBD of the digestive system. The tumor showed a glandular-predominant growth pattern, composed of columnar cells with clear glycogen-rich cytoplasm. The microcystic/reticular architecture or Schiller-Duval bodies were not found in the tumor. Immunohistochemically, the tumor cells were positive for p16, glypican-3 (GPC3), spalt-like transcription factor 4 (SALL4), CDX-2, and p53. TP53 mutation was identified by next-generation sequencing, and human papillomavirus (HPV) 35 was detected by HPV DNA polymerase chain reaction. In the present case, the adenocarcinoma cells in the superficial cervical glandular epithelium and the nonclear glandular components proved the existence of somatic components. The positivity of p16 and HPV also supports that the present case originates from an HPV-associated adenocarcinoma. The yolk sac tumor should be thought of as "germ cell differentiation" from a somatic carcinoma. This kind of yolk sac tumor arising from somatic-type adenocarcinoma in the female genital tract may be the counterpart of AEBD in the digestive tracts and adenocarcinomas with fetal gut-like morphology in other organs. The tumor might be more aggressive than conventional adenocarcinoma, pathologists should highlight the existence of the enteroblastic component in the pathologic report.

Bronchiolar adenoma-like tumour with monolayered component: Represent malignant transformation of bronchiolar adenoma? A series of five cases.

Pulmonary bronchiolar adenoma (BA) is a rare lung tumour, it is unclear whether BA can develop into a malignancy. We presented five cases of BA-like tumour with monolayered components. This type of tumour may represent the malignant transformation of BA. Histologically, these tumours showed acinar and lepidic growth patterns. The acinar components were well-differentiated. The glandular tumour cells in these tumours contained cuboidal to columnar cells resembling type II pneumocytes or club (Clara) cells. A small number of mucinous cells were found in two cases. A few scattered ciliated cells were detected in three cases. The ciliated cells only existed in the bilayered components. The basal cells were highlighted by CK5/6 and p40 in a partial region of the tumour rather than in the entire tumour. The glandular tumour cells, including those in the bilayered component, were diffusely positive for TTF-1 and napsin-A. EGFR Exon19 deletions were found in four cases, and BRAF V600E mutation was found in one case. These BA-like tumours have biphasic morphological and molecular characteristics of BA and lung adenocarcinoma, suggesting distal-type BA may develop into a malignancy. More cases should be studied and especially cases with metastasis should be searched to further prove the malignant transformation.

Simultaneous Quantitation of Seven Phenethylamine-Type Drugs in Forensic Blood and Urine Samples by UHPLC-MS-MS.

Abuse of new psychoactive substances (NPS) has become a health and social issue of global concern. p-Methoxyamphetamine (PMA)/p-methoxymethamphetamine (PMMA) with fluoro- or chloro-derivatives of amphetamine and methamphetamine were among the most common drugs found in specimens from fatal cases in Taiwan during the January 2011 to December 2018 period. A liquid-liquid extraction sample preparation protocol with highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry approach was developed for the simultaneous analysis of seven phenethylamine-type drugs-PMA, PMMA, p-methoxyethylamphetamine, 4-fluoroamphetamine (4-FA), 4-fluoromethamphetamine (4-FMA), 4-chloroamphetamine (4-CA) and 4-chloromethamphetamine (4-CMA)-in postmortem blood and urine specimens. Separation by liquid chromatography was performed by Agilent Zorbax SB-Aq column. Tandem mass spectrometry was operated in Agilent Jet Stream Technology electrospray ionization in positive-ion multiple reaction monitoring mode. An analytical methodology was evaluated using drug-free blood and urine after fortification with 100-2,000 ng/mL of the seven target analytes. Average extraction recoveries were >80%; slightly higher ion suppression was observed for PMA and 4-CA; intra-/inter-day precision (% coefficient of variation) and accuracy were in the ranges of 0.52-12.3% and 85-110%, respectively. Limit of detection and lower limit of quantitation for these seven analytes were both in the 0.5-5 ng/mL range. Interference and carryover were not significant. This relatively simple methodology was found effective and reliable for routine identification and quantitation of these seven analytes in postmortem and antemortem blood and urine specimens received in 2018. Analytical data obtained from these actual cases indicated the following: (i) compared to findings reported during the 2007-2011 period, the use of substituted phenethylamine-type drugs decreased in 2018; (ii) ketamine and 7-aminonimetazepam (the main metabolite of nimetazepam) were the most common co-ingested substances in specimens containing PMA/PMMA, 4-FA/4-FMA, or 4-CA/4-CMA; and (iii) in drug fatalities, the concentration of PMA was significantly higher than the concentration of PMMA in both urine and blood, while the reverse was true in urine specimens from antemortem cases.

Gastrointestinal Goblet Cell Adenocarcinomas Harbor Distinctive Clinicopathological, Immune, and Genomic Landscape.

Goblet cell adenocarcinoma (GCA) is a rare amphicrine tumor and difficult to diagnose. GCA is traditionally found in the appendix, but extra-appendiceal GCA may be underestimated. Intestinal adenocarcinoma with signet ring cell component is also very rare, and some signet ring cell carcinomas are well cohesive, having some similar morphological features to GCAs. It is necessary to differentiate GCA from intestinal adenocarcinomas with cohesive signet ring cell component (IACSRCC). The goal of this study is to find occurrence of extra-appendiceal GCA and characterize the histological, immunohistochemical, transcriptional, and immune landscape of GCA. We collected 12 cases of GCAs and 10 IACSRCCs and reviewed the clinicopathologic characters of these cases. Immunohistochemical stains were performed with synaptophysin, chromogranin A, CD56, somatostatin receptor (SSTR) 2, and Ki-67. Whole transcriptome RNA-sequencing was performed, and data were used to analyze differential gene expression and predict immune cell infiltration levels in GCA and IACSRCC. RNA-sequencing data for colorectal adenocarcinoma were gathered from TCGA data portal. Of the 12 patients with GCA, there were 4 women and 8 men. There were three appendiceal cases and nine extra-appendiceal cases. GCAs were immunohistochemically different from IACSRCC. GCA also had different levels of B-cell and CD8+ T-cell infiltration compared to both colorectal adenocarcinoma and cohesive IACSRCCs. Differential gene expression analysis showed distinct gene expression patterns in GCA compared to colorectal adenocarcinoma, with a number of cancer-related differentially expressed genes, including upregulation of TMEM14A, GOLT1A, DSCC1, and HSD17B8, and downregulation of KCNQ1OT1 and MXRA5. GCA also had several differentially expressed genes compared to IACSRCCs, including upregulation of PRSS21, EPPIN, RPRM, TNFRSF12A, and BZRAP1, and downregulation of HIST1H2BE, TCN1, AC069363.1, RP11-538I12.2, and REG4. In summary, the number of extra-appendiceal GCA was underestimated in Chinese patients. GCA can be seen as a distinct morphological, immunohistochemical, transcriptomic, and immunological entity. The classic low-grade component of GCA and the immunoreactivity for neuroendocrine markers are the key points to diagnosing GCA.

Taxonomy notes on Vandeae (Orchidaceae) from China: Five new species and two new records.

Five new species (Gastrochilus yei, Gastrochilus minimus, Luisia simaoensis, Taeniophyllum xizangense, Tuberolabium subulatum) and two newly recorded species (Cleisostoma tricornutum, Luisia inconspicua) of Vandeae (Orchidaceae) from China are described and illustrated. Gastrochilus yei is similar to G. affinis and G. nepalensis, but differs from them by having an epichile not lobed, the apex of the hypochile not bilobed, and a tine on the apex of the leaf. Gastrochilus minimus is similar to G. acinacifolius, but can be distinguished from the latter by having a flabellate epichile that is densely hirsute on the adaxial surface and an inconspicuous central cushion; in addition, the hypochile of G. minimus has a keel that extends to the apex of the epichile. Taeniophyllum xizangense is similar to T. stella and T. radiatum, but it is distinguished from them by having much bigger flowers, inflorescences densely covered with short-bristly hairs, papillae on the external surface of sepals, and bigger triangular-ovate viscidium. Luisia simaoensis is similar to L. magniflora and L. ramosii, but can be easily distinguished from them by having lateral sepals longer than dorsal sepals and petals, lip with irregular and waved margins, and lip with bilobed apex. Luisia inconspicua is moved from Gastrochilus to Luisia based on phylogenetic analyses of plastid matK sequence data. Tuberolabium subulatum is similar to T. carnosum, but it can be easily distinguished from the latter by having an inflorescence much shorter than the leaves, yellow sepals and petals, and many small papillae outside the lip lobes.

Three new species of Liparis s.l. (Orchidaceae: Malaxideae) from Southwest China based on morphological characters and phylogenetic evidence.

Liparis aureolabella and L. mengziensis, two new species from the karst region of southwestern China, and L. bingzhongluoensis, a new species from montane region in Yunnan, are described and illustrated. L. aureolabella is easily distinguished from its relatives by having abaxially purple leave with purple reticulate veins prominent adaxially, a lip auriculate at base, and falcate-lanceolate pollinia. Liparis mengziensis is closely related to L. petiolata and L. auriculata, but differs from them by having an ovate to broadly ovate leaf, purple lip and apex connate along the margins. Liparis bingzhongluoensis is similar to Liparis nanlingensis, but the new species is characterized by having a lip with two transparent ridges on its disc, longitudinally concave basal callus and triangular column wings. Phylogenetic analyses based on nuclear ribosomal ITS and plastid matK sequences showed that L. aureolabella and L. mengziensis are nested with L. petiolata or L. auriculata in a monophyletic clade. L. bingzhongluoensis is sister to a clade formed by L. nanlingensis, L. tsii, L. sasakii and L. krameri. Moreover, morphological comparisons strongly support that the three species as separated species newly to science.

Four new species and a new record of Orchidinae (Orchidaceae: Orchideae) from China.

Four new species of Orchidaceae from China, Heminium lijiangense, Peristylus fasciculatus, Platanthera milinensis, and Ponerorchis gongshanensis, together with a new country record, Peristylus tenuicallus, are described and illustrated based on morphological and/or phylogenetic analyses. Heminium lijiangense is closely related to H. elisabethae but differs from it by having the dorsal sepal ovate-orbicular and lip mid-lobe distinctly shorter than lateral lobes. P. fasciculatus is close to Peristylus tradescantifolius but is distinguished from it by having several fascicled and straight, root-like tubers (vs. one or two oblongoid tubers), old stems usually persistent, middle lobe of lip narrowly ligulate-lanceolate and half as long as the lateral lobes (vs. middle lobe deltoid, about a third as long as the lateral lobes or less), a raised callus at the base of each lateral lobe (vs. callus absent), spur gradually attenuate toward the apex (vs. spur clavate). Platanthera milinensis is similar to P. stenochila by sharing small green flowers and lip without a spur, but differs in having a creeping rhizome, a corymbose inflorescence, and a broadly ovate and slightly 3-lobed lip. Ponerochis gongshanensis is similar to P. faberi in its small flowers, but differs in having a linear leaf c. 3 mm wide (vs. leaf 5-13 mm wide), in the lip having collar-like raised margins on the sides of the spur entrance, and a mid-lobe which is notched at the apex but not divided into two divergent lobules that are nearly as large as the lateral lobes, as in P. faberi. All the proposed species obtained high support in phylogenetic analysis as new species. The recently described genus Apetalanthe is reduced to synonymy of Ponerorchis and a new combination is made.

Clinicopathological, Radiological, and Molecular Features of Primary Lung Adenocarcinoma with Morule-Like Components.

BACKGROUND: Morule-like component (MLC) was a rare structure in primary lung adenocarcinoma. We aimed to reveal the clinicopathological, radiological, immunohistochemical, and molecular features of lung adenocarcinoma with MLCs. METHODS: Twenty lung adenocarcinomas with MLCs were collected, and computed tomographic and histological documents were reviewed. Immunohistochemistry, targeted next-generation sequencing, and Sanger sequencing for ß-catenin gene were performed. RESULTS: There were 9 lepidic adenocarcinomas, 8 acinar adenocarcinomas, 2 papillary adenocarcinomas, and 1 minimally invasive adenocarcinoma. Most patients (16/17) were shown a pure solid nodule, and 1 patient was shown a partly solid nodule on chest computed tomography (CT). Nine cases were accompanied with micropapillary components, and 3 were with cribriform components in which 2 suffered a worse prognosis. No significant association was found between the MCLs and the overall survival of lung adenocarcinoma (P = 0.109). The MLCs were often arranged in whorled or streaming patterns. The cells in MLCs showed syncytial and mild appearance. The MLCs were positive for E-cadherin, CK7, TTF-1, napsin-A, vimentin, and ß-catenin (membrane), and negative for CK5/6, p40, p63, Synaptophysin, chromogranin A, and Cdx-2. EGFR mutation, ALK-EML4 fusion, HER2 amplification, and PIK3CA mutation were detected in 16 cases, 2 cases, 1 case, and 1 case, respectively. EGFR mutation was more frequent in adenocarcinomas with MLCs than those without MLCs (P = 0.040). ß-catenin gene mutation was not detected in any patients. CONCLUSIONS: MLC is often observed in the background of acinar, lepidic, and papillary adenocarcinomas. Lung adenocarcinomas with MLCs tend to appear as a solid mass on CT and harbor EGFR gene mutations. The micropapillary components and cribriform components may cause poor prognosis of lung adenocarcinomas with MLCs. Vimentin is always positive in MLCs, and it is a useful marker for the identification of MLCs.

Chromosome-Scale Genome Assembly of the Resurrection Plant Acanthochlamys bracteata (Velloziaceae).

Acanthochlamys bracteata (Velloziaceae) is a resurrection plant with cold tolerance. Herein, a chromosome-level reference genome of A. bracteata based on Nanopore, Illumina, and Hi-C data is reported. The high-quality assembled genome was 197.97 Mb, with a scaffold N50 value of 8.64 Mb and a contig N50 value of 6.96 Mb. We annotated 23,509 protein-coding genes. Eight contracted gene families and three expanded gene families were detected. Repeat sequences accounted for approximately 28.63% of the genome. The LEA1 and Dehydrin gene families, which are involved in desiccation resistance, expanded in A. bracteata. We identified genes involved in chilling tolerance, COLD1.

Indolent T cell lymphoproliferative disorder of the gastrointestinal tract: an uncommon case with lymph node involvement and the classic Hodgkin's lymphoma.

Here we describe an unusual case of an indolent CD8+ T-cell lymphoproliferative disorder in the gastrointestinal tract (ITCLD-GT) accompanied by neck lymph node infiltration and new onset of classic Hodgkin's lymphoma after two years follow-up. Previously, this 42-year-old Asian man suffered from diarrhea and abdominal pain for two years. Intestinal biopsies showed a group of small to intermediate-sized lymphocytes which were monomorphic as well as arranged in a nodular pattern with no clear boundary and were diagnosed as ITCLD-GT. He did not receive chemotherapy or have any disease progression in the gastrointestinal tract (GIT) during the follow-up until a development of neck lymphadenopathy, which led to an eventual mixed cellularity type of Hodgkin's lymphoma, one type of classic HL diagnosis. Interestingly, besides the Hodgkin's cells, the same pathological T-cells in the GIT were present in the Hodgkin's lymphoma lesions. These two pathological T cells in GIT and neck lymph node had the identical histopathological and molecular abnormalities that confirmed the abenteric distant infiltration of ITCLD-GT to the neck lymph node in this patient. This is the first case of ITCLD-GT that has a definite distant lymph node invasion. ITCLD-GT usually has a relatively good prognosis but patients with ITCLD-GT may have abenteric distant infiltration. Thus, long-term follow-up and further study of the underlying mechanisms of this process are necessary.

Pulmonary peripheral glandular papilloma and mixed squamous cell and glandular papilloma frequently harbour the BRAF V600E mutation.

AIMS: Pulmonary peripheral glandular papilloma (GP) and mixed squamous cell and glandular papilloma (MP) have very similar histological features to pulmonary ciliated muconodular papillary tumour (CMPT)/bronchiolar adenoma (BA). The underlying genetic relationships between GP/MP and CMPT/BA have rarely been characterised. We aimed to reveal the relationship between them. METHODS AND RESULTS: We performed a clinicopathological review and next-generation sequencing (NGS) study of two GPs and five MPs. Histologically, GPs/MPs showed similar cellular and architectural features to CMPTs/BAs, such as bilayered epithelium, bronchiole-associated lesions and skipping (discontinuous) growth pattern. One MP showed partial and inconspicuous endobronchiolar growth and more glandular structures, which was very similar to the appearance of CMPT/BA. BRAF V600E mutation was detected in four papillomas (57.1%, one GP and three MPs). CONCLUSIONS: Overlapping morphological features and comparable mutation profiles support that peripheral GPs/MPs and CMPTs/BAs are on the same disease spectrum. We propose expanding the concept of CMPT/BA and including GP and MP in the CMPT/BA family.

Extrapancreatic solid pseudopapillary neoplasm: report of a unique case of primary posterior mediastinum origin and review of the literature.

Solid pseudopapillary neoplasm (SPN) is a rare and low malignant potential neoplasm that traditionally occurs in pancreas. Herein, we report a mediastinal SPN in a 62-year-old woman. Clinically, the patient was asymptomatic. A mass in posterior mediastinum was detected by chest computerized tomographic (CT) scan during her annual checkup. The CT scan revealed a 30 mm solid nodule with well-defined outline in right posterior mediastinum. Histologically, the tumor was comprised of solid cellular nests as well as sheets of cells with an epithelioid appearance, and some pseudopapillary areas could also be identified. Immunohistochemically, the tumor cells were positive for ß-catenin (nuclear and cytoplasmic), cyclin D1, CD56, CD10, CD99 (paranuclear dot-like), SOX11 (weak) and TFE3, while negative for cytokeratin (AE1/AE3), E-cadherin, WT-1, synaptophysin, chromogranin and progesterone receptor. SPNs can occur in aberrant locations and this is the first one reported in mediastinum, pathologists should learn about the rare case for a better differential diagnosis. The patient underwent a video-assisted thoracoscope tumorectomy. She has been followed up for 5 months with no recurrence or metastasis.

Esophageal invasive acantholytic anaplastic Paget's disease: report of a unique case.

Paget's disease (PD) is an intraepithelial growth of neoplastic cells showing glandular differentiation. Primary esophageal PD is extremely rare, with only 14 cases reported to date. We report a case of esophageal PD in a 63-year-old man presenting with progressive dysphagia. On gross examination, the esophageal mucosa had a slightly mottled appearance and felt slightly thickened and indurated. Microscopically, the atypical tumor cells were mostly located in middle to basal cell layers of the squamous epithelium. Some tumor cells were difficult to be distinguished from normal squamous epithelium. Some regions of the lesion showed full-thickness cellular atypia with mitotic figures, and some tumor cells invaded through the basement membrane into the lamina propria, mimicking a squamous cell carcinoma. Acantholytic regions were prominent in the epithelium, and some gland-like clefts were formed. One recurrent laryngeal nerve lymph node showed metastatic foci. Immunohistochemically, tumor cells were positive for cytokeratin (CK) 7, CK8/18, carcinoembryonic antigen (CEA) and Her-2, but negative for CK5/6, p63, S-100 protein and HMB45, yielding the diagnosis of PD. This is the first case report of esophageal invasive Paget's disease (invPD) and the first case report of esophageal acantholytic anaplastic Paget's disease (AAPD).

Simultaneous Quantitation of Methamphetamine, Ketamine, Opiates and their Metabolites in Urine by SPE and LC-MS-MS.

Heroin, methamphetamine and ketamine have been the most commonly abused drugs in Taiwan. The presence of these drugs and their metabolites in postmortem specimens has been routinely monitored in our laboratory mostly by gas chromatographic-mass spectrometric methods. This study aimed to evaluate a more effective approach to simultaneously quantify these analytes (i.e., amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), morphine, codeine, 6-acetylmorphine, 6-acetylcodeine, ketamine and norketamine) in postmortem urine and blood specimens by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Samples (1 mL) were extracted via solid-phase extraction, evaporated and reconstituted in the mobile phase for injection into the LC-MS-MS system. Respective deuterated analogs of these analytes were used as internal standards. Chromatographic separation was achieved by an Agilent Zorbax SB-Aq analytical column at 50°C. Mass spectrometric analysis was performed by electrospray ionization in positive-ion dynamic multiple reaction monitoring mode with optimized collision energy for respective precursor ion selected for each analyte, and the monitoring of two transition ions. Performance characteristics were assessed using drug-free samples that were fortified with 50-1,000 ng/mL of the 10 analytes. Analytical parameters evaluated and resulting data are as follows: (i) average extraction recoveries (n= 3) were better than 80%, except for MDMA (71%) and morphine (74%); (ii) inter-day and intra-day precision ranges (%CV) were 1.59-8.80% and 0.57-3.89%, respectively; (iii) calibration linearity (r2), detection limit and quantitation limit for all analytes were >0.999, 1 and 5 ng/mL, respectively; (iv) matrix effects (ion suppression) were observed for three analytes, but were satisfactorily compensated for by the deuterated internal standards adopted in the analytical protocol. This method was successfully applied to the analysis of specimens collected from unknown death cases from various district prosecutors' offices in Taiwan, and was also found helpful to understanding whether the detected opiates were derived from heroin or legal morphine/codeine-containing medications.

Developing a UHPLC-QTOF-MS and Automated Library Search Method for Screening Drugs and Toxic Compounds in Postmortem Specimens.

Screening and confirming the presence of drugs and toxic compounds in various matrices are important and challenging tasks routinely faced by forensic and clinical laboratories. Recent advances in the liquid chromatographic and mass spectrometric technologies have provided an opportunity for the development of more specific and effective approaches to achieve the "screening" and "confirmation" goals in a single analytical step. The objectives of this study are: (i) the establishment of an ultra-high performance liquid chromatographic, quadrupole time-of-flight mass spectrometric mass spectrometric and MS-MS spectral database, including 1,200 compounds of interest; and (ii) the development of an effective protocol, using this database and three searching algorithms, for general unknown screening of these compounds. The established database and protocol were evaluated through the analysis of 30 external proficiency test and 100 postmortem samples and found to be significantly more effective than the LC-IT-MS and GC-MS approaches previously established in our laboratory.